BY NASIR ALI

CHARACTERISTICS OF HEAVY METAL UPTAKE AND

ACCUMULATION IN SELECTED PLANT SPECIES FOR

DEVELOPMENT OF PHYTOEXTRACTION TECHNOLOGY

BY

NASIR ALI

DEPARTMENT OF BIOTECHNOLOGY

UNIVERSITY OF MALAKAND,

CHAKDARA, DIR (L)

2016

CHARACTERISTICS OF HEAVY METAL UPTAKE AND

ACCUMULATION IN SELECTED PLANT SPECIES FOR

DEVELOPMENT OF PHYTOEXTRACTION TECHNOLOGY

BY

NASIR ALI

A thesis submitted to the Department of Biotechnology University of Malakand

for the partial fulfillment of the requirement for the degree of Doctor of

Philosophy (PhD) in Biotechnology

DEPARTMENT OF BIOTECHNOLOGY

UNIVERSITY OF MALAKAND,

CHAKDARA, DIR (L)

2016

DECLERATION

I declare that this work is original and I have not used other than the declared sources/

resources, and have explicitly marked all materials which have been quoted either literally or

by content from the used sources. I also declare that this work has so far neither been

submitted to Department of Biotechnology University of Malakand, Pakistan, for obtaining

the degree of PhD in Biotechnology or any other program.

Furthermore, the studies were carried out in the laboratory of the Department of

Biotechnology, University of Malakand Chakdara. Lower Dir, KPK Pakistan.

______________________________

Nasir Ali

ACKNOWLEDGEMENTS

I deem it highest pleasure to avail this opportunity to express my

heartiest gratitude to my supervisor, Dr. Fazal Hadi, Assistant Professor,

Department of Biotechnology, University of Malakand. His skillful guidance,

technical approach, art of making useful suggestions and inspiring attitude

made it very easy to undertake this work and to write this manuscript.

I am thankful to Dr. Syed Muhammad Jamal (Chairman Department of

Biotechnology) and other staff members of the Biotechnology Department,

for their sincere encouragement.

The Pakistan Science foundation is highly acknowledged for the financially

support of main part of my PhD research project, under the Pak-US linkage

program. The Directorate of Science and Technology, Khyber Pakthoonkhwa is

highly acknowledged for financially support of a part of my PhD research

project. The Higher Education Commission of Pakistan is acknowledged for

their support in term of Laptop provision Under Prime Minister Laptop

Scheme and reimbursement of the University fees Under Prime Minister fee

reimbursement scheme. Without their financial support it would be very

difficult for me to complete my PhD study.

I am very thankful to my parents, brother and sisters whose sincere

wishes accompanied me all the way my career. It was, in fact, their moral

support that gave me the confidence for taking this research work.

At last and not the least, I offer my thanks to all those who helped

me especially Dr. Ayaz Ahmad, Mr. Aminullah Jan (PhD Scholar) and Mr.

Altaf Hussain (MPhil Scholar) for their encouragement during the present

study.

In the last, all the errors that remain are mined alone.

NASIR ALI

Copyright Statement

This copy of the thesis has been supplied on condition that anyone who consults it is

understood to recognize that its copright rests with its author and that no quotation

from the thesis and no information derived from it may be published without the

author’s prior consent.

PREFACE

The thesis comprises of five chapters. A detail introduction to the problem has been given in

the first chapter. In second chapter the effect of chemical chelator (EDTA) and plant growth

regulator (Gibberellic acid) has been evaluated on the cadmium phytoextraction potential of

P. hysterophorus plant and include biochemical and physiological analysis. This chapter was

financially supported by Directorate of Science and Technology. In third chapter the effects

of various treatments of molybdenum (Mo) have been investigated on cadmium uptake and

accumulation in Ricinus communis and Cannabis sativa plants and studied the physiological

and biochemical changes occurred in the plants under cadmium stress. In fourth chapter, the

effect of Mo on expression of four CBF/DREB like genes in Ricinus communis and Cannabis

sativa plants under Cd stress have been investigated. The experimental work of chapter 3rd

and 4th

were financially supported by the Pakistan Science Foundation (PSF). The fifth

chapter gives a detailed discussion of the whole thesis and at the end of 5th

chapter

recommendations for further research has been given.

Table of contents

LIST OF FIGURES ...................................................................................................................................... I

LIST OF TABLES ...................................................................................................................................... III

LIST OF ABBREVATIONS ...................................................................................................................... V

LIST OF PUBLICATIONS ...................................................................................................................... VII

ABSTRACT .... ……………………………………………………..……………………………………………………………………………VIII

CHAPTER 1: GENERAL INTRODUCTION ............................. 1

1.1 Cadmium (Cd) as an environmental pollutant ........................................................................... 1

1.2 Sources of Cadmium pollution into the environment ............................................................... 1

1.3 Cadmium toxicity in humans ....................................................................................................... 2

1.4 Cadmium and plants ...................................................................................................................... 4

1.4.1 Cadmium uptake, transport and accumulation in plants ............................................... 4

1.4.2 Cadmium toxicity in plants ............................................................................................... 4

1.5 Remediation of toxic metals polluted soil ................................................................................ 10

1.5.1 Phytoremediation technologies ...................................................................................... 11

1.6 Enhanced phytoextraction .......................................................................................................... 13

1.6.1 Gibberellic acid (GA3) ..................................................................................................... 13

1.6.2 Ethylenediaminetetraacetic acid (EDTA) ..................................................................... 14

1.6.3 Molybdenum (Mo) ........................................................................................................... 14

1.7 Introduction to experimental plants........................................................................................... 15

1.7.1 Parthenium hysterophorus .............................................................................................. 15

1.7.2 Ricinus communis ............................................................................................................. 16

1.7.3 Cannabis sativa ................................................................................................................ 16

1.8 Aim and objectives ...................................................................................................................... 18

CHAPTER 2: THE EFFECT OF GIBBERELLIC ACID AND EDTA ON

CADMIUM PHYTOEXTRACTION: CORRELATIONS OF FREE

PROLINE, TOTAL PHENOLICS AND CHLOROPHYLL CONTENTS

WITH CADMIUM ACCUMULATION IN PARTHENIUM

HYSTEROPHORUS PLANT. ................................................................................... 20

ABSTRACT ........................................................................................................................................ 20

2.1 INTRODUCTION ....................................................................................................................... 21

2.1.1 Aim and objectives........................................................................................................... 23

2.2 MATERIALS AND METHODS .............................................................................................. 24

2.2.1 Preparation of soil and addition of cadmium................................................................ 24

2.2.2 Transplantation of seedlings and plant growth............................................................. 24

2.2.3 Treatments used ................................................................................................................ 24

2.2.4 Plant growth parameters analysis ................................................................................... 25

2.2.5 Analysis of free proline in plant root and leaves.......................................................... 25

2.2.6 Total phenolics estimation in roots and leaves ............................................................. 26

2.2.7 Chlorophyll estimation in leaves .................................................................................... 26

2.2.8 Cadmium (Cd) analysis in the plant .............................................................................. 27

2.2.9 Statistical analysis ............................................................................................................ 27

2.3 RESULTS ..................................................................................................................................... 28

2.3.1 Effect of EDTA and GA3 treatments on plant length (root and stem), biomass

(fresh and dry) and water contents of P. hysterophorus plant under Cd stress ........ 28

2.3.2 Effect of different treatments of GA3 and EDTA on plant Cd contents .................. 31

2.3.3 Effect of different treatments of GA3 and EDTA on total phenolics, free proline

and chlorophyll (a/b) contents of the plant under Cd stress ....................................... 33

2.3.4 Correlation among different parameters measured in plant ....................................... 35

2.4 DISCUSSION .............................................................................................................................. 40

2.4.1 Plant growth and biomass ............................................................................................... 40

2.4.2 Plant cadmium contents ................................................................................................... 41

2.4.3 Proline concentration ....................................................................................................... 41

2.4.4 Phenolics concentration within plant tissues ................................................................ 42

2.4.5 Chlorophyll contents ........................................................................................................ 42

Conclusions ................................................................................................................................. 42

CHAPTER 3: THE EFFECT OF MOLYBDENUM ON CADMIUM

PHYTOEXTRACTION AND PRODUCTION OF ENDOGENOUS

PHENOLICS, FREE PROLINE AND PHOTOSYNTHETIC PIGMENTS

IN RICINUS COMMUNIS AND CANNABIS SATIVA PLANTS. .................. 44

ABSTRACT ........................................................................................................................................ 44

3.1 INTRODUCTION ....................................................................................................................... 45

Aim and objectives .................................................................................................................... 47

3.2 MATERIALS AND METHODS .............................................................................................. 48

3.2.1 Preparation of soil and addition of cadmium................................................................ 48

3.2.2 Transplantation of seedlings and plant growth............................................................. 48

3.2.3 Molybdenum treatments .................................................................................................. 48

3.2.4 Plant growth parameters .................................................................................................. 50

3.2.5 Free proline analysis in root and leaves ........................................................................ 50

3.2.6 Total phenolics estimation in roots and leaves ............................................................. 50

3.2.7 Chlorophyll and carotenoids estimation in leaves ....................................................... 50

3.2.8 Cadmium (Cd) analysis in different plant parts ........................................................... 51

3.2.9 Statistical analysis ............................................................................................................ 51

3.3 RESULTS ..................................................................................................................................... 52

3.3.1 Ricinus communis plant ................................................................................................... 52

3.3.2 Cannabis sativa plant ...................................................................................................... 79

3.4 DISCUSSION ............................................................................................................................ 106

3.4.5 Conclusions ..................................................................................................................... 108

CHAPTER 4: EXPRESSION OF CBF/DREB LIKE TRANSCRIPTIONAL

FACTORS GENES IN RICINUS COMMUNIS AND CANNABIS SATIVA

PLANTS UNDER CADMIUM STRESS AND MOLYBDENUM FOLIAR

SPRAY ......................................................................................................................... 109

ABSTRACT ...................................................................................................................................... 109

4.1 INTRODUCTION ..................................................................................................................... 110

4.1.1. Aim and objectives ...................................................................................................... 112

4.2 MATERIALS AND METHODS ............................................................................................ 113

4.2.1 Plant materials and growth conditions ........................................................................ 113

4.2.2 Treatments during the experiment ............................................................................... 113

4.2.3 Genomic DNA extraction and amplification of DREB 1A, DREB 1B, DREB 1F

and CBF like genes sequences ..................................................................................... 113

4.2.4 Total RNA extraction .................................................................................................... 115

4.2.5 cDNA synthesis and Identification of DREB 1A, DREB 1B, DREB 1F and CBF

like genes ......................................................................................................................... 115

4.2.6. Sequence analysis.......................................................................................................... 116

4.2.7. Data analysis .................................................................................................................. 116

4.2.8. Experimental Design .................................................................................................... 117

4.3. RESULTS .................................................................................................................................. 118

4.3.1 Ricinus communis ........................................................................................................... 118

4.3.2 Cannabis sativa .............................................................................................................. 131

4.4 DISCUSSION ............................................................................................................................ 144

4.5 Conclusions ........................................................................................................................ 146

CHAPTER 5: GENERAL DISCUSSION .................................................................... 146

5.1. Phytoextraction as a promising green technology for heavy metals remediation ............ 146

5.2. CBF/DREB transcriptional factors (CBF regulon) can play role in phytoextraction of

cadmium ....................................................................................................................................... 146

5.3. Proline and phenolic compounds enhance plant defense and cadmium phytoextraction

....................................................................................................................................................... 147

5.4. High biomass and high concentration of toxic metals in the biomass greatly enhanced the

phytoextraction ability of plants ................................................................................................ 147

5.5. Conclusions ............................................................................................................................... 149

5.6. Recommendations .................................................................................................................... 150

REFERENCES ............................................................................................................................. 151

LIST OF FIGURES

Figure 1. 1: Effects of Cd on plant. ................................................................................................................ 5

Figure 1. 2 : Plants used during the experiments.. ....................................................................................... 17

Figure 2. 1: Effect GA3 and EDTA on the root and shoot length of P. hysterophorus................. 29

Figure 3. 1: Effect of different treatments of Mo on growth of Ricinus communis plant grown ... 56

Figure 3. 2: Overall effect of Mo on phenolic and proline concentration in Ricinus communis .... 62

Figure 3. 3: Overall effect of molybdenum on Cd accumulation and BCF in Ricinus communis. 68

Figure 3. 4: Effect of Mo on growth of Cannabis sativa plant under Cd stress. ............................ 83

Figure 3. 5: Overall effect of Mo on concentration of phenolic and proline Cannabis sativa. ...... 89

Figure 3. 6: Overall effect of Mo on Cd accumulation and Cd-bioconcentration in C. sativa. ...... 95

Figure 4.1: Total DNA extracted from Ricinus communis. .......................................................... 118

Figure 4.2: PCR product of CBF/DREB genes fragments from genomic DNA of R. communis.118

Figure 4.3: RT-PCR product of Actin gene fragment from Ricinus communis............................ 119

Figure 4. 4: RT-PCR product of DREB 1A and DREB 1B genes fragments of R. communis ..... 120

Figure 4. 5: RT-PCR product of DREB 1F and CBF like genes fragments of R. communis ...... 121

Figure 4. 6: Nucleotide sequence alignment of Ricinus communis DREB-1B............................. 123

Figure 4. 7: Nucleotide sequence alignment of Ricinus communis DREB-1F ............................ 124

Figure 4. 8: Nucleotide sequence alignment of Ricinus communis CBF like gene ...................... 125

Figure 4. 9: Multiple alignment CBF/DREB deduced amino acids sequence of R. communis .... 127

Figure 4. 10: Correlations of Cd accumulation with gene expression in Ricinus communis. ....... 129

Figure 4. 11: Correlations of genes expression with proline and phenolics in R. communis ....... 130

Figure 4. 12: Genomic DNA from Cannabis sativa plant. ........................................................... 131

Figure 4. 13: PCR product of DREB 1A, DREB 1B, DREB 1Fand CBF like transcription factor

of Cannabis sativa plant. ......................................................................................... 131

Figure 4. 14: RT-PCR product of Actin gene fragment ofCannabis sativa. ............................... 132

Figure 4. 15: RT-PCR product of DREB 1A and DREB 1B like genes of Cannabis sativa ...... 133

Figure 4. 16: RT-PCR product of DREB 1F and CBF gene of Cannasbis sative plant ............... 134

Figure 4. 17: Nucleotide sequence alignment of Cannabis sativa DREB 1B gene ...................... 136

Figure 4. 18: Nucleotide sequence alignment of Cannabis sativa DREB 1F gene ...................... 137

Figure 4. 19: Nucleotide sequence alignment of Cannabis sativa CBF like gene segment ......... 138

Figure 4. 20: Multiple alignment and comparison of the deduced amino acids sequence ........... 140

Figure 4. 21: Correlation of Cd accumulation with CBF/DREB genes expression in C. sativa .. 142

Figure 4. 22: Correlations of CBF/DREB genes expression with proline and phenolics in

Cannabis sativa plant. .............................................................................................. 143

LIST OF TABLES

Table 2. 1: Treatments done during the experiment. ............................................................................. 24

Table 2. 2: Effect of different treatments of GA3 and EDTA on growth of P. hysterophorus ............ 30

Table 2. 3: Effect of GA3 and EDTA on Cd contents within Parthenium hysterophorus plant ............ 32

Table 2. 4: Effect of EDTA and GA3 on free proline, total phenolics and chlorophyll contents. ........ 34

Table 2. 5: Correlations between different parameters in roots of P. hysterosphorus plant.................. 37

Table 2. 6: Correlations among different parameters measured in stem of P. hysterosphorus plant. ... 38

Table 2. 7: Correlation between the parameters measured in leaves of P. hysterosphorus plant. ......... 39

Table 3. 1: Treatments used during the experiment. .............................................................................. 49

Table 3. 2 Effect of Mo on plant growth in Ricinus communis plant under 25 ppm Cd polluted soil .. 53

Table 3. 3: Mo effect on growth of Ricinus communis plant in 50 ppm Cd polluted soil.. ................... 54

Table 3. 4: Effect of Mo on growth of R. communis plant grown in 100 ppm Cd contaminated soil. .. 55

Table 3. 5: Mo effect on proline, phenolic and photosynthetic pigments in R. communis plant grown in

25 ppm Cd contaminated soil. .......................................................................................... 59

Table 3. 6: Role of Mo treatments in proline, phenolic and photosynthetic pigments concentration in

Ricinus communis plant grown in 50 ppm Cd contaminated soil.. ................................... 60

Table 3. 7: Effect of Mo treatments on free proline, total phenolics and photosynthetic pigments in

Ricinus communis plant grown in 100 ppm Cd contaminated soil.. ................................. 61

Table 3. 8: Mo effect on Cd contents in Ricinus communis plant in 25 ppm Cd contaminated soil. ... 65

Table 3. 9 Effect of Mo on cadmium contents in R. communis plant in 50 ppm Cd contaminated soil.66

Table 3. 10: Mo effect on Cd contents in R. communis grown in 100 ppm Cd polluted soil. ............... 67

Table 3. 11: Different correlations in roots of R. communis grown in 25 ppm Cd contaminated soil. . 70

Table 3. 12: Correlations in roots of R. communis plant in 50 ppm Cd polluted soil. ........................... 71

Table 3. 13: Different correlations in roots of R. communis plant grown in 100 ppm Cd polluted soil.72

Table 3. 14: Correlations in stem of R. communis plant in 25 ppm Cd contaminated soil. ................... 73

Table 3. 15: Various correlations in stem of Ricinus communis grown in 50 ppm Cd polluted soil..... 74

Table 3. 16: Different correlations in stem of Ricinus communis plant under 100 ppm Cd stress. ....... 75

Table 3. 17: Correlations in Leaves of R. communis plant grown in 25 ppm Cd contaminated soil. .... 76

Table 3. 18: Different Correlations in Leaves of R. communis plant under 50 ppm Cd stress. ............. 77

Table 3. 19: Correlations in leaves of R. communis plant grown in 100 ppm Cd contaminated soil. ... 78

Table 3. 20: Effect of Mo on growth of Cannabis sativa plant under 25 ppm Cd stress ..................... 80

Table 3. 21: Mo effect on growth of Cannabis sativa plant in 50 ppm Cd contaminated soil. ............. 81

Table 3. 22: Effect of Mo on growth C. sativa plant under 100 ppm Cd stress .................................... 82

Table 3. 23: Effect of Mo on proline, phenolic, chlorophylls and carotenoids concentration in

Cannabis sativa plant grown in with 25 ppm Cd contaminated soil. ............................... 86

Table 3. 24: Mo effect on proline, phenolic chlorophyll (a, b) and carotenoids concentration in

Cannabis sativa plant grown in soil contaminated with 50 ppm Cd. ............................... 87

Table 3. 25: Effect of Mo on proline, phenolic, chlorophyll and carotenoids concentration in Cannabis

sativa plant grown in 50 ppm Cd soil. .............................................................................. 88

Table 3. 26: Cadmium contents in C. sativa plant under Mo treatments and 25 ppm Cd in soil. ......... 92

Table 3. 27: Effect of Mo on Cd contents in C. sativa plant grown in 50 ppm Cd polluted soil. ......... 93

Table 3. 28: Mo effect on Cd contents of C. sativa plant grown grown in 100 ppm Cd polluted soil. . 94

Table 3. 29: Correlations in roots of C. sativa plant grown under 25 ppm Cd stress . .......................... 97

Table 3. 30: Different Correlations in roots of C. sativa plant grown in 50 ppm Cd polluted soil. ...... 98

Table 3. 31: Correlations in roots of C. sativa plant grown in 100 ppm Cd contaminated soil . .......... 99

Table 3. 32: Correlations in stem of C. sativa plant under 25 ppm Cd stress ..................................... 100

Table 3. 33: Different correlations in stem of C. sativa plant grown in 50 ppm Cd containing soil

…………………………………………………………………………………………..101

Table 3. 34: Correlations in stem of C. sativa plant grown in 100 ppm Cd contaminated soil . ......... 102

Table 3. 35: Correlations in leaves of Cannabis sativa plant under 25 ppm Cd stress ...................... 103

Table 3. 36: Different correlations in leaves of Cannabis sativa plant under 50 ppm Cd stress. ........ 104

Table 3. 37: Correlations in leaves of C. sativa plant in 100 ppm Cd contaminated soil ………… .105

Table 4. 1: The following treatments were made during the experiment…………………......……..113

Table 4. 2: Primers used during the experiments. .............................................................................. 114

LIST OF ABBREVATIONS

Abbreviations Full Names

µL Microliter

0C Degree Centigrade

ABA Abscisic acid

ANOVA Analysis of variance

AP2 Activating Protein 2

BCF bio concentration factor

BLAST Basic Local Alignment Search Tool

Bp Base pair

CBF C-Repeat Binding Factor

Cd Cadmium

cDNA complementary DNA

Cm Centimeter

DW Dry weight

DNA Deoxyribonucleic acid

dNTP Deoxy Nucleotide triphosphate

DREB Dehydration Responsive Element Binding proteins

EDTA Ethylenediaminetetraacetic acid

ERF Ethylene-Responsive Factor

FC Folin-Ciocalteau reagent

G Gram

GA3 Gibberellic Acid

HSD Honestly Significant Difference

Mg Milligram

mg/L milligram per liter

mL Milli liter

Mo Molybdenum

N Nitrogen

Na+ Sodium ion

NCBI National Center for Biotechnological Information

Abbreviations Full Names

PAD Peripheral artery disease

PCR Polymerase Chain Reaction

pH Power of hydrogen ion concentration

Ppm Part per million

R2 Coefficient of Determination

RNA Ribonucleic acid

ROS Reactive Oxygen Species

rpm Revolution per minute

rpm Revolution per minute

SD Standard deviation

SPSS Statistical Package for Social Sciences

TF Transcriptional Factor

USEPA United State Environmental Protection Agency

μgg-1

Microgram per gram

LIST OF PUBLICATIONS

1. Nasir Ali and Fazal Hadi ―The effect of Gibberellic acid and EDTA on Cd

phytoextraction: correlation of free proline, total phenolics and chlorophyll content

with Cd contents of Parthenium hysterophorus plant. Environmental Science and

Pollution Research (2015) 22:13305–13318. (Impact factor 2.828).

2. Ayaz Ahmad, Fazal Hadi, and Nasir Ali ―Effective phytoextraction of cadmium

with increasing concentration of total phenolics and free proline in Cannabis sativa

plant under various treatments of fertilizers, plant growth regulators and sodium

salt‖ International Journal of Phytoremediation. 17: 56 – 65, 2015. (Impact factor

1.766)

3. Fazal Hadi, Nasir Ali, and Ayaz Ahmad ―Enhanced phytoremediation of Cd-

contaminated soil by Parthenium hysterophorus plant: Effect of gibberellic acid

(GA3) and synthetic chelator alone and in combinations‖ Bioremediation Journal,

18(1):46–55, 2014. (Impact factor 0.714)

4. Fazal Hadi, Sana Ullah, Fazal Hussain, Ayaz Ahmad, Amin Ullah Jan, Nasir Ali. ―Nitrogen fertilizer and EDTA effect on Cannabis sativa growth and

Phytoextraction of heavy metals (Cu and Zn) contaminated soil‖ International

Journal of Agronomy and Agricultural Research (IJAAR). 4 (6); 85-90. 2014.

(Impact factor 1.759).

5. Fazal Hadi, Ayaz Ahmad, Nasir Ali, ―Cadmium (Cd) removal from saline water

by Veronica anagallis and Epilobium laxum plants in hydroponic system‖

Agricultural Sciences, 5, 935-944 (2014). (Impact factor 0.117)

6. Fazal Hadi, Fazal Hussain, Muhammad Hussain, Sanaullah, Ayaz Ahmad, Saleem

Ur Rahman , Nasir Ali ―Phytoextraction of Pb and Cd; the effect of Urea and

EDTA on Cannabis sativa growth under metals stress” International Journal of

Agronomy and Agricultural Research (IJAAR). 5(3), 30-39. 2014. (Impact factor

1.759).

7. M. Tariq, G. Ali, F. Hadi, S. Ahmad, Nasir Ali and A. A. Shah. ―Callus induction

and invirto plant regeneration Rice (Oryza sativa L.) under various conditions‖

Pakistan Journal of Biological Sciences 11(2): 255-259, 2008.

8. Submission of two manuscripts are under process from chapter 3.

9. Submission of two manuscripts are under process from chapter 4.

Nasir Ali (2015) Characteristics of heavy metal uptake and accumulation in selected plant

species for development of phytoextraction technology. PhD Dissertation, Department of

Biotechnology, University of Malakand, pp: 1-177.

ABSTRACT

Metals are a group of highly toxic contaminants in the environment. Cadmium (Cd) is a

hazardous metal and its presence in soil is a serious threat to sustainable agriculture and to the

environment. Contaminated food is a major source of Cd entrance into the human body.

Cadmium can severely affect almost all the vital organs of human body, especially the liver

and kidney. Pollution of soil, especially agricultural fields contaminated with toxic metals,

has become a global problem and demands economic, efficient and environment friendly

remediation technologies. Phytoextraction is a potential plant-based technology for the

decontamination of polluted soil and water. It is an economic, solar driven, and environment

friendly technology. In the present study, physiological, biochemical and molecular

characteristics of cadmium uptake and accumulation in three plant species (Parthenium

hysterophorus, Ricinus communis and Cannabis sativa) were studied for the development of

phytoextraction technology.

In the first experiment, different treatments of Gibberellic Acid (GA3 10−2

, 10−4

and 10−6

M

as foliar spray) and Ethylenediaminetetraacetic acid (EDTA 40 mgKg-1

soil as single dose

and 10 mgKg-1

soil given in four doses) were studied for their effects on Cd phytoextraction,

and concentration of proline, phenolics and chlorophyll in Parthenium hysterophorus plants

grown in Cd (100 ppm) contaminated soil. The plants showed Cd hyperaccumulator potential

based on Cd bio-concentration factor (BCF > 1 in control plants). The GA3 and EDTA

application increased the extent of Cd phytoextraction by the plants. Most significantly

increase in Cd accumulation and bio concentration (BCF 9.75 ± 0.34) were found in plants

treated with GA3 (10−2

M) in combination with split doses of EDTA. Gibberellic acid

significantly increased the concentrations of phenolics and chlorophyll in the plants.

Cadmium accumulation in plant tissues showed positive correlation with free proline (R2

=

0.527, R2= 0.630) and total phenolics (R

2 = 0.554, R

2 = 0.723) in roots and leaves,

respectively.

In the second experiment, physiological and biochemical analyses were performed. The

objectives were to assess the effect of Molybdenum (Mo 0.5, 1.00 and 2.00 ppm) on Cd

phytoextraction, and concentration of endogenous proline, phenolics and photosynthetic in

Ricinus communis and Cannabis sativa plants grown in Cd (25, 50 and 100 ppm)

contaminated soil. Molybdenum was applied as a foliar spray, soil addition and seed soaking.

Foliar spray of Molybdenum highly increased Cd uptake and accumulation in both plants.

Molybdenum seed soaking and foliar spray highly increased the biomass, concentration of

free proline and total phenolics as compared to control plants. Positive correlations of proline

and phenolics with Cd accumulation were found in roots and leaves; suggesting a significant

role of proline and phenolics in Cd phytoaccumulation.

Molecular investigation was carried out with objectives: (1) To determine the presence and

then expression of DREB-1A, DREB-1B, DREB-1F and CBF like genes in Ricinus

communis and Cannabis sativa plants, (2) To evaluate the effect of molybdenum and

cadmium on expression of these genes, (3) To correlate the expression of genes with Cd

accumulation, and free proline and total phenolics concentrations in plants. Molybdenum was

applied as a foliar spray (0.5, 1, 2 ppm) while Cd (50 ppm) was added to soil. cDNA was

synthesized through reverse transcriptase Polymerase chain reaction (RT-PCR). PCR from

genomic DNA and cDNA with genes specific primers were performed. The PCR products

were sequenced and compared the nucleotide sequences and deduced amino acid sequences

for homology with other plants. Results confirmed the presence of DREB-1A, DREB-1B,

DREB-1F and CBF like genes in R. communis. In C. sativa the genes were identified for the

first time. Cadmium induced the expression of DREB-1B, DREB-1F and CBF like genes in

both the plants while molybdenum foliar spray further increased the expression of these

genes under Cd stress. The DREB-1A showed no expression in both the plants while its

presence was confirmed by the PCR product of genomic DNA with gene specific primers.

The CBF-like gene was expressed in both the plants sprayed with molybdenum (without Cd),

while the other genes were not expressed with Mo only. The DREB-1F and CBF like gene of

both the plants showed more than 80 % nucleotide sequence homology with these genes in

other plants. Predicted amino acid sequence of DREB-1F from both plant showed more than

75% homology with protein sequences of other plants, while the CBF like gene demonstrated

more than 80% homology with protein sequences of other plants species. The expression of

DREB-1B, DREB-1F and CBF-like genes (semi quantitative) was positively correlated with

Cd accumulation, free proline and total phenolics in Cannabis sativa plant, while these

correlations were significantly positive in Ricinus communis.

Application of GA3 and Mo increased concentration of Cd in plants tissues. A positive inter-

correlation was found between Cd concentration in plant tissues, production of prolin and

phenolics and the expression of DREB-1B, DREB-1F and CBF-like genes.

CHAPTER 1

GENERAL INTRODUCTION

CHAPTER 1 GENERAL INTRODUCTION

1

CHAPTER 1: GENERAL INTRODUCTION

1.1 Cadmium (Cd) as an environmental pollutant

Metals are a group of hazardous environmental contaminants. Their presence in soil and

water is a serious threat to sustainable agriculture, the environment and human health

(Ahmad et al 2015; Kevresan et al 1998). Out of 90 naturally occurring elements only 53 are

categorized as metals (Weast 1984). Among the metals Ni, Zn, Cu, Co, W, Cr, and V are

non-toxic at lower concentrations while As, Al, Hg, Sb, Ag, Cd and Pb are highly toxic to

humans and other living organisms even at lower concentration (Beak et al 2006; Sogut et al

2005). Cadmium (Cd) is a prevalent metal. It has relatively low occurrence (64th

among the

elements) in the earth's crust, and is found in water, soil, air as well as inside animals and

plants (Sarkari et al 2013). It does not occurs in a free-state, and almost always forms

compounds/complexes with other elements and molecules. Due to its highly toxic nature the

North Carolina National Toxicology Program (NCNTP) has categorized it as a potential

human carcinogen (Sarkari et al 2013). Cadmium is one of four metals of greatest global

concern because of its prevalence, hazardous effects on environment and impacts on human

health (di Toppi and Gabbrielli 1999). Its presence in water and soil can result in severe

health problems (Raskin et al 1997; di Toppi and Gabbrielli 1999).

1.2 Sources of Cadmium pollution into the environment

Cadmium enters into the environment through natural and anthropogenic (man-made)

activities. Natural processes such as erosion of parent rocks release 15,000 metric tons (mt) of

Cd per year, volcanic eruptions emit 820 mt Cd per annum and forest fires release 1-70 mt of

Cd per year in to the atmosphere (Sarkari et al 2013). Anthropogenic activities contribute 3-

10 times more Cd emission into the environment as compared to the natural processes

(Nriagu 1988). Anthropogenic contamination of cadmium into environment are from the

products in which Cd is an essential part (such as cadmium pigmented plastics, nickel-

cadmium batteries, glasses, ceramics, paints and enamels, cadmium coated ferrous and non-

ferrous products, cadmium stabilized polyvinylchloride (PVC) products, cadmium electronic

compounds and cadmium alloys) or those substances in which Cd occur as impurity (e.g.

non-ferrous alloys and metals of copper, lead and zinc, fossil fuels like oil, peat, coal and


2

wood, Iron and steel, cement and phosphate fertilizers) (Martelli et al 2006; Sarkari et al

2013).

1.3 Cadmium toxicity in humans

Due to high soil-to-plant transfer rates, food is considered as the major source of Cd exposure

(about 95%) in humans (McLaughlin et al 2006). It is estimated that most of the food-

cadmium (almost 80%) comes from vegetables, cereals and potatoes (McLaughlin et al 2006;

Berglund et al 1994). The average intake of cadmium in food usually varies from 8 - 25 μg

per day of which about 0.5-1.0 μg is retained in the body (Berglund et al 1994). Inside the

human body, Cd primarily accumulates in the liver and form complexes with

metallothioneins (MT - a low molecular weight protein) (Sarkari et al., 2013). The Cd-MT

complexes are released into the blood and transported into various organs and tissues of the

body (Sarkari et al., 2013). During prolonged exposure most of the Cd accumulates in the

cortical region of kidney (Curtis et al 1999; Gonick 2008). The chemical form of Cd

determines its distribution within the body. For example Cd accumulation in the liver mainly

occur in the form of CdCl2, whereas in kidney it accumulates in the form of CdMT. Cadmium

can accumulate almost in all organs of the human body such as kidney, liver, testis, heart,

spleen, thymus, lungs, central nervous system, epididymis, salivary glands, and prostate.

However, nearly 50% of the total body Cd accumulates in kidney and liver because of high

MT concentration in these organs (Sarkari et al 2013; Siddiqui 2010). Cadmium absorption is

increased by the acidic environment of the digestive tract and also by the wide-ranging

proton-metal co-transporter DMT1, Nramp2, DCT1, or SLC11A2 and the carrier MTP1

metal ions (metal transporter protein 1) at the enterocytes apical membrane (Ryu et al 2004).

Most of the ingested Cd is primarily excreted in urine, while small amounts of Cd is excreted

in feces by conjugating with metallothionein, glutathione or cysteine (Zalups and Ahmad

2003). The daily excretion of cadmium from the body (mainly by the kidneys) does not

exceed 0.01% of the amount of cadmium consumed in the diet (USEPA, 2007)

In different countries the daily intake of Cd in food ranges from 10 to 35 μg per person

(Sarkari et al 2013). Cadmium contents in food significantly affect its concentration within

human blood. Adult human body contains approximately 15 - 30 mg of Cd and this amount

increases with age due to the long half-life of cadmium (10-30 years) inside human body

(Martelli et al 2006). The suggested safe intake limit set for Cd is 7 μg Cd per week per kg


3

body weight or 25 μg Cd per kg body weight per month or 0.4 - 0.5 mg per week (WHO

1989).

Cadmium is serious threat to human health even at low concentration due its non-degradable

nature, poor excretion rate from human body and less tolerance of the body to this element

(Waalkes 2003). Cadmium mainly affects the organs like liver, lungs, kidney, testes, heart,

prostate, skeletal system, immune system and nervous system. Itai- itai disease is caused by

long term exposure to high doses of cadmium and mostly occurs in women. This disease is

characterized by severely reduced glomerular and tubular function of nephron, and

generalized osteoporosis and osteomalacia that cause multiple bone fractures (Inaba et al

2005). Several studies suggested an increased risk of peripheral artery disease (PAD) at low

dose Cd exposure (Navas-Acien et al 2005). In the stomach, Cd reacts with HCl and form

CdCl2, which produces severe inflammation of the digestive tract (Waisberg et al 2005).

Cadmium exposure in men can damage the leydig, sertoli and vascular endothelial cells of

testes and thus result in the inhibition of testosterone synthesis and impairment of

spermatogenesis (Goyer et al 2004). Prolonged exposure of Cd can lead to malfunctioning of

the immune system because target cells for Cd are T cells, B cells, macrophages and natural

killer cells. It seems that the direct immune-toxicity by Cd is the alteration in immune

responses of both cell-mediated and humoral immunity (Krocova et al 2000; Marth et al

2000). Some reports also suggest an association of eosinophilia and anemia with cadmium

toxicity (Sarkari et al 2013).

Cadmium is a potential carcinogen. It can effect gene expression by reducing DNA

methylation, interferes with DNA damage-repair system, inhibit apoptosis and induce

oxidative stress (Takiguchi et al 2003; Huang et al 2008). Cadmium has been considered as a

Class-1 human carcinogen by the International Agency for Research on Cancer (IARC 1993).

Cadmium exposure can cause cancer in organs such as kidneys (Pesch et al 2000), lungs

(Nawrot et al 2006), liver (Waalkes and Misra, 1996), hematopoietic system (Waalkes and

Misra, 1996), endometrium (Akesson et al 2008), mammary glands (McElroy et al 2006),

pancrease (Kriegel et al 2006), urinary bladder (Kellen et al 2007), prostate (Jarup et al 1998;

Zeng et al 2004) and stomach (Waalkes and Misra 1996).

Major symptoms of severe Cd toxicity, such as fever, general weakness and shortness of

breath, generally appear 24 hrs after exposure. Acute Cd exposure can also leads to

pneumonia, pulmonary oedema and in severe circumstances results in respiratory failure and

even death (Jarup et al 1998). Women possess higher concentration of Cd in their body as


4

compared to men (Vahter et al 2007; Jarup and Akesson 2009) because of intestinal

absorption of dietary Cd in females is higher than males (Berglund et al 1994).

1.4 Cadmium and plants

1.4.1 Cadmium uptake, transport and accumulation in plants

Of the different properties of soil known to affect Cd bio-availability, pH is considered to be

the most significant. Several studies revealed a linear trend between pH of soil and uptake of

Cd by plants i.e. decreasing pH of the soil will increase Cd uptake and concentration with in

plant tissues (Kirkham 2006). Another factor that determines Cd accumulation is the plant

species (Li et al 2005). It is believed by some investigators that uptake of Cd from soil occurs

through active transport (in which energy is consumed), but most researchers points towards

the passive uptake hypothesis of Cd by plants (Larcher 1995; Marschner 1995). Cadmium

ions have been found to compete with many elements like copper (Kudo et al 2011), chlorine

(Oporto et al 2009), phosphorus (Dheri et al 2007), calcium (Choi and Harada 2005) and zinc

(Zhao et al 2002) for uptake and transport within plants. Inside the xylem Cd form bonds

with sulphydryl, nitrogen and oxygen ligand groups and is transported upward with the

movement of water (Tran and Popova 2013). Transpiration is thought to have important role

in Cd uptake and transport (Hardiman and Jacoby, 1984; Salt et al 1995), but some scientists

have provided contradictory evidence (Perfus-Barbeoch et al 2002). Transport of Cd in

phloem occurs through phytochelatins and phytometallophores, for example metallothionein,

nicotinamine, cysteine, glutathione and molecules bearing sulphydryl groups (Tran and

Popova 2013). It is also supposed that phytometallophores and phytochelatins play a role in

Cd accumulation within grains and seeds of a plant (Tran and Popova 2013).

1.4.2 Cadmium toxicity in plants

Cadmium is a phytotoxic element. It reduces plant growth even at very low concentration

(Aery and Rana 2003). Cadmium inhibits photosynthesis in plants by damaging

photosynthetic apparatus (especially the photosystems), inhibiting the activity of the enzyme

Fe (III) reductase (causing serious deficiency of Fe) and also causing closure of stomata in

higher plants (Vassilev et al 2005; Chugh and Sawhney 1999; Siedlecka and Krupa 1996).

Cadmium also targets two important enzymes involved in CO2 fixation i.e.

phosphoenolpyruvate carboxylase and ribulose-1,5-bisphosphate carboxylase (Stiborova

1988). Overall decrease in protein levels has been found in plants subjected to Cd stress


5

(Tamas et al 1997). Cadmium negatively affects the absorption, translocation and distribution

of many essential nutrients such as Mg, Fe, Ca, K, P, Zn, Mn, B and S in plants, thus

disturbing plant growth and development (Metwally et al 2005; Guo et al 2007). Cadmium

has been found to cause H+ATPase inhibition in roots cells of maize plant. The H

+ATPase is

an ion transporter across the plasma membrane and its inhibition by Cd results in the

decreased uptake of some essential nutrients (Astolfi et al 2005).

Figure 1. 1: Effects of Cd on plant. (http://www.nature.com/articles/srep14078)

1.4.2.1 Generation of ROS under heavy metals stress and plant defense

Reactive oxygen species (ROS) are toxic by-product of incompletely reduced oxygen species.

They are produced mostly in mitochondria and chloroplast of plant cells. At low

concentration, under normal physiological condition, plant cells are able to manage their

negative impacts (Michalak 2006). Increases in the levels of ROS have been observed in

plant cells under stress conditions, such as wounding, pathogen attacks, herbivore feeding,

metals, UV light and others (Diaz et al 2001; Wojtaszek 1997). Normally cells try to maintain


6

lower concentration of ROS, as they are highly reactive (Wojtaszek 1997). They can react

with virtually every organic component of a living cell. The most common ROS are

superoxide radical (.O2-), hydroxyl radical (.OH) and hydrogen peroxide (H2O2) which

originate from the transfer of one, two or three electrons to di-oxygen (O2). Heavy metals

cause oxidative stress by generation of free radicals, disturbance in metabolic pathways,

inactivation of antioxidant enzymes (catalases, peroxidases and superoxide dismutases) and

destruction of low molecular weight antioxidants (such as glutathione) (Sahw et al 2004).

Cadmium is thought to induce oxidative stress in plant by indirect mechanisms, such as

disturbance of the electron transport chain, interference with the anti-oxidative defence or

initiation of lipid peroxidation through the stimulation of lipoxygenase (an enzyme

responsible to initiate lipid peroxidation) (Michalak 2006; Smeets et al 2005).

Cadmium is induces oxidative stress in plants (Somashekaraiah et al 1992; Hendy et al 1992)

by stimulating the production of oxygen free radicals (Balaknina et al 2005; Demirevska-

Kepava et al 2006) and/or by reducing the concentration of anti-oxidants both enzymatic and

non-enzymatic (Mohan and Hosetti 2006; Cho and Seo 2004). Cadmium stimulates lipid

peroxidation by decreasing the activities of catalase, superoxidase, dehydroascorbate

reductase, ascorbate peroxidase and glutathione reductase while enhanced the activity of

lipoxygenases in plant leaves (Panda and Khan 2003; Khan et al 2002). Several investigators

have reported the induction of peroxidase (POX) activity by Cd in many plants species such

as Calamus tenuis, Brassica juncea, Cicer arietinum, Bacopa monniera (Khan and Patra

2007; Hayat et al 2007; Hasan et al 2007; Mishra et al 2006).

Damage to plants occurs when the capability of antioxidant system and detoxification process

decrease as compared to the production of ROS. Several antioxidant mechanisms are present

to defend plants from the damage caused by oxidative stress (Pal et al 2006; Smeets et al

2005). These antioxidant systems include metabolites like tocopherol, glutathione and

ascorbate, and enzymatic scavengers of reactive oxygen for example catalases, superoxide

dismutases and peroxidases (Mandhania et al 2006; Demiral and Turkan 2005; Panda and

Khan 2003; Khan et al 2002). The most significant low molecular weight antioxidants

include glutathione, ascorbic acid, α-tocopherol, thiols and protective pigments for example

carotenoids (Devi and Prasad 1998; Tausz et al 2003). These non-enzymatic ROS scavengers

are important in defending different components of a cell from most of ROS, however they

cannot deal with reducing radicals like metastable hydroperoxides or superoxide (Chaudiere

and Ferrari-Iliou 1999).


7

1.4.2.2 Antioxidant Action of Phenols

A variety of secondary metabolites are produced in the plants. Among which phenolic

compounds holds an important position. These compounds possess at least one aromatic ring

with one or more hydroxyl (OH-) groups. The antioxidant activity of phenolic compounds

has been known for many years (Bors et al 1990). Several reports demonstrated the

accumulation of phenolic compounds and increase in peroxidase activity in many plants

subjected to high concentrations of toxic metals. Antioxidant activity of phenolics is due to

their high capability to chelate toxic metals (Jung et al 2003). Metal ions initiate free radical

oxidative chain reaction by converting lipid hydroperoxide (COOH) into lipid alkoxyl

radicals. Phenolic compounds prevent lipid peroxidation of the lipid alkoxyl radicals by

trapping them. This process depends on the molecular structure, and the position and number

of the OH-group in the molecules (Millic et al 1998). It has been demonstrated that phenolic

compounds (particularly flavonoids) alter the kinetics of lipid peroxidation through

modification of the lipid packing order (Arora et al 2000). Phenolic compounds increase the

stability of membranes by reducing fluidity of membrane (in a concentration-dependent way)

and alter the rate of free radicals diffusion and inhibit peroxidative reaction (Blokhina et al

2003; Arora et al 2000). In addition to the capacity of phenolic compounds (procyanidins and

flavanols) to bind with proteins, they can also bind to the phospholipids (through H-bonding)

in the membranes and accumulate on both sides of the membrane; thus protecting membrane

from oxidation stress of ROS (Verstraeten et al 2003). In-vitro studies revealed that active

oxygen species such as O2-2

superoxide, peroxyl radical, 1O2 -singlet oxygen or H2O2 -

hydrogen peroxide are directly scavenge by flavonoids, mainly because of their ability to

donate hydrogen atoms or electrons (Khan et al 2000; Arora et al 2000; Sakihama et al

2000). Antioxidant activity of phenolics (especially flavonoids) are due to three structural

features; (1) the 2, 3-double bond with conjugation with 4 – oxo group in C-ring, (2) the orto

3,4-dihydroxy structure in B-ring and (3) the presence of a 3 – OH group in C ring and a 5 –

OH group in the A ring (Michalak 2006). Among these features, the most important electron

donating activity is shown by 3– OH group (Takahama and Oniki 2000).

1.4.2.3 Effect of free proline

Proline is an amino acid which accumulates within plant tissues during environmental stress

from salinity, drought, frost, toxic heavy metals and others (Sun et al 2007; Khatamipour et al

2011; Handique and Handique 2009). High concentration of free proline is found in plants


8

under cadmium stress (Ahmad et al 2015; Sun et al 2007) and It can be an indicator of heavy

metal stress in plants (Khatamipour et al 2011). Prolines protect plants from toxic metal stress

by acting as a chelator of metals (Farago and Mullen, 1979), osmoprotectant (Hartzendorf

and Rolletschek 2001), membrane stabilizer (Bandurska 2001), scavenger of hydroxyl ions

(Smirnoff and Cumbes 1989), inhibitor of lipid peroxidation (Mehta and Gaur 1999),

protective shield for enzymes (Paleg et al 1984), source of nitrogen and carbon (Fukutaku

and Yamada 1984), stabilizer of the protein synthesis machinery (Kadpal and Rao 1985), and

regulater of cytosolic acidity (Venekamp 1989; Venekamp et al 1989). Under cadmium

stress, accumulation of free proline occurs mainly due to deficiency of water within cells

(Nikolic et al 2008).

1.4.3 Plant stresses and CBF/DREB transcriptional factors (CBF regulon)

Plants combat environmental stresses by activation of set of genes playing diverse functions.

These genes can be classified into two groups. The first group comprise of functional proteins

(i.e. late embryogenesis abundant proteins, molecular chaperones, antifreeze proteins,

enzymes involved in biosynthesis of important osmolytes such as sugar, proline and sugar

alcohols, enzymes involved in detoxification reactions, betaines, membrane transporters and

water channel proteins) that play a direct role in plant defence against harsh environmental

conditions. The second group consisted of regulatory proteins that control transduction of

signals and expression of stress related genes, including several transcription factors (TF),

enzymes that catalyze metabolism of phospholipids, protein kinases and other stress

signalling molecules. Understanding the molecular mechanism behind the abiotic stress

tolerance is very important for further enhancement of stress tolerance in crop plants through

genetic manipulation (Lata and Prasad 2011; Shinozaki and Yamaguchi-Shinozaki 2007;

Agarwal et al 2006).

Expression of several abiotic stress responsive genes can be regulated by controlling the

expression of a single regulatory gene encoding TF/regulon, for the betterment of crops under

various environmental stress conditions (Yang et al 2011; Century et al 2008). A regulon

consist of two or more structural genes each having its own promotor and are coordinately

regulated by a common regulator molecule/transcription protein. Genes in a single regulon

share a common or related regulatory sequence which can each be recognized by the

transcriptional factor/ regulator molecule. Gene for transcriptional factors can be grouped

into many large families including AP2/ERF, MYC, MYB, NAC, zincfinger, WRKY and


9

Cys2His2 (Umezawa et al 2006). Members of the same family share homologous DNA-

binding domains encoding closely related proteins. Each member of a family respond in a

different way to a stress stimulus.

One group of proteins, i.e. AP2/ERF transcription factors, that exist only in plants and play

significant role in combating biotic and abiotic stresses (Agarwal et al 2006). This super

family of genes (encoding TF proteins) has been further divided into three main groups i.e.

the ERF, RAV and AP2 families on the basis of numbers of AP2/ ERF domains and sequence

similarities (Lata and Prasad 2011; Nakano et al 2006). Dehydration responsive element

binding (DREB) also called C-repeat binding factor (CBF) proteins belongs to the ERF group

of TF and have received much attention during the past few decades because of their vital

role in plants under stress conditions. The DREB subfamily proteins can further be divided

into six groups denoted as A-1, A-2, A-3, A-4, A-5 and A-6, among these A-1 and A-2 are

the two largest groups (Sakuma et al 2002). These genes are involved in the ABA-

independent pathways and triggers stress responsive genes in plants. For the first time

DREB1A and DREB2A cDNA were identified in Arabidopsis plant through yeast one hybrid

screening (Liu et al 1998; Stockinger et al 1997). After that, several DREB genes have been

identified and isolated from other plants. DREBs proteins bind specifically to the regulatory

DRE sequences (5'-TACCGACAT-3') of several downstream genes and thus regulate their

expression. The DRE-sequences were first identified in the promoter of gene rd29A

responsible for drought tolerance (Yamaguchi-Shinozaki and Shinozaki 1993). The

DREB1B/ CBF1, DREB1C/ CBF2 and DREB1A/ CBF3 genes are present on chromosome 4

of Arabidopsis (Liu et al 1998; Gilmour et al 1998). Highly conserved AP2/ERF DNA–

binding domain in the DREB proteins have been found throughout the plant kingdom which

consist of one a α - helix and three-stranded β - sheet running nearly parallel to it that binds

with DNA via arginine and tryptophan residues within the β - sheet (Magnani et al 2004).

The DNA-binding domain contain two highly conserved amino acids (glutamic acid and

valine) at residue number 14th

and 19th

respectively and these amino acids residues are

important sites for DREBs and DRE core sequences binding to DNA (Liu et al 1998). These

proteins also have an acidic C-terminal region that might involve in trans-activation process

(Stockinger et al 1997) and an alkaline N–terminal region that function as a nuclear

localization signal (NLS) and mostly contain a conserved Serine/Threonine-rich region which

is responsible for the phosphorylation of nearby AP2/ERF DNA-binding domain (Agarwal et

al 2006; Liu et al 1998). The expression of these genes are specific to organ and the extent of


10

stress given to the plant. For example, expression of AhDREB1 gene was higher in roots as

compared to stem and leaves under salt stress (Shen et al 2003). The OsDREB1F showed

constitutive expression throughout the plant with highly significant expression in callus and

panicles as compared to the other tissues in the plant (Wang et al 2008). Over expression of

DREB1A/CBF3 or DREB1B/CBF1 in transgenic Arabidopsis plants demonstrated strong

tolerance to drought, high salinity and frost which implies that DREB/CBF regulate a wide

range of genes under stress conditions (Kasuga et al 1999; Liu et al 1998; Jaglo-Ottosen et al

1998). Over expression of DREB1A/CBF3 transgenics are reported to accumulate amino acid

proline and different sugars under normal physiological (non-stressed) conditions (Gilmour et

al 2000). The OsDREB1F over expression highly increase plant tolerance to low temperature,

drought and high salinity both in Arabidopsis and rice, thus playing an important role in

signal transduction under stress conditions (Wang et al 2008). The effect of CBF/DREB

genes have not been studied under the stress caused by toxic heavy metals and in present

study we have investigated the effect to molybdenum on expression level of DREB1A,

DREB1B, DREB1F and CBF like transcriptional factors in three plants (Ricinus communis,

Cannabis sativa and Parthenium hysterophorus) under Cd stress.

1.5 Remediation of toxic metals from polluted soils

Soil contaminated with toxic metals is a potential threat to the sustainable agriculture and

human health. Heavy metal polluted soil can be remediated by physical (such as excavation

and soil washing), chemical (e.g. chemical extraction and oxidation/reduction process), or

biological (phytoremediation) methods (Hadi et al 2014; Jadia and Fulekar, 2009). The first

two methods (chemical and physical) are very expensive and laborious while the

phytoremediation method is not only cheaper but also environment friendly and solar driven

(Ahmad et al 2015; Hadi et al 2014). Phytoremediation (―phyto‖ means plant, and

―remedium‖ means to restore or clean) is a diverse collection of techniques in which plants

(naturally occurring or genetically engineered) are employed to remediate/clean polluted

water and soil (Revathi et al 2011; Jadia and Fulekar 2009). Several plans have been

reported to accumulate and tolerate higher concentrations of toxic heavy metals in their

tissues without symptoms of toxicity (Entry et al 1999; Baker and Brooks 1989). For example

Fronds of Pteris vitatta (a fern) can accumulate nearly 14,500 mgKg-1

arsenic without any

toxic symptom (Ma et al 2001). More than 400 different plant species have been documented

to have potential for toxic metals phytoremediation, while the mostly studied species belongs

to Arabidopsis, Brassica, Sedum and Thlaspi (lone et al 2008). Two important factors


11

determining the phytoremediation potential of a plant are its biomass and concentration of

toxic metals in the biomass i.e. plants having high biomass and high concentration of

pollutant in the biomass are most suitable for the phytoremediation potential (Fritioff et al

2005).

1.5.1 Phytoremediation technologies

Phytoremediation is a broad term used for several remediation techniques based on green

plants. These techniques are given below.

1.5.1.1 Phytoextraction

Phytoextraction is based on the extraction and accumulation of contaminants by plants.

Ideally, the contaminants are translocated to the above ground parts of the plants. Plants

usually show tolerance to heavy metals but most of them do not have the ability to

accumulate these metals to significant amount in above-ground parts of the plants. Some

plants have the natural ability to accumulate higher concentration of heavy metals without

developing any symptoms of toxicity and such plants are called hyperaccumulator of that

metal. Such hyperaccumulator plants should be used for the phytoextraction purposes (Baker

and Brooks 1989; Zhou and Song 2004; Sun et al 2008). Plants used for phytoextraction

purposes should have the following properties: (a) the concentration of metal in the shoots

should be higher than 100 mg kg-1

for cadmium, 1000 mg kg-1

for Ni, Cu, As, Se and Co and

10,000 mg kg-1

for Mn and Zn (Baker and Brooks 1989; Ma et al 2001; Zhou and Song 2004;

Sun et al 2008); (b) shoot to root metal concentration (translocation factor) should be greater

than 1.0 (Wei and Zhou, 2004); (c) the ratio of metal concentration in plant to soil

(bioconcentration factor) should be higher than 1.0, occasionally touching 50-100 (Cluis,

2004; Sun et al 2008); (d) the plant used should have high biomass and have the potential to

translocate contaminant from roots to aerial parts of the plant (Sun et al 2011; Evangelou et

al 2007). Most of the hyperaccumulator do not show all the desirable characteristics required

for efficient phytoextraction of toxic heavy metals.

1.5.1.2 Rhizofilteration/ phytofilteration

Rhizofilteration is a phytoremediation technique in which contaminants from polluted water

is absorbed, concentrate and accumulate in roots of green plants (Ghosh and Singh 2005; Salt

et al 1995). This technique can be used for Cd, Pb, Cu, Zn, Ni and Cr, which mostly retained

in roots of a plants (Jadia and Fulekar 2009; Ghosh and Singh 2005). In this technique, plants


12

are first grown in clean water until a long root system is formed and then transferred into

polluted water for acclimatization and pollutant (metal) uptake by roots of the plants. After

saturation with heavy metals, the roots of plants are harvested and disposed of safely.

Repetition of the process several times can reduce the pollutant in the water to a safe limit.

Several plant (such as Helianthus annus, Brassica juncea, Zea mays and Secale cereale) have

been screened for their ability to absorb and concentrate toxic metals in their roots from

solutions (Dushenkov et al 1995). Water hyacinth was found to effectively remove trace

elements from waste water streams (Zhu et al 1999).

1.5.1.3 Phytostabilization

In this technique, roots of a plant stabilize the pollutant either by absorption, precipitation,

forming complex with metal or binding the metal with organic materials (Gwozdz and

Kopyra 2003). The main idea behind phytostabilization is to transform toxic metals to an

inert form in the soil (Cunningham et al 1997). Plants used for phytostabilization purposes

should have the properties like tolerance to high concentrations of toxic metals, ability of

immobilizing toxic metals in soil through root absorption, reduction or precipitation and low

metal translocation rate from root into aerial parts of the plant to eliminate the need of

treating harvested shoot as toxic waste. Stabilization is a technical and logistical stance in the

areas where remediation of soil from toxic metals is not possible. Through this method

vegetation can be restored that has been removed as a result of high concentrations of toxic

metals in the site (Tordoff et al 2000).

1.5.1.4 Phytotransformation/ phytodegradation

Pollutant absorption by plants and subsequent transformation into non-toxic substances

through the metabolic reactions within plants or externally by plant exudates (such as

enzymes) is known as Phytotransformation/ phytodegradation. This technique cannot be used

for heavy metals due to the non-biodegradable nature of metals. It is mostly used for complex

organic compounds that are degraded by plants into non-toxic form (Prasad and Freitas

2003). Presently this technique is being employed (demonstration-scale) on groundwater

contaminated with explosive materials like RDX (Research Department explosive) and TNT

(Trinitro toluene) at Milan Army Ammunition Plant by the Army Corps Engineers in

Tennessee, United States of America (Miller 1996).


13

1.5.1.5 Phytovolatilization

Phytovolatilization is the transformation of toxic pollutant into volatile non-toxic form within

the plants, which then evaporate into air. The pollutant may modified in to less toxic volatile

form as it travels with water along the vascular system of the plant from roots to leaves,

whereby the pollutants evaporate into the air along with water. For example mercury in the

form of Hg (II) when absorbed by a plant is converted into less toxic form Hg (0) and then

evaporated from leaves of the plant (Kozuchowski and Johnson 1978).

1.6 Enhanced phytoextraction

There are two strategies to remediate metal contaminated soil using green plants. First

strategy is the use of metal hyperaccumulators plant species while second method is the use

of plants having fast growth and high biomass (Griga and Bjelkova 2013). Reduction in plant

biomass under metal stress and non-bioavailability of heavy metals in soil is often a problem

for metal phytoextraction. Scientists have tried to overcome the problem by application of

various chemicals either to the plants directly or into the soil (Tassi et al 2008; Falkowska et

al 2011). In the present research, we have used gibberellic acid (GA3),

ethylenediaminetetraacetic acid (EDTA) and molybdenum (Mo) to enhance the Cd

phytoextraction potential of our experimental plants.

1.6.1 Gibberellic acid (GA3)

Gibberellic acid (GA3) is one of the plant growth regulators, which was first time reported by

Kurusawa in Gibberella fujikuroi infected rice plant in 1962. Gibberellic acids comprises a

vast group of hormones derived from the ent-gibberellane skeleton and are produced in

different plant species. Gibberellic acids play many important roles in growth and

development of a plant, including stimulation of cell division and cell elongation, promotion

of seed development and germination (by α-amylase enzyme synthesis), delay senescence in

leaves (Falkowska et al 2011). During stress conditions a decline in concentration of plant

growth promoters (gibberellic acids and cytokinins) and an up rise in concentration of plant

growth inhibitor (such as abscisic acid) occur in plants. To compensate for the decrease in

GA3 concentration, the exogenous application of gibberellic acid could be used as an

alternative mechanism (Falkowska et al 2011). We have used GA3 in our experiments for two

purposes; to compensate for the decrease in its concentration due to stress and to increase

biomass of the plant.


14

1.6.2 Ethylenediaminetetraacetic acid (EDTA)

Ethylenediaminetetraacetic acid(EDTA) is a colourless and water soluble solid having

chemical formula (HO2CCH2)2N CH2 CH2 N(CH2 CO2 H)2 (Holleman et al 2001). EDTA was

synthesized for the first time by Ferdinand Munz in 1935, from ethylenediamine and

chloroacetic acid. EDTA is produced as several salts, notably disodium EDTA and calcium

disodium EDTA. It is an excellent synthetic chelator of metals and form strong complexes

with metals (especially divalent and trivalent metals) through four carboxylate and two amine

groups (Holleman et al 2001). After binding with EDTA, the metal ions remain in solution

with low reactivity. In the present research EDTA was used in soil to chelate Cd and increase

its bio-availability to the plant.

1.6.3 Molybdenum (Mo)

Plant need several nutrients for normal growth and development. These mineral nutrients can

be divided into two groups on the basis of the quantity required by plants i.e. macronutrients

and micronutrients. Macronutrients (such as C, H, O, N, P, K, S, Ca, and Mg) are required by

plants in relatively large amount while micronutrients (like Fe, Zn, Mn, Ni, Cu, and Mo) are

needed by plant in very minute quantity. Molybdenum (Mo) is a transition element having

many oxidation states (from Zero to VI) but mostly occur in soils in VI oxidative state

Molybdenum mostly occur as an important component of molybdenum cofactor called Moco,

which binds to molybdoenzymes (molybdenum-requiring enzymes) found in animals, plants

and microorganisms (Williams and Frausto da Silva 2002). Arnon and Stout (1939)

demonstrated that Mo is required by plants, while they were working on tomato plant grown

hydroponically. Molybdenum occurs in the soil normally up to 2 – 3 mg kg-1

but can reach

as high as 300 mg kg-1

in shales having substantial amount of organic matter (Reddy et al

1997; Fortescue 1992). In agricultural soils, Mo occurs in several different forms such as

ferrimolybdenite [Fe2 (MoO4)], wulfenite (PbMoO4) and molybdenite (MoS2) (Reddy et al

1997). Mo bio-availability in soil for plants strongly depends on the pH of soil and the

concentration of adsorbing oxides like Fe oxides, also on the water drainage and organic

substances in the soil (Kaiser et al 2005). In alkaline soils the availability of Mo to plants

becomes higher mainly in MoO4 anion form while in acidic soil (pH < 5) bio-availability

decreases due to increased adsorption of oxides anions to soil particles (Reddy et al 1997).

Plants develop many phenotypic variation under Mo deficiency and most of these symptoms

http://en.wikipedia.org/wiki/Ethylenediamine

http://en.wikipedia.org/wiki/Chloroacetic_acid


15

are associated with reduction in activity of molybdoenzymes. These symptoms are mostly

related to reduce nitrogen availability mainly when nitrate is the major nitrogen source

available for plant. Failure to synthesize the molybdenum cofactor (Moco) decreases the

activity of the important nitrogen reducing and assimilatory enzymes such as Nitrate

reductase (NR) and xanthine dehydrogenase/oxidase (XDH) (Agarwala et al 1978; Jones et al

1976). Mo catalyses other enzymes such as aldehyde oxidase (AO) involved in Abscisic acid

biosynthesis and sulfite oxidase (SO) catalyse the conversion of sulfite to sulfate, an essential

step in the catabolism amino acids containing sulfur (Williams and Frausto da Silva 2002;

Mendel and Haensch 2002). Molybdoenzymes are involved in the synthesis of indole-3-

acetic acid (IAA) and ABA (Hesberg et al 2004; Sagi et al 2002). In present research we

investigated the effect of Mo on growth, biomass and Cd phytoextraction potential of Ricinus

communis and Cannabis sativa plant under cadmium stress. Also the effect of Mo on

expression of CBF/DREB genes in these plants grown in Cd contaminated soil.

1.7 Introduction to experimental plants

Three plants were used during the experiments. They are Parthenium hysterophorus, Ricinus

communis and Cannabis sativa.

1.7.1 Parthenium hysterophorus

Parthenium hysterophorus belongs to the family Asteraceae. It is an annual herb having tap

roots grows deep into soil and erect soft stem which progressively become semi-woody with

maturity (figure 1.1). The stem usually attains a height of about 1 - 2 meter. Leaves are pale

green, bi-pinnatfied and soft fine hairs covered its surface (Bhawmilk and Sarkar 2005). It is

native to the tropics and subtropics of America (Parsons and Cuthbertson 1992). It is

common invasive species in Australia, India, Pakistan and some parts of Africa that invades

all disturbed land including pastures, farms and roadsides. In some places, it is found almost

in epidemic proportions, affecting crops, human health (causes allergy) and livestock.

Parthenium hysterophorus have the ability to grow and reproduce throughout the year. Under

favorable conditions, 4 or 5 consecutive generations of seedlings could arise at the same

place. Photosynthetic properties of this plant leaf is mostly associated to C3 type pathway and

the plant possess high rate of photosynthesis at 25 - 35 0C while low temperature showed

negative effect of plant growth, flowering and seed production (Navie et al 1996).

http://en.wikipedia.org/wiki/Neotropic_ecozone


16

1.7.2 Ricinus communis

Ricinus communis belongs to family Euphorbiaceae in plant kingdom. It is known as castor

bean and is distributed across the world, mostly in the tropical regions (Rana et al 2012).

Ricinus is a perennial shrub which reaches a height of more than 3 meter. It is a fast growing

C3 plant. Glossy alternate leaves surround the stem which is covered with light brown bark.

The leaves appear like palm leaves, containing 5 – 9 finger lobes. Small monoecious flowers

appear in the form of panicle inflorescence (figure 1.2). The fruit is in the form of spiny

epicarp enclosing bean like seeds. Ricinus is an economical plant due the quantity and quality

of its oil which is used in making eco-friendly coatings and paints (Rajkumar and Freitas

2008). Ricinus got attention due to its natural ability to grow in polluted soil as well as its

potential for toxic metals accumulation (Shi and Cai 2009; Rajkumar and Freitas 2008). Stem

of the plant possess antiprotozoal, antidiabetic and anticancer activities (Singh et al 2010).

Oil extracted from roots, leaf and seed of the plant is used in the treatment of liver disorder

and inflammation (Kensa and Yasmi 2011).

1.7.3 Cannabis sativa

Cannabis sativa is an annual herb and belong to Cannabaceae family in plant kingdom

(Figure 1.3). Mostly the male and female plants are separate i.e. dioecious except the fiber

hemp varieties which are monoecious (Debruyne et al 1994). Male (staminate) plans are

generally taller but less healthy than female (pistillate) plants. Plant stem is erect and ranges

in length from 0.2m to 4.0 m. Though, most Cannabis plants attain heights of about 1 – 3m.

This plant is highly suitable for growing in heavy metal polluted soil because of its fast

growth, high biomass and non-palatable nature (Citterio et al 2003).


17

Figure 1. 2 : Plants used during the experiments. (A) Parthenium hysterophorus plant

(http://www.durbaninvasives.org.za/target-list/Parthenium-hysterophorus), (B) Ricinus

communis (http://luirig.altervista.org/pics/index4.php?search=Ricinus+communis&page=1)

and (C) Cannabis sativa (http://www.photomazza.com/?Cannabis-sativa).

http://www.durbaninvasives.org.za/target-list/parthenium-hysterophorus

http://luirig.altervista.org/pics/index4.php?search=Ricinus+communis&page=1

http://www.photomazza.com/?Cannabis-sativa

CHAPTER # 1 AIM AND OBJECTIVES

18

1.8 Aim and objectives

Aim:

This physiological, biochemical and molecular investigation was carried out with aim to find

out the cadmium phytoextraction potential of selected plant species and to evaluate the effect

of molybdenum (Mo), Gibberellic acid (GA3) and ethylenediaminetetraacetic acid (EDTA)

on cadmium uptake and translocation into plant tissues, for the development of

phytoextraction technology. The main aim of this study was to investigate the role of

CBF/DREB like genes in Cd accumulation in selected plants species. Three plants i.e.

Parthenium hysterophorus, Cannabis sativa, and Ricinus communis were selected for present

research study.

Objectives:

1. To investigate the effect of GA3 and EDTA on biomass, Cd uptake and accumulation in

Parthenium hysterophorus plant.

2. To find out the role of EDTA and GA3 on free proline and total phenolics concentration

in P. hysterophorus plant under Cd stress.

3. To evaluate the effect of GA3 and EDTA on chlorophyll and carotenoids contents in P.

hysterophorus plant in Cd contaminated soil.

4. To evaluate the effect of Mo on plant height, biomass and water content in Ricinus

communis and Cannabis sativa under Cd stress.

5. To investigate the role of Mo on Cd uptake, translocation and accumulation in Ricinus

communis and Cannabis sativa plants.

6. To evaluate the effect of Mo and Cd on the concentration of free proline and total

phenolics in Ricinus communis and Cannabis sativa plants.

7. To study the concentration of chlorophyll and carotenoids in Ricinus communis and

Cannabis sativa plants under different treatments of Mo and Cd.

8. To identify the presence of DREB 1A, DREB 1B, DREB 1F and CBF like gene

sequences in Ricinus communis and Cannabis sativa plants.

9. To study the expression of DREB 1A, DREB 1B, DREB 1F and CBF like genes in

Ricinus communis and Cannabis sativa plants.

10. To find out the correlations of:

a. Free proline and total phenolics with the expression of DREB 1A, DREB 1B,

DREB 1F and CBF like genes in Ricinus communis and Cannabis sativa

plants.

b. Cadmium accumulation with the expression of DREB 1A, DREB 1B, DREB

1F and CBF like genes in Ricinus communis and Cannabis sativa plants.

c. Cadmium concentration and accumulation with the concentration of free

proline and total phenolics in all the three plants.

d. Biomass with free proline and total phenolics concentration in all the selected

plants.

e. Chlorophyll and carotenoids concentration with biomass, total phenolics, free

proline and cadmium accumulation in all the three plants.

CHAPTER # 2

THE EFFECT OF GIBBERELLIC ACID AND EDTA ON Cd

PHYTOEXTRACTION: CORRELATIONs OF FREE PROLINE, TOTAL

PHENOLICS AND CHLOROPHYLL CONTENTs WITH CADMIUM

ACCUMULATION IN PARTHENIUM HYSTEROPHORUS PLANT

CHAPTER # 2 ABSTRACT

20

CHAPTER 2: The effect of gibberellic acid and EDTA on cadmium

phytoextraction: correlations of free proline, total phenolics

and chlorophyll contents with Cd accumulation in


ABSTRACT

A pot experiment was conducted to evaluate the effects of gibberellic acid (GA3) and

ethylenediaminetetraacetic acid (EDTA) on plant growth (measured as length, biomass and

water content), cadmium (Cd) up-take, total phenolics, free proline and chlorophyll content

of P. hysterophorus plant in Cd contaminated (100 mg Kg-1

) soil. GA3 (10-2

, 10-4

and 10-6

M)

was applied as foliar spray while EDTA (40 mg Kg-1

soil single dose, 10 mg Kg-1

four doses)

was added to soil. Results demonstrated a significant decrease in growth parameters of the

plant due to Cd stress. Lower concentration of GA3 (10-6

M) showed highest significant

increase in the growth parameters while Cd concentration, accumulation (1.97 ± 0.11 mg per

DW) and bioconcentration (9.75 ± 0.34) was significantly higher in the treatment T11 (GA3

10-2

+ four split doses of 10mg EDTA). Cadmium induced increased free proline levels in

roots, while total phenolics concentration was significant in all parts of the plant. Chlorophyll

contents were significantly reduced by Cd while GA3 showed significantly increase the

chlorophyll concentration. Cadmium contents of the plant showed negative correlation with

FW, DW, TWC and chlorophyll contents while positive correlation with free proline (R2

=

0.527, R2

= 0.630) and total phenolics (R2

= 0.554, R2

= 0.723) in roots and leaves of the

plant respectively. Proline and phenolics also showed positive correlation with DW of the

plant.

Published in Journal “Environmental Science and Pollution Research‖

Thomsons Impact factor: 2.826.

Authors: Nasir Ali and Fazal Hadi

Title of paper: ―The effect of Gibberellic acid and EDTA on Cd phytoextraction:

correlation of free proline, total phenolics and chlorophyll content with Cd contents of


Year of publication: (2015),

Volume and PP: 22(17): 13305-13318.

DOI: 10.1007/s11356-015-4595-3

CHAPTER # 2 INTRODUCTION

21

2.1 INTRODUCTION

Cadmium is a heavy metal of great environmental concern which enters the agricultural soil

mostly through anthropogenic activities such as mining, sewage effluents, pesticides, chemical

fertilizers application to fields and industrial waste disposal (Kidd et al 2007; Adewole et al

2010; Hadi et al 2014). From soil and water it can easily absorbed and accumulated into plants

tissues due to its high bio-availability in soil and consequently reaches the human bodies through

food chain (Liu et al 2009; Ambedkar and Muniyan 2013). Crops cultivated in polluted soil may

accumulate Cd in different parts mainly root, leaf and grain. Consumption of Cd polluted plants

may develop a number of Cd-related chronic diseases such as cancer, oxidative stress (by

displace Ca2+

or Zn2+

in proteins), tissue necrosis and impairment of kidney and liver (Kafel et al

2014; John et al 2008; Liu et al 2005). As heavy metals are not bio-degradable (by

microorganisms and plants) so, they are continuously accumulating in soil and their presence in

soil (especially agricultural soil) is of great concern for both plants and animals health (Mubeen

et al 2010). For clean and sustainable environment the removal of such heavy metal form soil

and water is very important and need the development of an effective, affordable and

environment friendly technology for this purpose. Various conventional methods (including both

chemical and physical) have been used for the restoration of heavy metal contaminated soil but

these methods are very costly, laborious and adversely affect both the soil structure and

ecosystem. The discovery of some plants ability to accumulate and tolerate high concentrations

of heavy metals, led to the development of a new plant based technology, known as

phytoremediation (Entry et al 1999; Baker and Brooks 1989). Phytoremediation technology is

cost-effective, solar driven, aesthetically pleasing and environment friendly (Schwitzguebel et al

2009; Chai et al 2012). More than 400 species of plants have been investigated for their heavy

metals phytoremediation potential and most of these plants belong to Arabidopsis, Brassica,

Sedum and Thlaspi species (lone et al 2008). In the present research Parthenium hysterophorus

was studied at its reproductive stage for its Cd phytoextraction potential. This plant belong to

Asteraceae family and is native species of America, which invaded Australia, India, Pakistan and

some parts of Africa (Dhawan and Dhawan 1996). It is a fast growing, stress tolerant perennial

herb, which is unpalatable to herbivores, thus prevent metal entrance into food chain.

Plants grown on metal contaminated soil often show slow growth, low biomass and/ or lower

metal concentration within the biomass (Li et al 2003). For efficient heavy metals


22

phytoremediation plant must have high biomass and can also tolerate and accumulate high

concentration of toxic heavy metal within their tissues. Heavy metal tolerant plants commonly

have low biomass or most plants showing high biomass do not show tolerance to high metals

concentration of heavy metals in soil. To increase biomass as well as metal phytoextraction

potential of plants, several chemical amendments (applied to plant or added to soil/ water) have

been done by different scientists, such as the application of hormones (foliar spray) and addition

of metal chelator to soil (Falkowska et al 2011; Hadi and Bano 2009; Chen and Cutright 2001).

In the present experiment a plant hormone ―Gibberellic acid‖ (GA3) and a synthetic chelator

EDTA (ethylenediaminetetraacetic acid) was used for increasing the Cd phytoremediation

potential of the plant. GA3 enhance plant growth and biomass while EDTA increase the metal

bioavailability in soil by forming complexes with metals (Broughton and McComb 1971;

Benjerano and Lips 1970; Chen et al 2004; Hadi and Bano 2009; Hadi et al 2010).

Plant under stress conditions produce and accumulate a variety of metabolic products including

amino acids (such as proline) and phenolic compounds (Diaz et al 2001). Many investigators

have reported accumulation of free proline under conditions of salinity, drought, intense light

and ultraviolet radiation, heavy metals, and in response to oxidative stress and biotic stresses

(Haudecoeur et al 2009; Yang et al 2009; Choudhary et al 2005). Proline not only take part in

protein synthesis but also showed a positive correlation with plant stress because it can act as a

metal chelator, signaling molecule, maintaining osmotic or cell turgor pressure; reducing

electrolyte leakage by stabilizing membranes; and protecting plant from oxidative stress by

reducing concentration of reactive oxygen species (ROS) (Xu et al 2009). Similarly phenolic

compounds are also produced during heavy metal stress, which not only act as metal chelator but

also act as antioxidant and directly scavenge reactive oxygen specie (ROS) (Michalak 2006).

ROSs can destroy lipids, DNA, proteins and chlorophyll by producing highly reactive (nascent)

oxygen (Ramadevi and Parsad, 1998). High concentrations of phenolic compounds has been

reported in different plants such as wheat in response to nickel toxicity (Diaz et al 2001),

Phaseolus vulgaris when exposed to cadmium and Phyllantus tenellus leaves in response to

copper sulphate (Diaz et al 2001) and maize due to aluminum (Winkel-shirley 2002).

CHAPTER # 2 AIM AND OBJECTIVES

23

2.1.1 Aim and objectives

Aim:

The aim of this chapter was to evaluate the effect of a plant growth regulator (gibberellic acid,

GA3) and a chelating agent (ethylenediaminetetraacetic acid, EDTA) on the phytoextraction

potential of Parthenium hysterophorus plants.

Objectives:

1. To study the growth and biomass of the P. hysterophorus plant in Cd contaminated soil.

2. To investigate the concentration of proline and phenolics in different parts of the plant.

3. To find out the effects of GA3 and EDTA on Cd uptake, its translocation into plant shoot

and accumulation in different parts of the plant.

4. To evaluate the effect of Cd on contents of chlorophyll in leaves.

5. To study the correlations of total phenolics and free proline with the DW and Cd contents

of the plant.

CHAPTER # 2 MATERIALS AND METHODS

24

2.2 MATERIALS AND METHODS

2.2.1 Preparation of soil and addition of cadmium

Soil was collected from fields nearby the University of Malakand at Chakdara, Pakistan. The soil

was grounded into powdered form after drying in sunlight. Water holding capacity (300 ml water

per kg soil ± 3) and pH (6.5 ± 0.3) of the soil was calculated. The dried soil was then poured in

to plastic pots (size ‘18 height x15 diameter cm‘) at the rate of 1 kg soil per pot. Cadmium (100

mg kg-1

soil) was added to each pot as cadmium acetate dihydrate (CH3COO)2 Cd·2H2O (Merck,

Germany) solution. No cadmium was added to the control (C) pots.

2.2.2 Transplantation of seedlings and plant growth

Each pot was watered a day before transplantation of plantlets. Seedlings (P. hysterophorus

plantlets) of uniform size (height 3 in) were collected for fields and single plantlet was

transferred to each pot. Natural condition of light and temperature (35/25°C) was provided for

growth of plants. Replicate of three pots were used for each treatment and the controls. Two

controls were used; one without cadmium (C) and the other with cadmium only (C1). Plants

were watered, at three days interval.

2.2.3 Treatments used

Table 2. 1: Treatments done during the whole experiment.

Treatment Treatment

code

Treatment Treatment

code

control (No Cd) C Cd + GA3 10-6

M + EDTA 40 mg T6

control (only Cd) C1 Cd + GA3 10-6

M + EDTA 10 mg T7

Cd + GA3 10-2

M T1 Cd + GA3 10-4

M + EDTA 40 mg T8

Cd + GA3 10-4

M T2 Cd + GA3 10-4

M + EDTA 10 mg T9

Cd + GA3 10-6

M T3 Cd + GA3 10-2

M + EDTA 40 mg T10

Cd + EDTA 40 mg T4 Cd + GA3 10-2

M + EDTA 10 mg T11

Cd + EDTA 10 mg T5

Note: Foliar spray of GA3 was applied to plants in four split doses, while EDTA was added to

soil as 40 mg single dose and 10 mg EDTA in four split doses.

CHAPTER # 2 RESULTS

25

2.2.3.1 Exogenous application of GA3

Three different concentrations (10-2

, 10-4

and 10-6

M) of GA3 were applied to the plants in the

form of foliar spray (10 ml solution per plant) in four doses (each dose at 10 d intervals). First

treatment was made 15 days after transplantation. Polythene bags were used to cover soil in

pots during application of GA3 so that hormones droplets do not reach the root zone.

2.2.3.2 Ethylenediaminetetraacetic acid (EDTA) addition into soil

A total of 40 mg of EDTA was added per Kg soil (i.e. single pots) in the form of aqueous

solution in two different ways i.e. single dose of 40 mg EDTA per pot (kg soil) and in four

split doses, each of 10 mg EDTA per dose, at 10 days interval. First treatment of EDTA

(single or split dose) was made 10 days after transplantation

2.2.3.3 Combination treatments of GA3 and EDTA

Some plants were treated with both GA3 and EDTA in combination. The three different

concentrations of GA3 (10-6

, 10-4

and 10-2

M) and the two different ways of EDTA (40 mg per

pot) application (single and split dose) form a total of six types of different combination

(3×2). In the combination treatments, both the GA3 and EDTA applications were made as

mentioned earlier.

2.2.4 Plant growth parameters analysis

Plant shoot length was measured on weekly basis. After two and a half months from

transplantation (at reproductive stage) the plants were harvested and length of the plant roots

and shoots were measured in cm. Plants were washed with a 5 mM solution of EDTA and

Tris-HCl (pH 6.0), and then rinsed with distilled water to remove any surface bounded metals

(Genrich et al 2000). After washing each plant was cut into three parts i.e. roots, stem and

leaves and fresh weight of each part was measured with the help of analytical balance. The

parts of each plant were packed in separate paper envelopes and then kept in oven for 48 h at

800C for drying. The dry weight of each part was measured by analytical balance and then

grinded into powdered form.

2.2.5 Analysis of free proline in plant root and leaves

Bates et al (1973) method was used for the quantification of free proline within different parts

(root and leaves) of the plant. Fresh plant tissue (100 mg from each part) was homogenized/

crushed in 2 ml tubes containing 1.5 ml of 3 % sulfosalicylic acid. The homogenate was then

CHAPTER # 2 RESULTS

26

centrifuged for 5 minutes at 13000 rpm. The supernatant (only 300 µL) was transferred into

new tube and then 2 ml each of acid ninhydrin (containing 1.25 g of ninhydrin heated in 20

ml phosphoric acid (6 M) and 30 ml glacial acetic acid until dissolved completely) and

glacial acetic acid were added to it. The mixture was kept in water bath (100 0C) for one hour.

The tubes were immediately dipped into ice after removing from water bath. Toluene (1 ml

per tube) was added to the reaction mixture and then vigorously mixed for 10-30 seconds.

Toluene containing chromophore layer was removed from the aqueous phase with the help of

micropipette and warmed to the room temperature. Spectrophotometer was used (250 nm

wavelength) to measure the absorbance of each sample. Toluene was used as a blank

(control). The standard curve was used to calculated the concentration of proline in different.

Three replicates were used for each sample.

2.2.6 Total phenolics estimation in roots and leaves

Total phenolic were calculated in roots, stems and leaves of each plant. Dried sample (200

mg each) was mixed with 10 ml of methanol (80 %) and then shake for at least 30 minutes in

close vessel (flask) to prevent evaporation of solvent. From each extract 2ml was taken in

separate tubes and centrifuged at 13000 rpm for 3 to 5 minutes. Singleton and Rossi (1965)

method with slight modifications was used to analysis total phenolics in extract. A 250 µL of

Folin-Ciocalteau (FC) reagent was mixed with 100 µL gallic acid standard solutions or

methanolic extract and the mixture was kept in dark (at room temperature) for 3-5 minutes.

Then 7 % of 500 µL sodium carbonate (Na2CO3) solution was added to the mixture and the

dH2O was used to raise the net volume up to 5 mL with. The mixture was kept in dark at

room temperature for 2 hrs. A spectrophotometer was used to measure the absorbance of the

samples at 760 nm. Different standard solutions (10, 30, 50, 100, 150 mgL-1

) of gallic acid

were prepared in methanol (80 %) and their absorbance were used as standard for measuring

total phenolics in each samples. 80 % methanol solution was used as blank (control). Three

replicates were used for each sample.

2.2.7 Chlorophyll measurment in leaves

Concentration of chlorophyll a and b were calculated by using the method of Arnon, (1949).

First of all fresh leaves were obtained from the plants (both control and treated plants). Then

2ml (80%) acetone was mixed with 200 mg of fresh leaves and properly grinded. After

grinding the mixtures were shifted into ependorf tubes and then centrifuged for five minutes

at 10,000 rpm. The supernatant (after centrifugation) were poured into clean test-tubes and

6ml acetone (80 %) was added to it. The samples were then analyzed for absorbance at 645

CHAPTER # 2 RESULTS

27

nm and 663 nm with the help of spectrophotometer. The following formulas were used for

calculating the concentration of chlorophyll a and b:

Chlorophyll a (µg ml-1

) = 12.7 (A663) - 2.69 (A645)

Chlorophyll b (µg ml-1

) = 22.9 (A645) - 4.68 (A663)

2.2.8 Cadmium (Cd) analysis in plants

Oven dried samples (root, stem and leaves) were first grounded into powdered form and then

subjected to acid digestion using Allen (1974) method. Dried powder (0.25 g) from each

sample was taken into separate flasks (50 ml). A 6.5 ml of three acids mixture containing

sulfuric acid, nitric acid and perchloric acid (1, 5 and 0.5 ml respectively) was added to each

flask. For complete digestion each flask (sample) was kept on electric hot plates until

completely digested. The digested samples were then filtered into another volumetric flask

(50 ml) and with the help of dH2O the volume was raised up to 50 ml. Each filtrate sample

was then stored in small plastic bottles. The samples were then analyzed for Cd concentration

with the help of Atomic Absorption/Flame Spectrophotometer (model Hitachi Z-8000,

Japan). Analysis was carried out under the conditions: wavelength (228.8 nm), Lamp current

(4.0 mA), Flow rate (Argon gas), 200 ml per min.

Translocation factor (TF) is the ratio of metal concentration in aerial parts (stem and leaves)

of the plant to the metal concentration in roots. Translocation factor of Cd was calculated

using the formula given below:

TF (roots into stem) = [metal] shoot / [metal] root

TF (roots into leaves) = [metal] leaves / [metal] root

Bio-concentration Factor (BCF) was defined as the accumulated concentration of heavy

metals in root divided by concentration to that in respective soil (Yoon et al., 2006).

BCF= [metal] root / [metal] soil

2.2.9 Statistical analysis

The data was further analyzed for mean values, analysis of variance (ANOVA) and

correlations between different parameters using software such as SPSS 16 (Statistical

Package for Social Sciences – 16) and MS Excel 2007.

CHAPTER # 2 RESULTS

28

2.3 RESULTS

2.3.1 Effect of EDTA and GA3 treatments on plant length (root and stem), biomass

(fresh and dry) and water contents of P. hysterophorus plant under Cd stress

Plant length, biomass and water content were significantly reduced by the Cd contaminated

(100 mg Cd Kg-1

) soil when both the controls C (without Cd) and C1 (with Cd only) were

compared, except the dry weight (DW) of stem and total water content (TWC) of leaves

(Figure 2.1 and Table 2.2). The effect of all the treatments (except T4, T5 and T11 in root,

while T4 and T11 in stem) were statistically significant on root and shoot length of the plant

as compared to C1 (control with Cd only) (Table 2.2). The highest significant root length

(24.33 ± 1.00 cm) and stem length (44.33 ± 4.73 cm) was demonstrated by the treatment T3

(GA3 10-6

M). There was a significant increase in Fresh weight (FW) and TWC of plant roots

in all the treatments (except T10 and T11) when compared with C1. , However, FW of plant

stem and leaves were statistically significant only in GA3 alone treatments (T1, T2 and T3) as

compared to C1 (Table 2.2). Among the treatments highest FW and TWC of entire plant were

produced by the treatment T3. All the treatments significantly increased DW of root, stem,

leaves and entire plant as compared to C1, except root DW in T6 (GA3 10-6

M + EDTA 40

mg kg-1

) and T7 (GA310-6

M + EDTA 10 mg kg-1

), stem DW in T10 (GA3 10-2

M + EDTA

40 mg kg-1

) and T11 (GA3 10-2

M + EDTA 10 mg kg -1

) and leaf DW in T11. The highest

significant DW in roots (1.65 ± 0.02 g), stem (2.40 ± 0.05 g), leaves (2.40 ± 0.05 g) and

entire plant (6.45 ± 0.12 g) was recorded in the treatment T3 as given in Table 2.2.

29

Figure 2. 1: Effect of different treatments of GA3 and EDTA on the root and shoot length of Parthenium hysterophorus plant, in cadmium contaminated

soil (100 mg Cd kg-1

soil).

30

Table 2. 2: Effect of different treatments of GA3 and EDTA on different growth parameters (length, biomass and water content) of Parthenium hysterophorus plant, in cadmium

contaminated soil (100 mg Cd/kg soil). GA3 was applied in four split doses, and 40 mg EDTA was added in a single dose while four doses of 10 mg EDTA added to a pot. SD denote

standard deviation and different letters shows significant different among values of different parameters. While ‗R‘ stands for Roots, ‗S‘ for Stem, ‗L‘ for leaf and EP for Entire Plant

Treatment length (cm) ±

SD

Fresh weight (g) ± SD Dry biomass (g) ± SD Total water content (g) ± SD

R S R S L EP R S L EP R S L EP

C No Cd 24.00 ± 1.00 ab

33.00 ± 1.00 cd

8.70 ± 0.44 a

10.00 ± 1.00 bc

8.50 ± 0.50 bc

27.20 ± 1.61 bc

1.92 ± 0.07 a

1.67 ± 0.06 ef

2.40 ± 0.10 a

5.99 ± 0.23 b

6.78 ± 0.37 ab

8.33 ± 0.94 bcd

6.10 ± 0.46 c

21.21 ± 1.39 bcd

C1 Cd only 12.00 ± 0.98 hi

24.10 ± 0.85 ef

4.23 ± 0.20 f

6.40 ± 0.98 def

6.50 ± 0.60 de

17.13 ± 1.78 ef

0.68 ± 0.03 ij

1.53 ± 0.03 f

1.40 ± 0.03 e

3.75 ± 0.09 f

3.55 ± 0.17 e

4.73 ± 0.95 efg

5.10 ± 0.57 cde

13.38 ± 1.69 fgh

T1 Cd + GA3 10-2

M 21.37 ± 1.09 bc

33.50 ± 1.50 cd

6.50 ± 0.19 c

11.50 ± 1.09 ab

10.10 ± 0.56 ab

28.10 ± 1.84 b

1.30 ± 0.03 d

2.20 ± 0.03 b

2.27 ± 0.03 a

5.77 ± 0.09 b

5.20 ± 0.16 c

9.30 ± 1.06 abc

7.83 ± 0.53 ab

22.33 ± 1.75 bc

T2 Cd + GA3 10-4

M 22.00 ± 0.78 abc

40.30 ± 4.59 ab

7.80 ± 0.19 b

12.67 ± 0.78 a

10.40 ± 0.56 a

30.87 ± 1.53 ab

1.45 ± 0.01 c

2.29 ± 0.05 ab

2.34 ± 0.05 a

6.08 ± 0.11 b

6.35 ± 0.18 b

10.38 ± 0.73 ab

8.06 ± 0.51 a

24.79 ± 1.42 ab

T3 Cd + GA3 10-6

M 24.33 ± 1.00 a

44.33 ± 4.73 a

8.70 ± 0.25 a

13.53 ± 1.00 a

11.20 ± 0.75 a

33.43 ± 2.00 a

1.65 ± 0.02 b

2.40 ± 0.05 a

2.40 ± 0.05 a

6.45 ± 0.12 a

7.05 ± 0.23 a

11.13 ± 0.95 a

8.80 ± 0.70 a

26.98 ± 1.88 a

T4 Cd + EDTA 40 mg 10.00 ± 0.97 i

18.00 ± 1.00 fg

3.40 ± 0.32 g

5.20 ± 0.97 ef

5.30 ± 0.96 e

13.90 ± 2.24 f

0.43 ± 0.02 k

1.32 ± 0.02 g

1.10 ± 0.02 f

2.85 ± 0.06 g

2.97 0.30 ef

3.88 ± 0.95 fg

4.20 ± 0.94 de

11.05 ± 2.19 gh

T5 Cd + EDTA 10 mg 10.50 ± 1.05 i

15.00 ± 1.32 g

3.33 ± 0.17 g

4.70 ± 1.05 f

5.20 ± 0.50 e

13.23 ± 1.72 f

0.50 ± 0.01 k

1.43 ± 0.05 g

1.20 ± 0.05 f

3.13 ± 0.11 g

2.83 ± 0.16 f

3.27 ± 1.00 g

4.00 ± 0.45 e

10.10 ± 1.61 h

T6 Cd + GA3 10-6

M + EDTA 40 mg

20.00 ± 0.99 cd

35.00 ± 1.00 bc

5.40 ± 0.09 d

8.90 ± 0.99 bcd

8.20 ± 0.26 cd

22.50 ± 1.34 cd

0.67 ± 0.01 j

1.96 ± 0.04 c

1.95 ± 0.04 b

4.58 ± 0.09 c

4.73 ± 0.08 cd

6.94 ± 0.95 cde

6.25 ± 0.22 bc

17.92 ± 1.25 cde

T7 Cd + GA3 10-6

M + EDTA 10 mg

18.00 ± 1.00 de

31.07 ± 2.11 cd

5.32 ± 0.02 d

8.70 ± 1.00 cd

8.00 ± 0.06 cd

22.02 ± 1.08 d

0.76 ± 0.03 hi

1.87 ± 0.03 cd

1.89 ± 0.03 bc

4.52 ± 0.09 c

4.56 ± 0.01 d

6.83 ± 0.97 cde

6.11 ± 0.03 c

17.50 ± 0.99 def

T8 Cd + GA3 10-4

M + EDTA 40 mg

17.60 ± 0.87 def

32.50 ± 1.32 cd

5.20 ± 0.10 d

8.43 ± 0.87 cd

7.50 ± 0.30 cd

21.13 ± 1.27 de

0.84 ± 0.03 gh

1.77 ± 0.03 de

1.84 ± 0.03 bc

4.45 ± 0.09 cd

4.36 ± 0.07 d

6.66 ± 0.84 de

5.66 ± 0.27 cde

16.68 ± 1.18 def

T9 Cd + GA3 10-4

M + EDTA 10 mg

16.30 ± 0.65 efg

28.33 ± 1.15 de

5.16 ± 0.14 de

8.10 ± 0.65 cd

7.34 ± 0.43 cd

20.60 ± 1.22 de

0.87 ± 0.03 fg

1.72 ± 0.03 e

1.78 ± 0.03 c

4.37 ± 0.09 cd

4.29 ± 0.11 d

6.38 ± 0.62 def

5.56 ± 0.40 cde

16.23 ± 1.13 ef

T10 Cd + GA3 10-2

M + EDTA 40 mg

15.00 ± 0.45 fg

29.67 ± 1.53 cde

4.50 ± 0.33 ef

7.87 ± 0.45 cd

7.26 ± 0.98 cd

19.63 ± 1.76 de

0.95 ± 0.02 ef

1.66 ± 0.06 ef

1.56 ± 0.06 d

4.17 ± 0.14 de

3.55 ± 0.31 e

6.21 ± 0.39 def

5.70 ± 0.92 cde

15.46 ± 1.62 efg

T11 Cd + GA3 10-2

M + EDTA 10 mg

14.20 ± 0.43 gh

23.67 ± 1.53 ef

4.43 ± 0.25 f

7.76 ± 0.43 cde

7.21 ± 0.75 cd

19.40 ± 1.43 de

0.99 ± 0.03 e

1.58 ± 0.03 f

1.46 ± 0.03 de

4.03 ± 0.09 ef

3.44 ± 0.22 ef

6.18 ± 0.40 def

5.75 ± 0.72 cd

15.37 ± 1.34 efg

CHAPTER # 2 RESULTS

31

2.3.2 Effect of different treatments of GA3 and EDTA on plant Cd contents

Cadmium concentrations in the Roots of the plant increased significantly in all the treatments

when compared with C1 (control with Cd only) and the highest significant root Cd

concentration (1267.00 ± 12.60 and 1245 ± 16.20 ppm) was recorded for the treatment T4

and T5 respectively (Table 2.3). The treatments showed significant increase in Cd

concentration of the plant stem (except T1, T2 and T3) and leaves (except T1) as compared to

C1. The treatment T11 produced the highest significant effect on the concentration of stem

and leaf Cd concentrations (166.33 ± 18.00 and 570.00 ± 23.45 ppm respectively).

Accumulation of Cd (mg per DW) in different parts of the plant was statistically significant

in all the treatments (except in T1 plant stem) as compared to C1 (Table 2.3). The table

shows that the highest significant Cd accumulation in roots (0.84 ± 0.04, 0.86 ± 0.03 and 0.87

± 0.04 mg Cd per DW) of the plant was found in the treatments T9, T10 and T11

respectively, while the treatment T11 also possessed the highest Cd accumulation in stem

(0.26 ± 0.03 mg Cd per DW), leaves (0.83 ± 0.04 mg Cd per DW) and entire plant (1.97 ±

0.11 mg Cd per DW). The treatments showed an increase of 4.07-9.79 folds Cd contents in

roots, 1.61-3.21 in stem, 1.86-4.71 in leaves and 2.37-5.65 folds the entire plant Cd

accumulation respectively as given in Table 2.3. The highest increase in Cd accumulation

within roots (9.79 times), stem (3.21 times), leaves (4.71 times) and entire plant (5.65 times)

was demonstrated by the treatment T11. The results also showed that the highest Cd

accumulation percentage in the treatments was found within roots followed by leaves of the

plant while the lowest Cd accumulation percentage was noted in the plant stem. The control

plant showed more than 50% of Cd with in its leaves. Cadmium translocation in treated

plants was significantly lower than the control C1. Cadmium bio-concentration (BCF) of the

plant was found higher than one (i.e. 1.85 ± 0.22) in the control C1 plants which clearly

shows that the P. hysterophorus is a hyper accumulator of Cd. The treatment further

increased the Cd BCF and the increase was found statistically significant as compared to the

control C1. The highest significant Cd BCF (9.75 ± 0.34) was demonstrated by the treatment

T11.

CHAPTER # 2 RESULTS

32

Table 2. 3: Effect of different treatments of GA3 and EDTA on Cd concentration, accumulation, translocation and Bioconcentration of Parthenium hysterophorus plant, in cadmium

contaminated soil (100 mg Cd kg-1

soil). GA3 was applied in four split doses, and 40 mg EDTA was added in a single dose while four doses of 10 mg EDTA added to a pot. SD

denote standard deviation and different letters shows significant different among values of different parameters. while ‗R‘ stands for Roots, ‗S‘ for Stem, ‗L‘ for leaf, EP for Entire

Plant, TF for translocation factor, BCF for Bioconcentration Factor.

Treatment Cd concentration (ppm) ± SD Cd accumulation (mg Kg-1

) ± SD Fold increase in Cd

accumulation

compared to C1*

Cd accumulation % Cadmium TF Cadm-

ium

BCF

R S L R S L EP R S L EP R S L R to

S

R to

L

C1 Cd only 129.67 ± 12.00

h 49.00 ± 9.40

d 126.00 ± 12.00

i 0.09 ± 0.01

g 0.08 ± 0.02

d 0.18 ± 0.02

g 0.35 ± 0.05

g 0.09 mg

0.08 mg

0.18 mg

0.35 mg

25.59 23.45 50.96 0.38 ± 0.04

a 0.97 ± 0.01

a 1.85 ± 0.22

h

T1 Cd + GA3 10-2

M 278.00 ± 9.20

g 60.00 ± 12.00

d 145.00 ± 9.20

hi 0.36 ± 0.02

f 0.13 ± 0.03

cd 0.33 ± 0.03

f 0.82 ± 0.07

f 4.07 1.61 1.86 2.37 44.01 15.95 40.04 0.22 ±

0.04 b

0.52 ± 0.02

d 2.85 ± 0.21

g

T2 Cd + GA3 10-4

M 346.33 ± 13.40

f 72.00 ± 9.20

d 167.00 ± 13.40

gh 0.50 ± 0.02

e 0.17 ± 0.02

bc 0.39 ± 0.04

ef 1.06 ± 0.09

ef 5.65 2.01 2.21 3.05 47.54 15.55 36.92 0.21 ±

0.02 b

0.48 ± 0.02

e 3.48 ± 0.22

fg

T3 Cd + GA3 10-6

M 432.33 ± 11.20

e 80.50 ± 13.40

cd 189.00 ± 11.20

g 0.71 ± 0.03

c 0.19 ± 0.04

abc 0.45 ± 0.04

de 1.36 ± 0.10

d 8.03 2.36 2.57 3.92 52.51 14.15 33.34 0.19 ±

0.03 b

0.44 ± 0.01

f 4.22 ± 0.23

f

T4 Cd + EDTA 40 mg 1267.00 ± 12.60

a 143.00 ± 11.20

ab 425.00 ± 12.60

cd 0.54 ± 0.03

de 0.19 ± 0.02

abc 0.47 ± 0.02

de 1.20 ± 0.07

de 6.13 2.30 2.65 3.46 45.36 15.70 38.94 0.11 ±

0.01 b

0.34 ± 0.01

hi 8.43 ± 0.32

bcd

T5 Cd + EDTA 10 mg 1245.00 ± 16.20

a 149.00 ± 12.60

ab 456.00 ± 16.20

c 0.62 ± 0.02

d 0.21 ± 0.03

ab 0.55 ± 0.04

d 1.38 ± 0.09

cd 7.01 2.60 3.10 3.98 45.05 15.39 39.56 0.12 ±

0.01 b

0.37 ± 0.01

gh 8.84 ± 0.25

bc

T6 Cd + GA3 10-6

M + EDTA 40 mg

1123.00 ± 16.00

b 112.00 ± 16.20

bc 345.00 ± 12.90

f 0.75 ± 0.02

bc 0.22 ± 0.04

ab 0.67 ± 0.04

c 1.65 ± 0.10

bc 8.47 2.68 3.81 4.74 45.78 13.31 40.91 0.10 ±

0.01 b

0.31 ± 0.01

i 7.18 ± 0.28

e

T7 Cd + GA3 10-6

M + EDTA 10 mg

1098.00 ± 15.20

b 119.00 ± 12.90

b 365.00 ± 15.20

f 0.83 ± 0.04

ab 0.22 ± 0.03

ab 0.69 ± 0.04

bc 1.75 ± 0.11

ab 9.40 2.72 3.91 5.03 47.79 12.71 39.50 0.11 ±

0.01 b

0.33 ± 0.01

i 7.73 ± 0.34

de

T8 Cd + GA3 10-4

M + EDTA 40 mg

992.33 ± 16.00

c 120.00 ± 15.20

b 379.00 ± 9.60

ef 0.83 ± 0.04

ab 0.21 ± 0.03

abc 0.70 ± 0.03

bc 1.74 ± 0.10

ab 9.39 2.59 3.95 5.02 47.82 12.15 40.02 0.12 ±

0.01 b

0.38 ± 0.03

g 7.83 ± 0.30

de

T9 Cd + GA3 10-4

M + EDTA 10 mg

968.00 ± 12.00

c 129.00 ± 9.60

ab 413.00 ± 9.20

de 0.84 ± 0.04

a 0.22 ± 0.02

ab 0.74 ± 0.03

abc 1.80 ± 0.09

ab 9.48 2.71 4.16 5.18 46.81 12.32 40.87 0.13 ±

0.01 b

0.43 ± 0.02

f 8.23 ± 0.24

cd

T10 Cd + GA3 10-2

M + EDTA 40 mg

908.00 ± 9.00

d 147.33 ± 9.20

ab 510.00 ± 18.00

b 0.86 ± 0.03

a 0.24 ± 0.02

ab 0.80 ± 0.06

ab 1.90 ± 0.11

ab 9.71 2.99 4.51 5.48 45.36 12.84 41.80 0.16 ±

0.01 b

0.56 ± 0.01

c 9.13 ± 0.22

ab

T11 Cd + GA3 10-2

M + EDTA 10 mg

878.00 ± 9.00

d 166.33 ± 18.00

a 570.00 ± 23.45

a 0.87 ± 0.04

a 0.26 ± 0.03

a 0.83 ± 0.04

a 1.97 ± 0.11

a 9.79 3.21 4.71 5.65 44.27 13.36 42.37 0.19 ±

0.02 b

0.65 ± 0.01

b 9.75 ± 0.34

a

* For C1 (control with Cd only), actual values of extracted Cd (mg) are given.

CHAPTER # 2 RESULTS

33

2.3.3 Effect of different treatments of GA3 and EDTA on total phenolics, free proline

and chlorophyll (a/b) contents of the plant under Cd stress

Free proline and total phenolics in roots, stem and leaves while chlorophyll (a/b) contents in

leaves of the plant are presented in Table 2.4. cadmium contaminated soil significantly

increased free proline contents only in plant roots while total phenolics were significantly

higher in all parts (root, stem and leaves) of the plant (comparing the controls C and C1). The

increase in proline content of the plant roots were statistically non-significant in the

treatments (compared to C1) while the treatment T3 in stem and treatments T10 and T11 in

leaves of the plant significantly increased free proline concentration when compared with

control C1. The increase in total phenolics in roots of the plant was significantly increased by

the treatments except the EDTA alone treatments (T4 and T5) where increase was

statistically non-significant (compared to the control C1) as given in Table 2.4. In plant stem

total phenolic concentration was significantly increased by the treatments (except T1, T4, T5,

T8 and T9) as compared to C1 while in leaves all the treatments showed significant increase

in total phenolics (compared to C1). The highest significant total phenolic content in roots

(79.00 ± 3.94 ppm) was found in treatment T7 while in stem (34.00 ± 2.50 ppm) and leaves

(156.00 ± 13.20 ppm) it was recorded in the treatment T11 (Table 2.4). The table also shows

that the concentration of chlorophyll (a and b) was reduced significantly by the cadmium

polluted soil (comparing C with C1). The treatments containing GA3 foliar spray significantly

increased chlorophyll (a/b) concentration in leaves of the plant as compared to C1, except the

chlorophyll a contents of T9, T10 and T11 (where the increase is non-significant compared to

C1). The EDTA alone treatments showed significant decrease in chlorophyll content (except

chlorophyll a concentration in T4) as compared to C1.

CHAPTER # 2 RESULTS

34

Table 2. 4: Effect of Gibberellic acid and EDTA treatments on free proline, total phenolics and chlorophyll (a/b) contents of Parthenium

hysterophorus plant grown in Cd contaminated soil (100 mg Cd/kg soil). GA3 was applied in four split doses, and 40 mg EDTA was added in a

single dose while four doses of 10 mg EDTA added to a pot.

Treatments Free proline (ppm) ± SD Total Phenolics ± SD Chlorophyll ± SD

Roots Stem leaf Root stem leaf a b total

chlorophyll

C control (without Cd) 12.00 ±

2.80 b

15.00 ±

3.40 c

10.00 ±

1.88 d

15.00 ±

4.20 g

8.50 ±

3.50 d

27.00 ±

3.50 d

5.10 ±

0.36 bc

4.67 ±

0.15 a

9.77 ±

0.31 b

C1 Cd only 57.60 ±

3.33 a

24.00 ±

3.33 bc

28.57 ±

5.40 cd

29.23 ±

3.33 f

20.00 ±

3.33 c

90.80 ±

9.40 c

3.65 ±

0.20 gh

2.81 ±

0.03 h

6.46 ±

0.23 f

T1 Cd + GA3 10-2

M 58.00 ±

3.00 a

25.00 ±

3.00 abc

39.00 ±

5.06 abc

50.00 ±

3.00 cd

29.00 ±

3.00 abc

120.00 ±

6.80 b

5.20 ±

0.19 bc

4.32 ±

0.03 b

9.52 ±

0.22 b

T2 Cd + GA3 10-4

M 64.30 ±

5.96 a

25.80 ±

5.96 ab

45.00 ±

9.00 abc

57.00 ±

2.96 bc

33.00 ±

2.96 a

139.00 ±

9.20 ab

5.70 ±

0.19 ab

4.54 ±

0.05 a

10.24 ±

0.24 a

T3 Cd + GA3 10-6

M 62.50 ±

3.60 a

35.00 ±

3.60 a

48.57 ±

6.75 abc

65.00 ±

3.60 b

36.00 ±

3.60 a

145.00 ±

9.80 ab

5.98 ±

0.25 a

4.70 ±

0.05 a

10.68 ±

0.30 g

T4 Cd + EDTA 40 mg 60.00 ±

2.50 a

24.00 ±

2.50 bc

32.00 ±

8.61 bcd

33.00 ±

2.50 f

23.00 ±

2.50 bc

138.00 ±

5.90 ab

3.05 ±

0.32 hi

2.35 ±

0.02 i

5.40 ±

0.34 g

T5 Cd + EDTA 10 mg 59.70 ±

2.70 a

27.00 ±

2.70 ab

30.00 ±

12.66 cd

36.00 ±

2.70 ef

22.00 ±

2.70 bc

123.00 ±

6.50 b

2.95 ±

0.17 i

2.27 ±

0.05 i

5.22 ±

0.22 c

T6 Cd + GA3 10-6

M + EDTA 40 mg 63.00 ±

2.25 a

32.00 ±

2.25 ab

43.40 ±

9.45 abc

79.00 ±

2.25 a

29.95 ±

2.25 ab

123.00 ±

11.00 b

4.76 ±

0.09 cd

3.87 ±

0.04 c

8.63 ±

0.13 cd

T7 Cd + GA3 10-6

M + EDTA 10 mg 59.00 ±

3.94 a

26.00 ±

3.94 ab

44.29 ±

8.10 abc

76.00 ±

3.94 a

30.00 ±

3.94 ab

143.00 ±

12.90 ab

4.67 ±

0.02 cde

3.59 ±

0.03 d

8.26 ±

0.05 de

T8 Cd + GA3 10-4

M + EDTA 40 mg 63.00 ±

2.50 a

32.00 ±

2.50 ab

46.43 ±

5.06 abc

65.00 ±

2.50 b

28.00 ±

2.50 abc

130.00 ±

10.40 ab

4.37 ±

0.10 def

3.36 ±

0.03 e

7.73 ±

0.13 de

T9 Cd + GA3 10-4

M + EDTA 10 mg 60.00 ±

3.58 a

31.00 ±

3.58 ab

49.29 ±

9.00 abc

60.00 ±

3.58 b

28.90 ±

3.58 abc

145.00 ±

9.60 ab

4.30 ±

0.14 defg

3.31 ±

0.03 ef

7.61 ±

0.17 e

T10 Cd + GA3 10-2

M + EDTA 40 mg 63.00 ±

3.38 a

29.00 ±

3.38 ab

54.29 ±

8.86 ab

47.00 ±

3.38 d

31.00 ±

3.38 ab

130.00 ±

9.20 ab

4.10 ±

0.33 efg

3.15 ±

0.06 fg

7.25 ±

0.39 ef

T11 Cd + GA3 10-2

M + EDTA 10 mg 61.67 ±

2.50 a

32.00 ±

2.50 ab

57.14 ±

6.75 a

44.00 ±

2.50 de

34.00 ±

2.50 a

156.00 ±

13.20 a

3.95 ±

0.25 fg

3.12 ±

0.03 g

7.07 ±

0.28 ab

CHAPTER # 2 RESULTS

35

2.3.4 Correlation among different parameters measured in plant

Correlations among different parameters measured in roots of the plants are presented in

Table 2.5. The table shows significant correlations among plant growth parameters for roots

(length, FW, DW and TWC). Conversely the correlation of roots Cd concentration with

length (R2 = -0.452), FW (R

2 = - 0.588), DW (R

2 = -0.674) and TWC (R

2 = -0.546) of the

plant roots was found negative and mostly significant. Cadmium concentration in roots

showed highly significant positive correlation (R2 = 0.661) with the accumulation of Cd

within roots. The concentration of free proline and total phenolics in roots showed a positive

significant correlations (R2 = 0.527 and R2 = 0.554 respectively) with Cd accumulation but

their correlation with the concentration of Cd in roots was statistically non-significant. The

total phenolics of roots also showed positive significant correlations with length (R2 =

0.728), FW (R2 = 0.537) and TWC (R2 = 0.590) while its correlation with roots DW was

also positive but statistically non-significant. The correlations of free proline contents of roots

also demonstrated positive correlation with the growth parameters but the correlation were

found to be statistically non-significant. Correlations between the parameters measured in

stems of the plant are given in Table 2.6. The table shows positive significant correlations

between length, FW, DW and TWC of the stem while the correlations of these parameters

with stem Cd concentration were negative and statistically significant. Stem Cd accumulation

also showed negative correlations with above growth parameters but these correlations were

non-significant statistically. Free proline and total phenolics concentration of stem showed

positive correlation with all the parameters measured in plant stem but the correlations of

proline concentration was statistically significant only with phenolics content (R2 = 0.577)

and Cd accumulation (R2 = 0.867) while the correlations of total phenolics were significant

with all the parameters except Cd concentration and accumulation with in stem. The table 2.7

shows correlations among different parameters measured in plant leaves. Like roots and stem,

the leaves of plant also showed positive significant correlations among the growth parameters

(FW, DW and TWC). Cadmium concentration and accumulation of leaf demonstrated

negative correlations with the growth parameters and only the correlations of Cd

concentration were found statistically significant with the growth parameters. Correlation

between free proline and total phenolics was positive and statistically significant but their

CHAPTER # 2 RESULTS

36

(proline and phenolics) correlations with all the other parameters were positive but non-

significant (except the negative correlation of proline and chlorophyll b). Chlorophyll a and b

showed positive correlation with all the other parameters of leaf (except its negative

correlation with Cd contents of leaf). The correlations of chlorophyll a were statistically

significant with growth parameters (positive) and Cd concentration (negative).

CHAPTER # 2 RESULTS

37

Table 2. 5: Correlations among different parameters measured in roots of P. hysterophorus plant

Length FW DW TWC Cd conc Cd accumulation Proline Phenolics

Length Pearson Correlation 1 0.945**

0.833**

0.950**

-0.452 0.131 0.415 0.728**

Sig. (1-tailed) 0.001 0.001 0.001 0.070 0.343 0.090 0.004

FW Pearson Correlation 0.945**

1 0.921**

0.994**

-0.588* -0.010 0.397 0.537

*

Sig. (1-tailed) 0.001 0.001 0.001 0.022 0.488 0.101 0.036

DW Pearson Correlation 0.833**

0.921**

1 0.872**

-0.674**

-0.001 0.371 0.299

Sig. (1-tailed) 0.001 0.001 0.001 0.008 0.499 0.117 0.173

TWC Pearson Correlation 0.950**

0.994**

0.872**

1 -0.546* -0.012 0.393 0.590

*

Sig. (1-tailed) 0.001 0.001 0.001 0.033 0.485 0.103 0.022

Cd concentration

Pearson Correlation -0.452 -0.588* -0.674

** -0.546

* 1 0.661

** 0.132 0.163

Sig. (1-tailed) 0.070 0.022 0.008 0.033 0.010 0.341 0.306

Cd accumulation

Pearson Correlation 0.131 -0.01 -0.001 -0.012 0.661**

1 0.527* 0.554

*

Sig. (1-tailed) 0.343 0.488 0.499 0.485 0.010 0.039 0.031

Proline

Pearson Correlation 0.415 0.397 0.371 0.393 0.132 0.527* 1 0.423

Sig. (1-tailed) 0.090 0.101 0.117 0.103 0.341 0.039 0.085

Phenolics

Pearson Correlation 0.728**

0.537* 0.299 0.590

* 0.163 0.554

* 0.423 1

Sig. (1-tailed) 0.004 0.036 0.173 0.022 0.306 0.031 0.085

**. Correlation is significant at the 0.01 level (1-tailed). *. Correlation is significant at the 0.05 level (1-tailed).

CHAPTER # 2 RESULTS

38

Table 2. 6: Correlations among different parameters measured in stem of P. hysterophorus plant.

Length FW DW TWC Cd conc Cd accumulation Proline Phenolics

Length Pearson Correlation 1 0.944**

0.937**

0.941**

-0.543* -0.070 0.437 0.745

**

Sig. (1-tailed) 0.001 0.001 0.001 0.034 0.415 0.078 0.003

FW Pearson Correlation 0.944**

1 0.972**

0.999**

-0.572* -0.132 0.333 0.771

**

Sig. (1-tailed) 0.001 0.001 0.001 0.026 0.341 0.145 0.002

DW Pearson Correlation 0.937**

0.972**

1 0.964**

-0.689**

-0.257 0.267 0.651*

Sig. (1-tailed) 0.001 0.001 0.001 0.007 0.210 0.201 0.011

TWC Pearson Correlation 0.941**

0.999**

0.964**

1 -0.553* -0.114 0.341 0.785

**

Sig. (1-tailed) 0.001 0.001 0.001 0.031 0.362 0.139 0.001

Cd concentration Pearson Correlation -0.543* -0.572

* -0.689

** -0.553

* 1 0.867

** 0.275 0.040

Sig. (1-tailed) 0.034 0.026 0.007 0.031 0.001 0.193 0.451

Cd accumulation Pearson Correlation -0.070 -0.132 -0.257 -0.114 0.867**

1 0.577* 0.481

Sig. (1-tailed) 0.415 0.341 0.210 0.362 0.001 0.025 0.057

Proline Pearson Correlation 0.437 0.333 0.267 0.341 0.275 0.577* 1 0.640

*

Sig. (1-tailed) 0.078 0.145 0.201 0.139 0.193 0.025 0.013

Phenolics


0.771**

0.651* 0.785

** 0.040 0.481 0.640

* 1

Sig. (1-tailed) 0.003 0.002 0.011 0.001 0.451 0.057 0.013


CHAPTER # 2 RESULTS

39

Table 2. 7: Correlation among different parameters measured in leaves of P. hysterophorus plant.

FW DW TWC Cd

conc.

Cd

accumul-

ation

Proline Phenolics Chlorophyll

a

Chlorophyll b Total

Chlorophyll

FW Pearson

Correlation

1 0.971**

0.998**

-0.614* -0.223 0.402 0.219 0.986

** 0.136 0.146

Sig. (1-tailed) 0.001 0.001 0.017 0.243 0.098 0.247 0.001 0.337 0.326

DW Pearson

Correlation

0.971**

1 0.952**

-0.608* -0.166 0.377 0.189 0.984

** 0.043 0.052

Sig. (1-tailed) 0.001 0.001 0.018 0.303 0.114 0.279 0.001 0.447 0.436

TWC Pearson

Correlation

0.998**

0.952**

1 -0.609* -0.238 0.405 0.226 0.976

** 0.162 0.171

Sig. (1-tailed) 0.001 0.001 0.018 0.228 0.096 0.24 0.004 0.308 0.297

Cd

concentration

Pearson

Correlation

-0.614* -0.608

* -0.609

* 1 0.859

** 0.399 0.494 -0.576

* -0.398 -0.337

Sig. (1-tailed) 0.017 0.018 0.018 0.001 0.101 0.051 0.025 0.101 0.142

Cd

accumulation

Pearson

Correlation

-0.223 -0.166 -0.238 0.859**

1 0.723**

0.630* -0.141 -0.458 -0.39

Sig. (1-tailed) 0.243 0.303 0.228 0.001 0.004 0.014 0.331 0.067 0.105

Proline Pearson

Correlation

0.402 0.377 0.405 0.399 0.723**

1 0.693**

0.448 -0.147 -0.087

Sig. (1-tailed) 0.098 0.114 0.096 0.101 0.004 0.006 0.072 0.324 0.394

Phenolics Pearson

Correlation

0.219 0.189 0.226 0.494 0.630* 0.693

** 1 0.252 0.013 0.106

Sig. (1-tailed) 0.247 0.279 0.240 0.051 0.014 0.006 0.215 0.484 0.371

Chlorophyll a Pearson

Correlation

0.986**

0.984**

0.976**

-0.576* -0.141 0.448 0.252 1 0.137 0.147

Sig. (1-tailed) 0.001 0.001 0.001 0.025 0.331 0.072 0.215 0.336 0.324

Chloropyll b Pearson

Correlation

0.136 0.043 0.162 -0.398 -0.458 -0.147 0.013 0.137 1 0.993 **

Sig. (1-tailed) 0.337 0.447 0.308 0.101 0.067 0.324 0.484 0.336 0.001

Total

chlorophyll

0.146 0.052 0.171 -0.337 -0.39 0.017 0.106 0.147 0.993** 1

0.326 0.436 0.297 0.142 0.105 0.394 0.371 0.324 0.001


CHAPTER # 2 DISCUSSION

40

2.4 DISCUSSION

Soils polluted with metals not only effect plants growth and yield but can also negatively

affect animals and humans, when reach their bodies through food chain. Cadmium is toxic

metal, which is a potential threat to human health, must be removed from the soil. In our

research, the effects of chemical chelator (EDTA) and plant growth regulator (Gibberellic

acid) was evaluated on the cadmium phytoextraction potential of P. hysterophorus plant.

2.4.1 Plant growth and biomass

Plant growth parameters such as biomass have been reported to be highly sensitive to heavy

metals exposure (Hadi and Bano, 2009; Hadi et al 2010; John et al 2009; Arun et al 2005).

Heavy metals like cadmium when present in large amounts with in plant tissues reduction in

plant development due to their toxicity (Khatamipour et al 2011). In this research study,

cadmium demonstrated significant reduction in plant length and biomass of Parthenium

hysterophorus plant (comparing C with C1). Decrease in biomass and growth of plants in

cadmium contaminated soil is often observed and this reduction in length (root and shoot)

and biomass could be directly related to the negative affect of heavy metal on the division

meristematic cells and also on the cell elongation and cell expansion growth of these cells

(Houshmandfar and Moraghebi, 2011). One of the reasons for inhibition of cell elongation

growth is the increase cross-linking of cell wall components by the heavy metal cadmium

(Poschenrieder et al 1989). Our plant also showed reduction in fresh weight and consequently

in the water contents with in different its different parts under cadmium stress (comparing C

with C1). Similar reduction in fresh weight under cadmium stress was reported by Zheng et al

(2010) and Khatamipour et al (2011) in Glycyrrhiza uralensis plant. Cadmium has also been

found to cause physiological drought by altering water content in plant tissues (Barcelo and

Poschenrieder, 1990).

Addition of EDTA into the cadmium contaminated soil further reduced the plant growth

while application of GA3 foliar spray enhanced the growth and biomass of the Parthenium

hysterophorus plant. This effect of EDTA on decreasing plant growth and biomass could be

due to increase in mobility of cadmium by EDTA in soil (Epelde et al 2008; Lou et al 2007).

The increase in growth and biomass might be due to the role of GA3 in promotion of cell

enlargement (Buchanan et al 2000) and on the rate of cell division (Moore, 1989; Arteca,

1996) which are the two main processes for the increase in growth and biomass. GA3 also

enhance the synthesis of DNA, RNA and protein (Benjerano and Lips, 1970; Broughton and


41

McComb, 1971) and ribose and polyribosome multiplication (Evins and Varner, 1972) would

increase biomass of a plant.

On the other hand GA3 treatment also increases permeability of cell membrane (Crozier and

Turnbull, 1984) that would enhance absorption of mineral nutrients, their transport and

utilization (Khan et al 1998; Crozier and Turnbull, 1984; Al-Wakeel et al 1995). Thus

enhancing the capability of the GA3 treated plants for high biomass production as

demonstrated in our experiment (Table 2.2). Increase in biomass of due to GA3 application

was observed tomato plants by Masroor et al (2006) and in in maize by Hadi et al (2010).

2.4.2 Plant cadmium up take

The results showed that Parthenium hysterophorus is a hyperaccumulator of cadmium at its

reproductive stage as obvious from its high Bioconcentration factor (1.85). GA3 and EDTA in

combination greatly increase cadmium concentration with in different parts of the plant. The

reason might be that EDTA increase the bioavalibility in soil solution (Mamindy-Pajany et al

2014; Meers et al 2005; Chen and Cutright 2001) while the GA3 increased absorption and

translocation of cadmium into different parts of the plant (Hadi et al 2014; Tassi et al 2008).

2.4.3 Proline concentration

Accumulation of proline in plant tissue is often considered as an indicator of environmental

stress such as drought, salinity and heavy metal stress. It has been found that free proline

chelates cadmium ion in plant tissues and convert them into nontoxic complex of Cd-proline

(Sharma et al 1998). Our results also showed a strong correlation between free proline and

cadmium accumulation within different tissues of the Parthenium hysterophorus plant. This

suggests that free proline may play an important role in Cd accumulation and also in the

reduction of cadmium toxicity within plants. Several plant have been reported to accumulate

high concentration of free proline under heavy metal stress such as S. nigrum, sunflower,

wheat, tomato and Igna unguiculata (Khatamipour et al 2011; Sun et al 2007; Zengin and

Munzuroglu, 2006; Costa and Morel, 1994). Higher concentration of free proline was

recorded in the roots of P. hysterophorus plant as compared to the stem and leaves. Similarly

high root proline concentration in Vigna unguiculata plant was found by Bhattacharjee and

Mukherjee (1994). GA3 treatment demonstrated high concentrations of free proline, which

suggested an important role of GA3 in the synthesis of proline.


42

2.4.4 Phenolics concentration within plant tissues

Phenolic compounds play an important role in protection, restoration and degradation

processes caused by toxic chemicals (Rice-Evans et al 1997). High concentrations of total

phenolics have been found in different plants under various environmental stresses (Diaz et al

2001; Lavola et al 2000). Soluble phenolic compounds showed important antioxidant activity

and are thus considered to be closely related to stress situations (Wild and Schmitt, 1995).

Schutzendubel et al (2001) reported that scots pine accumulate high concentration of soluble

phenolics subjected to Cd stress. Phenolic compounds act as metal chelators and also as

antioxidant during heavy metal stress (Michalak, 2006). High concentration of phenolics was

recorded in leaves of the P. hysterophorus plant compared to roots and stem. Similarly high

concentration of phenolic compounds in leaves of Crotalaria juncea was reported by

Uraguchi et al (2006).

2.4.5 Chlorophyll contents

Excess of cadmium in soil decreases content of chlorophyll (Ngayila et al 2008), its synthesis

rate (Vajpayee et al 2000), efficiency of photosystems (Chugh et al 1997), photosynthetic

enzymes (Mobin and Nafees, 2007; Thapar et al 2008), plant water balance and consequently

on plant growth and biomass (Zhou and Qiu, 2005). Our results also demonstrated that a

negative correlation existed between the cadmium contents of leaf and the chlorophyll

contents (Table 2.7) which are in agreement with earlier reports (Mobin and Nafees, 2007;

Sun et al 2008; Ekmekci et al 2008), who found that heavy metal suppressed the

photosynthetic activity of plants. Faller et al (2005) demonstrated that Cd2+

has inhibitory

effect on the photoactivation of photosystem II as result of its competitive binding with the

Ca2+

site. The net photosynthetic rate has been shown to decrease conspicuously with high

concentrations of cadmium (Lakshaman and Surinder, 1999). Different physiological

activities influence the metabolization of chlorophyll in plants. The chlorophyll-a firs

synthesized and transformed into Chlorophyll-b (Guo et al 2006). Therefore, higher content

of Chl-a was found in our plant as compared to the Chl-b (Table 2.4) which is in complete

agreement with the work of Mobin and Nafees (2007).

Conclusions

Phytoextraction capabilities of Parthenium hysterophorus highly increased at reproductive

stage and at this it can be considered as hyperaccumulator of cadmium at this stage. GA3

increased cadmium content of the plant but the effect of GA3 was more pronounced at higher

concentration in combination split doses of EDTA at low concentrations. It has also been


43

found that concentration of free proline and total phenolics significantly increased with the

increase in Cd concentration of plant tissues, especially in the GA3 treated plants. This

suggests that GA3 has some role in the synthesis of these compounds. Proline and phenolics

showed positive correlation with the plant dry weight as well as with the Cd accumulation in

different parts of the plant. Further study is recommended to find the biochemical basis of

proline and phenolics synthesis and the molecular mechanism through which GA3 enhance

their biosynthesis in plant during Cd stress.

CHAPTER # 3

THE EFFECT OF MOLYBDENUM ON CADMIUM PHYTOEXTRACTION AND

PRODUCTION OF ENDOGENOUS PHENOLICS, FREE PROLINE AND

PHOTOSYNTHETIC PIGMENTS IN RICINUS COMMUNIS AND CANNABIS

SATIVA PLANTS


44

CHAPTER 3: The effect of molybdenum on cadmium phytoextraction and

production of endogenous phenolics, free proline and

photosynthetic pigments in Ricinus communis and

Cannabis sativa plants.

ABSTRACT

Purpose of the present study was to investigate the effect of molybdenum (Mo 0.5, 1.0 and

2.0 ppm) on Cd phytoextraction, total phenolics, free proline, biomass and photosynthetic

pigments in Ricinus communis and Cannabis sativa plants grown in cadmium (Cd 25, 50 and

100 ppm) contaminated soils. Mo was applied as seed soaking, soil addition and foliar spray.

Foliar applications of Mo significantly increased biomass, Cd accumulation and Cd-

bioconcentration in R. communis and C. sativa plants. Total phenolics and free proline in

roots and leaves of the plants were highly increased by foliar as well as seed soaking

treatments of Mo. Significantly positive correlations existed between Cd accumulation and

concentration of total phenolics and free proline in roots and leaves of both the plants. In R.

communis the correlations of Cd accumulation with concentration of total phenolics (in roots

and leaves) and free proline (in leaves) were higher than 70 % (R2 = 0.70). In C. sativa plant,

the roots demonstrated significant correlations (R2 > 0.75) of Cd accumulation with proline

and phenolic concentrations under 25 ppm and 50 ppm Cd contaminated soil. Total phenolics

concentration in leaves of both the plants showed highly significant correlations (R2 > 0.70)

with chlorophylls and carotenoid concentration. Foliar spray of molybdenum was found more

significant as compared to seed soaking and soil addition treatments, in terms of increase in

plants growth, Cd concentration, endogenous production of total phenolics and free proline.

Manuscripts submitted from this chapter:

Nasir Ali and Fazal Hadi. (2015). ‗‗Molybdenum (Mo) increased the phytoremediation

potential of industrially important plant (Ricinus communis) for removal of hazardous metal

(cadmium) from contaminated soil, along with increase in endogenous phenolics, proline and

photosynthetic pigments‘‘ Journal of Hazardous Materials. (Submitted).

.


45

3.1 INTRODUCTION

Cadmium is one of the toxic heavy metal that enters agricultural soil mostly through

industrial effluents, mining operations, municipal runoff and application of phosphate

fertilizers (Rogers et al 2007). It can easily be absorb by plant roots and translocated into

aerial parts; inhibiting plant growth, uptake of micro- and macronutrients and reduction in

rate of photosynthesis; thus reduce crops yield and also compromise the quality of food

(Ahmad et al 2015; Zadeh et al 2008). Consumption of Cd contaminated food results in

serious health problems (Ahmad et al 2015; Zadeh et al 2008). In human body, Cd can affect

gene expression, interferes with DNA damage repair system, inhibit apoptosis and induce

oxidative stress; resulting damage to different organs such as kidneys, liver, lung and bone

(Takiguchi et al 2003; Huang et al 2008; Krocova et al 2000; Joseph 2009). Safe restoration

of Cd polluted soil is utmost important for sustainable agriculture, environment and human

health. Phytoremediation is an environment friendly remediation technology that uses green

plants for the decontamination of polluted soil and water. This is an economical, environment

friendly and aesthetically pleasing technology (Hadi et al 2014). Plants under heavy metals

stress often showed decrease in growth and biomass which in turn reduce their

phytoremediation potential (Tassi et al 2008; Falkowska et al 2011). To combat the toxic

metals, increase in concentration of endogenous free proline and total phenolic compounds

have been reported in many plants (Ahmad et al 2015; Ali and Hadi 2015). Phenolic

compounds protect cellular components from oxidative stress caused by reactive oxygen

species while free proline has been reported to protect some important enzymes from

deactivation by toxic heavy metals (Michalak 2006; Handique and handique 2009).

Micronutrients are required by plants in very minute quantity for normal physiological

activities. Molybdenum (Mo) is one of the micronutrients required by plants for normal

growth and its deficiency reduces the activities of nitrate reductase and glutamine synthetase:

enzymes catalyzing the initial steps of nitrate metabolism (Hristozkova et al 2006). Mo have

also been reported to catalyse other enzymes such as aldehyde oxidase (AO) involved in

Abscisic acid biosynthesis and sulfite oxidase (SO) catalyse the conversion of sulfite to

sulfate, an essential step in the catabolism amino acids containing Sulphur (Williams and

Frausto da Silva 2002). It is also found that molybdoenzymes are involved in the synthesis of

indole-3-acetic acid (IAA) and abscisic acid (Hesberg et al 2004; Sagi et al 2002).

Ricinus communis (Castor bean) and Cannabis sativa plants were used as experimental plant

which belongs to Euphorbiaceae and Cannabaceae families respectively of the plantae


46

kingdom (Rana et al 2012). Both the plants are highly suited for metal phytoremediation

purpose due to their high biomass, fast growth, deep roots and non-palatable nature to

herbivores (prevent entrance of metals into food chain) (Linger et al 2005; Citterio et al

2003).

CHAPTER # 3 AIMS AND OBJECTIVES

47

Aims and objectives

Aims:

Aim of this chapter was to study the role of molybdenum (Mo) in cadmium phytoextraction

by Ricinus communis and Cannabis sativa plants and biochemical changes occurred in these

plants under molybdenum treatments and cadmium stress.

Objectives:

1. To evaluate the effect of different concentrations of cadmium in soil on plant growth

and biomass.

2. To study the effect of molybdenum on plant length, biomass and water content under

cadmium stress.

3. To find out the role of molybdenum on cadmium concentration and accumulation in

different parts of the selected plants.

4. To evaluate the effect of molybdenum treatments on translocation of cadmium from

roots into the aerial parts of the selected plants.

5. To assess the effect of molybdenum and cadmium on the production of endogenous

production of free proline and total phenolics in roots and leaves of the plants.

6. To find out the role of molybdenum and cadmium on chlorophyll and carotenoids

contents in leaves of the selected plants.

7. To study the inter-correlations between plant length, biomass, proline, phenolics, Cd

concentration, Cd accumulation and photosynthetic pigments.


48


3.2.1 Preparation of soil and addition of cadmium

Fertile soil was collected from fields near the University of Malakand at Chakdara, Pakistan.

The soil was dried in sunlight and grounded into powdered form. Water holding capacity

(300 ml water per kg soil ± 3) and pH (6.5 ± 0.3) of the soil was calculated. From the soil,

0.25 kg was poured into plastic pots (20 × 12 cm). Cadmium (Cd) in the form of cadmium

acetate dihydrate (CH3COO)2 Cd·2H2O (Merck, Germany) solution was added to soil in the

pots. Three different concentrations (25, 50 and 100 ppm) cadmium were used in soil (Table

3.1). Cadmium was allowed to equilibrate in soil for one month.

3.2.2 Transplantation of seedlings and plant growth

Each pot was watered a day before transplantation of seedlings. Seeds of Ricinus communis

and Cannabis sativa were obtained from Herbarium University of Malakand, and sown in

normal soil beds in green house. After germination uniform sized (7 cm) seedlings were

collected from the green house and transplanted into pots (single seedling per pot). Plants

were allowed to grow under natural condition of light and temperature (35/25°C). Three

replicate pots were used for each treatment and control. Plants were watered at three days

interval, according to the water holding capacity of the soil.

3.2.3 Molybdenum treatments

Three concentrations (0.5, 1.0 and 2.0 ppm) of molybdenum were applied in three different

ways i.e. seed soaking, soil addition and foliar spray (Table 3.1). Ammonium molybdate

pentahydrate was used as a source of molybdenum. Stock solution of Mo was prepared and

then treatments solutions were made through serial dilution. In case of seed soaking

treatments, seeds were kept in respective Mo solutions for 24 hrs before sowing. In soil

addition treatments, Mo (0.5, 1.0 and 2.0 ppm) solution were added into soil once after 15

days of transplantation. The solutions of Mo were applied according to the water holding

capacity of soil. Six foliar treatments were done, each at one week interval. First foliar

treatment was done 15 days after transplantation. During foliar spray, soil in the pots were

covered with plastic bags to avoid entrance of Mo droplets into soil.


49

Table 3. 1: The following treatments were used during the experiment. C act as control for

C1, C2 and C3. The C1 is control for treatments T1 to T9, C2 is control for treatments T10 to

T18 while C3 is control for treatments T19 to T27.

Treatments

Sym

bols

Treatments

Sym

bols

Treatments

Sym

bols

Without Cd and Mo C

25 ppm Cd C1 50 ppm Cd C2 100 ppm Cd C3

0.50 ppm Mo [S.S] T1 0.50 ppm Mo [S.S] T10 0.50 ppm Mo [S.S] T19



0.50 ppm Mo [A.S] T4 0.50 ppm Mo [A.S] T13 0.50 ppm Mo [A.S] T22



0.50 ppm Mo [F.S] T7 0.50 ppm Mo [F.S] T16 0.50 ppm Mo [F.S] T25



Note: ‗S.S‘ stands for seed soaking, ‗A.S‘ stands for added to soil ‗F.S‘ stands for foliar

spray, ‗C‘ stands for control, ‗T‘ denotes treatment, ‗Cd‘ symbolize cadmium and Mo denote

molybdenum. Treatments T1-T9 possess 25 ppm Cd, T10- T18 contain 50 ppm Cd while

T19-T27 contain 100 ppm Cd concentration.


50

3.2.4 Plant growth parameters

Plant were harvested after two months of seedling transplantation. Roots and shoot lengths of

each plant were measured using a centimeter ruler. Plants were washed with a solution of 5

mM EDTA and 5 mM Tris-HCl (pH 6.0), and then with distilled water to remove metal ion

bounded to the plant surface (Genrich et al 2000). After washing each plant was cut into

three parts i.e. roots, stem and leaves and fresh weight of each part was measured

immediately with the help of analytical balance. Each part was packed in separate paper

envelopes and dried in oven for 48 h at 80 0C. The dry weight of each part was measured by

analytical balance and then grinded into powdered form for further analysis of metal

concentration.

3.2.5 Free proline analysis in root and leaves

Free proline quantification in plant tissues (root and leaves) were made by the method of

Bates et al (1973). The detailed method has been mentioned in previous chapter 2, Section

Materials and Methods on page 25 under the title ―Analysis of free proline in plant root and

leaves‖.

3.2.6 Total phenolics estimation in roots and leaves

Total phenolic were estimated in roots and leaves of the plants using the method of Singleton

and Rossi (1965). The detailed method has already been given in previous chapter 2, section

material and methods on page 26 under title ―Total phenolics estimation in roots and leaves‖.

3.2.7 Chlorophyll and carotenoids estimation in leaves

Concentration of chlorophylls (a and b) were estimated by the method of Arnon (1949). A

detail of the method is already given in previous chapter 2, Section Materials and Methods on

page 26 under the title ― Chlorophyll estimation in leaves‖.

Carotenoids concentrations in fresh leaves were estimated by using the method of Sumanta et

al (2014). Fresh leaf samples (0.5 g) were homogenized in 10 ml of 80% acetone, centrifuged

at 10000 rpm for 15 minutes. The supernatants were transferred into clean test tubes

containing 4.5 ml of 80% acetone. Three replicates were used for each treatment. Carotenoids

were estimated by measuring absorbance of the samples at 470 nm wavelength. The

following formulas were used for calculation of carotenoids contents:


51

Carotenoids contents = A480 x volume of extract x 10 x 100/2500 x weight of plant material

(g).

3.2.8 Cadmium (Cd) analysis in different plant parts

Oven dried samples (root, stem and leaves) were first grounded into powdered form and then

subjected to acid digestion using Allen (1974) method. Detailed method has already been

given in previous chapter 2, section material and methods on page 27 under title ―Cadmium

(Cd) analysis in the plant‖. After digestion the samples were analyzed for Cd concentration

with the help of Atomic Absorption/Flame Spectrophotometer (model Hitachi Z-8000,

Japan).

3.2.9 Statistical analysis

The data was further analyzed for mean values, analysis of variance (ANOVA) using

software such as SPSS 16 and MS Excel 2007. Significant differences among the treatments

for different parameters were analyzed through Tukey‘s Honestly Significant Difference

(HSD) test.

CHAPTER # 3 RESULTS

52

3.3 RESULTS

3.3.1 Ricinus communis plant

3.3.1.1 Plant length, biomass and water contents

Plant length, biomass and water content in different parts of Ricinus communis under various

treatments of molybdenum and Cd are shown in Tables 3.2, 3.3 and 3.4. In table 3.2 and

figure 3.1. The control C (without Cd and Mo) is compared with C1 (25 ppm Cd), C2 (50

ppm Cd) and C3 (100 ppm) for the effect of Cd on plant growth. In the same table, C1 is

compared with treatments T1 – T9 for the effect of Mo on plant growth under Cd stress. A

gradual decrease in plant growth parameters was noted with increasing concentration of Cd in

soil i.e. C1 (25 ppm Cd) > C2 (50 ppm Cd) > C3 (100 ppm Cd). Treatments of Mo increased

the growth and biomass of Ricinus communis plant as compared to C1 (Table 3.2). Highest

significant increase in roots and stem length were found in T7 (1 ppm Mo foliar spray) as

given in table 3.2 and figure 3.1X. It was found that 2 ppm Mo foliar treatment most

significantly increased dry weight (DW) of the plant (Table 3.2).

The table 3.3 shows the effect of Mo treatments on growth parameter of Ricinus communis

plants grown in 50 ppm Cd contaminated soil. Highest significant increase in roots and stem

length were demonstrated by T10 (0.5 ppm Mo seed soaking) and T18 (2.0 ppm Mo foliar

spray) respectively, as compared to the C2 (Table 3.3 and figure 3.1Y). Dry weight in roots

and stem were most significantly increased by 2 ppm Mo foliar spray (T18) while the same

concentration of Mo (2 ppm) in the form of seed soaking (T12) highly increased dry weight

in leaves.

The effect of Mo treatments on plant growth parameters in 100 ppm Cd contaminated soil is

presented in Table 3.4. Roots and stem lengths were increased significantly by 2 ppm Mo in

the form of seed soaking and foliar spray respectively as compared to C3 (Table 3.4 and

figure 3.1 Z). Biomass (fresh and dry) in all parts of the plant were highly increased by 2 ppm

Mo foliar treatment (T27).

CHAPTER # 3 RESULTS

53

Table 3. 2: Effect of different treatments of molybdenum (Mo) on plant length, biomass and water content in different parts of Ricinus communis plant grown in

25 ppm Cd contaminated soil. C1 (25 ppm Cd only) is used as control for the treatments (T1 – T9). Table also compares C1 (25 ppm Cd), C2 (50 ppm Cd) and

C3 (100 ppm Cd) with each other and also with control C (without Cd and Mo). Note: ‗SD‘ stands for ‗standard deviation‘ and the different alphabets in

superscript represent significant difference between the values within a single column.

Treatments Length (cm) ± SD FW (g) ± SD DW (g) ± SD TWC (g) ± SD

Roots Stem Roots Stem Leaves Entire plant

Roots Stem Leaves Entire plant


C Control (without Cd and Mo)

12.50 ± 1.00 abcd

31.25 ± 3.13 abc

3.75 ± 0.30 cde

7.81 ± 0.70 cde

9.77 ± 0.78 abcd

21.33 ± 1.71 abc

1.13 ± 0.09 de

2.34 ± 0.19 bcd

2.93 ± 0.23 abc

6.40 ± 0.51 cde

2.63 ± 0.21 bcd

5.47 ± 0.44 abcd

6.84 ± 0.55 bcd

14.93 ± 1.19 abc

C1 Control (with Cd 25 ppm)

9.73 ± 0.78 def

24.32 ± 2.43 cde

2.92 ± 0.26 ef

6.08 ± 0.49 ef

6.99 ± 0.63 efg

15.99 ± 1.44 cde

0.79 ± 0.07 fg

1.64 ± 0.15 ef

1.89 ± 0.15 ef

4.32 ± 0.35 fg

2.13 ± 0.17 cde

4.44 ± 0.36 cdef

5.10 ± 0.41 ef

11.67 ± 0.93 cde


8.12 ± 0.65 ef

20.30 ± 2.03 de

2.78 ± 0.22 ef

5.08 ± 0.51 ef

6.54 ± 0.65 fg

14.40 ± 1.15 de

0.66 ± 0.05 fg

1.19 ± 0.11 fg

1.58 ± 0.13 fg

3.42 ± 0.27 gh

2.12 ± 0.17 cde

3.89 ± 0.31 ef

4.96 ± 0.40 ef

10.97 ± 0.88 de


6.81 ± 0.54 f

17.02 ± 1.70 e

2.04 ± 0.14 f

4.26 ± 0.34 f

4.89 ± 0.39 g

11.19 ± 1.12 e

0.55 ± 0.06 g

1.02 ± 0.08 g

1.05 ± 0.08 g

2.62 ± 0.21 h

1.49 ± 0.12 e

3.24 ± 0.26 f

3.84 ± 0.31 f

8.57 ± 0.69 e

T1 Cd 25 ppm+ Mo 0.5 ppm (Seed soaking)

15.00 ± 1.50 a

27.23 ± 2.45 cd

3.68 ± 0.40 cde

6.81 ± 0.75 cde

8.71 ± 0.78 cdef

19.20 ± 1.54 bcd

1.25 ± 0.13 cde

2.11 ± 0.17 cde

2.70 ± 0.22 bcd

6.06 ± 0.48 de

2.43 ± 0.19 bcd

4.70 ± 0.42 bcde

6.01 ± 0.54 cde

13.14 ± 1.05 bcd


14.21 ± 1.42 ab

35.00 ± 3.15 ab

5.25 ± 0.47 ab

8.75 ± 0.70 ab

10.97 ± 0.77 abcd

24.97 ± 3.00 a

1.52 ± 0.11 bc

2.71 ± 0.30 bc

3.47 ± 0.28 a

7.70 ± 0.62 abc

3.73 ± 0.30 a

6.04 ± 0.48 a

7.50 ± 0.60 abc

17.27 ± 1.38 a

T3 Cd 25 ppm+ Mo 2 ppm (Seed soaking)

13.00 ± 1.30 abc

28.00 ± 2.52 bc

5.46 ± 0.55 a

8.45 ± 0.85 a

11.12 ± 1.22 abc

25.03 ± 2.25 a

1.64 ± 0.13 ab

2.77 ± 0.22 b

3.24 ± 0.26 ab

7.65 ± 0.61 abc

3.82 ± 0.31 a

5.68 ± 0.45 ab

7.88 ± 0.63 ab

17.38 ± 1.39 a

T4 Cd 25 ppm+ Mo 0.5 ppm (Soil addition)

12.00 ± 1.20 abcd

26.31 ± 2.37 cd

3.16 ± 0.25 de

6.58 ± 0.59 de

9.24 ± 0.83 bcde

18.97 ± 1.33 cd

0.98 ± 0.09 ef

1.91 ± 0.15 de

2.14 ± 0.17 def

5.02 ± 0.40 ef

2.18 ± 0.17 cd

4.67 ± 0.37 bcde

7.10 ± 0.57 bcd

13.95 ± 1.12 abcd


11.00 ± 0.99 bcde

29.45 ± 2.36 abc

3.98 ± 0.32 cd

7.36 ± 0.44 cd

8.94 ± 0.72 cdef

20.28 ± 1.62 abc

1.23 ± 0.10 cde

2.14 ± 0.19 cde

2.39 ± 0.22 cde

5.76 ± 0.52 def

2.74 ± 0.25 bc

5.23 ± 0.42 abcd

6.55 ± 0.65 bcde

14.52 ± 1.16 abc

T6 Cd 25 ppm+ Mo 2 ppm (Soil addition)

10.45 ± 0.94 cde

27.32 ± 2.19 cd

3.99 ± 0.36 cd

7.02 ± 0.77 cd

8.54 ± 0.68 def

19.55 ± 1.56 bcd

1.47 ± 0.12 bc

1.98 ± 0.16 de

2.87 ± 0.23 abc

6.32 ± 0.51 cde

2.52 ± 0.20 bcd

5.04 ± 0.40 abcde

5.67 ± 0.45 de

13.23 ± 1.06 bcd

T7 Cd 25 ppm+ Mo 0.5 ppm (Foliar spray)

10.90 ± 0.98 cde

26.94 ± 2.16 cd

3.31 ± 0.23 de

6.68 ± 0.53 de

8.97 ± 0.90 cdef

18.96 ± 1.71 cd

1.28 ± 0.12 cde

2.42 ± 0.22 bcd

2.96 ± 0.27 abc

6.66 ± 0.60 bcd

2.03 ± 0.18 de

4.26 ± 0.38 def

6.01 ± 0.54 cde

12.30 ± 1.11 cd

T8 Cd 25 ppm+ Mo 1.0ppm (Foliar spray)

15.76 ± 1.33 a

36.00 ± 2.88 a

4.43 ± 0.40 bc

9.00 ± 0.81 a

12.15 ± 1.09 a

25.58 ± 2.30 a

1.45 ± 0.13 bcd

3.42 ± 0.27 ab

3.34 ± 0.27 ab

8.21 ± 0.66 ab

2.98 ± 0.24 b

5.58 ± 0.45 abc

8.81 ± 0.70 a

17.37 ± 1.39 a


12.71 ± 1.14 abcd

31.00 ± 2.48 abc

4.68 ± 0.37 abc

8.42 ± 0.67 abc

11.45 ± 0.92 ab

24.55 ± 1.96 ab

1.91 ± 0.19 a

3.95 ± 0.29 a

3.41 ± 0.34 a

8.27 ± 0.83 a

2.77 ± 0.28 bc

4.48 ± 0.55 cdef

8.04 ± 0.80 ab

16.29 ± 1.63 ab

CHAPTER # 3 RESULTS

54

Table 3. 3: Role of Mo treatments on length, biomass and water content of Ricinus communis plant in 50 ppm Cd polluted soil. Note: ‗SD‘ denote ‗standard

deviation‘, and the different letter in superscript present significant difference among the values within a column.


Roots Stem Roots Stem Leaves

Entire plant


Roots Stem Leaves

Entire plant


8.12 ± 0.97 c

20.30 ± 2.44 ab

2.78 ± 0.33 d

5.08 ± 0.61 d

6.54 ± 0.78 b

14.40 ± 1.73 c

0.66 ± 0.08 c

1.19 ± 0.14 b

1.58 ± 0.19 c

3.42 ± 0.41 c

2.12 ± 0.25 c

3.89 ± 0.47 b

4.96 ± 0.60 ab

10.97 ± 1.32 b


13.46 ± 1.21 a

26.05 ± 2.34 ab

4.68 ± 0.42

6.11 ± 0.55 c

7.82 ± 0.70 ab

18.61 ± 1.68 bc

1.12 ± 0.10 ab

1.89 ± 0.17 ab

2.12 ± 0.19 bc

5.14 ± 0.46 abc

3.56 ± 0.32 ab

4.21 ± 0.38 ab

5.70 ± 0.51 ab

13.48 ± 1.21 ab


12.75 ± 1.53 ab

25.32 ± 3.04 ab

6.20 ± 0.74 a

7.85 ± 0.94ab

9.84 ± 1.18 a

23.90 ± 2.87 a

1.24 ± 0.15 ab

2.52 ± 0.30 a

2.86 ± 0.34 ab

6.62 ± 0.79 a

4.96 ± 0.60 a

5.33 ± 0.64 ab

6.98 ± 0.84 a

17.28 ± 2.07 a


11.67 ± 1.63 abc

18.00 ± 2.52 b

6.14 ± 0.86ab

7.29 ± 1.02 bc

9.98 ± 1.40 a

23.41 ± 3.28 a

1.38 ± 0.19 a

2.32 ± 0.32 a

3.14 ± 0.44 a

6.84 ± 0.96 a

4.76 ± 0.67 a

4.97 ± 0.70 ab

6.84 ± 0.96 a

16.57 ± 2.32 a


8.98 ± 1.08 bc

20.14 ± 2.42 ab

4.02 ± 0.48 cd

5.45 ± 0.65 cd

8.29 ± 0.99 ab

17.76 ± 2.13 bc

0.87 ± 0.10 bc

1.20 ± 0.14 b

1.69 ± 0.20 c

3.76 ± 0.45 bc

3.15 ± 0.38 bc

4.25 ± 0.51 ab

6.60 ± 0.79 a

14.00 ± 1.68 ab


9.87 ± 1.28 abc

24.02 ± 3.12 ab

5.07 ± 0.66 b

6.61 ± 0.86 bc

7.45 ± 0.97 ab

19.12 ± 2.49 b

1.11 ± 0.14 ab

1.92 ± 0.25 ab

2.03 ± 0.26 bc

5.05 ± 0.66 abc

3.96 ± 0.51 ab

4.69 ± 0.61 ab

5.42 ± 0.70 ab

14.07 ± 1.83 ab


10.88 ± 1.31 abc

21.41 ± 2.57 ab

5.33 ± 0.64 b

7.85 ± 0.94ab

7.40 ± 0.89 ab

20.58 ± 2.47 ab

1.21 ± 0.15 ab

2.08 ± 0.25 a

3.12 ± 0.37 a

6.41 ± 0.77 a

4.12 ± 0.49 ab

5.77 ± 0.69 a

4.28 ± 0.51 b

14.17 ± 1.70 ab


9.79 ± 1.47 abc

25.00 ± 3.75 ab

4.21 ± 0.63 c

6.54 ± 0.98 bc

8.02 ± 1.20 ab

18.77 ± 2.82 bc

1.42 ± 0.21 a

2.17 ± 0.33 a

2.65 ± 0.40 ab

6.25 ± 0.94 a

2.79 ± 0.42 bc

4.37 ± 0.66 ab

5.37 ± 0.80 ab

12.53 ± 1.88 ab


12.56 ± 2.01 ab

25.23 ± 4.04 ab

4.87 ± 0.78 bc

8.08 ± 1.29 a

7.89 ± 1.26 ab

20.84 ± 3.33 ab

1.30 ± 0.21 ab

2.45 ± 0.39 a

2.44 ± 0.39 abc

6.19 ± 0.99 a

3.57 ± 0.57 ab

5.63 ± 0.90 ab

5.45 ± 0.87 ab

14.64 ± 2.34 ab


12.02 ± 1.44 abc

28.14 ± 3.38 a

6.32 ± 0.61 a

7.56 ± 0.91ab

6.23 ± 0.75 ab

20.11 ± 2.27 ab

1.49 ± 0.13 a

2.55 ± 0.30 a

1.98 ± 0.24 bc

6.02 ± 0.67 a

4.98 ± 0.48 a

5.05 ± 0.61 ab

4.25 ± 0.51 ab

14.19 ± 1.59 ab

CHAPTER # 3 RESULTS

55

Table 3. 4: Effect of Mo treatments on growth parameter of Ricinus communis plant grown in 100 ppm Cd contaminated soil. Note: ‗SD‘ denote ‗standard

deviation‘ and the different letter in superscript present significant difference among the values within a column.


Roots Stem Roots Stem Leaves

Entire plant

Roots Stem Leaves

Entire plant

Roots Stem Leaves

Entire plant


6.81 ± 0.82 b

17.02 ± 2.04 bc

2.04 ± 0.25 e

4.26 ± 0.51 e

4.89 ± 0.59 b

11.19 ± 1.34 b

0.55 ± 0.07 e

1.02 ± 0.12 c

1.05 ± 0.13 c

2.62 ± 0.31 d

1.49 ± 0.18 d

3.24 ± 0.39 c

3.84 ± 0.46 b

8.57 ± 1.03 b


7.68 ± 0.69 b

21.77 ± 1.96 ab

3.45 ± 0.31 cde

5.11 ± 0.46 cde

6.54 ± 0.59 ab

15.09 ± 1.36 ab

0.80 ± 0.07 cde

1.58 ± 0.14 abc

1.77 ± 0.16 bc

4.15 ± 0.37 bcd

2.65 ± 0.24 bcd

3.52 ± 0.32 bc

4.76 ± 0.43 ab

10.94 ± 0.98 ab


8.48 ± 1.02 ab

21.16 ± 2.54 ab

5.18 ± 0.62 ab

5.55 ± 0.65 bc

8.23 ± 0.99 a

18.86 ± 2.26 a

1.00 ± 0.12 abcd

2.11 ± 0.25 a

2.54 ± 0.30 a

5.65 ± 0.68 ab

4.18 ± 0.50 a

3.34 ± 0.40 c

5.69 ± 0.68 a

13.21 ± 1.59 a


11.35 ± 1.59 a

19.21 ± 2.69 ab

5.54 ± 0.78 a

6.09 ± 0.85 bc

7.34 ± 1.17 a

18.97 ± 2.80 a

1.13 ± 0.18 ab

1.94 ± 0.27 ab

2.55 ± 0.39 a

5.62 ± 0.84 ab

4.41 ± 0.59 a

4.15 ± 0.58 bc

4.79 ± 0.78 a

13.35 ± 1.96 a


7.51 ± 0.90 b

16.83 ± 2.02 c

3.10 ± 0.37 de

4.56 ± 0.55 de

6.93 ± 0.83 ab

14.58 ± 1.75 ab

0.73 ± 0.09 de

1.11 ± 0.13 bc

1.32 ± 0.16 c

3.16 ± 0.38 cd

2.37 ± 0.28 cd

3.45 ± 0.41 bc

5.61 ± 0.67 a

11.42 ± 1.37 ab


8.25 ± 1.07 ab

16.98 ± 2.21 c

4.54 ± 0.59 abcd

5.35 ± 0.71 cd

6.23 ± 0.81 ab

16.22 ± 2.11 ab

0.92 ± 0.12 bcd

1.60 ± 0.21 abc

1.70 ± 0.22 bc

4.22 ± 0.55 bcd

3.62 ± 0.47 abc

3.85 ± 0.50 abc

4.53 ± 0.59 ab

11.99 ± 1.56 ab


10.23 ± 1.23 ab

17.89 ± 2.15 bc

5.01 ± 0.60 abc

6.36 ± 0.79 ab

6.18 ± 0.74 ab

17.76 ± 2.13 a

1.10 ± 0.13 abc

1.74 ± 0.21 ab

2.65 ± 0.32 a

5.49 ± 0.66 ab

3.91 ± 0.47 ab

4.82 ± 0.58 a

3.53 ± 0.42 b

12.27 ± 1.47 ab


8.18 ± 1.23 ab

20.90 ± 3.13 ab

3.87 ± 0.58 bcd

5.47 ± 0.82 cd

6.70 ± 1.01 ab

16.04 ± 2.41 ab

1.19 ± 0.18 ab

1.82 ± 0.27 a

2.22 ± 0.33 ab

5.22 ± 0.78 ab

2.68 ± 0.40 bcd

3.65 ± 0.55 bc

4.48 ± 0.67 ab

10.82 ± 1.62 ab


9.47 ± 1.52 ab

22.45 ± 3.59 a

4.01 ± 0.64 abcd

6.75 ± 1.08 a

6.59 ± 1.06 ab

17.35 ± 2.78 a

1.09 ± 0.17 ab

2.05 ± 0.33 a

1.98 ± 0.32 ab

5.12 ± 0.82 ab

2.92 ± 0.47 abc

4.70 ± 0.75 a

4.61 ± 0.74 ab

12.24 ± 1.96 a


11.55 ± 1.21 a

23.52 2.82 a

5.96 ± 0.51 a

7.38 ± 0.79 a

6.18 ± 0.62 ab

19.52 ± 1.91 a

1.44 ± 0.11 a

2.53 ± 0.27 a

1.92 ± 0.18 ab

5.89 ± 0.55 a

4.52 ± 0.40 a

4.85 ± 0.52 a

4.26 ± 0.44 b

13.63 ± 1.36 a

CHAPTER # 3 RESULTS

56

Figure 3. 1: Effect of different treatments of Mo on growth of Ricinus communis plant grown

in soil contaminated with 25 ppm (X), 50 ppm (Y) and 100 ppm (Z) cadmium. In figure X,

the control C (without Cd and Mo) was compared with C1 (25 ppm Cd), C2 (50 ppm Cd) and

C3 (100 ppm Cd) while the treatments T1 –T9 were compared with C2. In figure Y, the

treatments T10 – T18 are compared with C2 while in figure Z, the treatments T19 –T27 are

compared with C3.

X

Y

Z

CHAPTER # 3 RESULTS

57

3.3.1.2 Biochemical variation in plants under various treatments of Mo and Cd

Variation in concentrations of free proline, total phenolics and photosynthetic pigments

(chlorophylls and carotenoids) in Ricinus communis plant under various treatments of Mo and

in Cd contaminated soil are given in table 3.5, table 3.6 and table 3.7. In table 3.5, the control

C (without Cd and Mo) is compared with C1 (25 ppm Cd), C2 (50 ppm Cd) and C3 (100

ppm) for the Cd effect on free proline, total phenolics, chlorophyll and carotenoids

concentration in the Ricinus communis plant. The treatments T1 –T9 are compared with the

C1 for the effect of Mo on the biochemical parameter under Cd stress in table 3.5. Increase in

concentration of free proline and total phenolics were recorded with increasing Cd

concentration in control soils (C3 > C2 > C1 > C). Highest significant increase in

concentration of total phenolics and free proline concentration in roots and leaves were

recorded in 1.00 and 2.00 ppm Mo foliar treatments (T8 and T9) respectively, as compared to

C1 (Table 3.5). Photosynthetic pigments were significantly increased by the treatments T8

and T9 as compared to C1.

Table 3.6 presents the effect of Mo treatments on the concentration of free proline, total

phenolics, chlorophyll and carotenoids in Ricinus communis plant in 50 ppm Cd

contaminated soil. Plants treated with 2 ppm Mo as seed soaking (T12) and foliar spray (T18)

most significantly increased concentration of proline and phenolics concentrations

(respectively) in roots as compared to C2. Leaves demonstrated highest concentration of

proline and phenolics with the treatment T18 (Table 3.6). Chlorophylls concentration in

leaves were most significantly high in the treatment T12 (2 ppm Mo foliar spray) as

compared to C2, while concentration of carotenoid in leaves was highly significant in T16 (1

ppm Mo foliar spray).

Effect of Mo on free proline, total phenolics, chlorophyll and carotenoids concentrations in

Ricinus communis plant grown in 100 ppm Cd contaminated soil is given in table 3.7. Highly

significant increase in concentration of proline in roots and leaves were recorded in plants

treated with 1.00 ppm Mo as seed soaking (T20) and foliar spray (T26) respectively. Foliar

treatments T25 (0.50 ppm Mo) and T27 (2.00 ppm Mo) highly increased concentration of

total phenolics in leaves and roots respectively (Table 3.7). Carotenoids concentration within

leaves were significantly increased (compared to C3) by the foliar treatments of Mo (T25,

CHAPTER # 3 RESULTS

58

T26 and T27) and highest significant increase in carotenoids was recorded in plants treated

with foliar spray of 2.00 ppm Mo (T27).

The overall effect of Mo treatments on free proline and total phenolics under different

concentrations of Cd in soil is given in figure 3.2. It was found that Mo treatments increased

the concentration of free proline and total phenolics as the soil Cd concentration increased

from 25 to 50 ppm and then decrease as the Cd concentration reaches 100 ppm.

CHAPTER # 3 RESULTS

59

Table 3. 5: Effect of various Mo treatments on concentrations of free proline, total phenolic compounds and photosynthetic pigments in Ricinus

communis plant grown in 25 ppm Cd contaminated soil. The table also shows effect of different Cd concentrations (25, 50 and 100 ppm) in soil on the

above parameters with in the plant. Note: ‗SD‘ denote ‗standard deviation‘ and the different letter in superscript present significant difference among the

values within a column.

Treatments Free proline (ppm) ± SD Total Phenolics (ppm) ± SD

Chlorophyll contents (ppm) ± SD Carotenoids (ppm) ± SD

Roots Leaves Roots leaves a b total chlorophyll C Control (without Cd and Mo) 28.80 ±

2.30 e 20.70 ± 1.66 e

26.45 ± 2.12 f

36.80 ± 2.94 e

36.61 ± 2.93 a

19.37 ± 1.55 a

55.98 ± 4.48 a

40.23 ± 3.22 ab

C1 Control (with Cd 25 ppm) 40.20 ± 4.82 de

37.08 ± 4.45 d

35.00 ± 4.20 ef

58.54 ± 7.02 d

6.99 ± 0.84 de

5.76 ± 0.69 cde

12.75 ± 1.53 de

29.00 ± 3.48 cde

C2 Control (with Cd 50 ppm) 43.54 ± 3.48 de

40.86 ± 3.27 d

39.00 ± 3.12 de

67.74 ± 5.42 cd

6.62 ± 0.53 de

5.32 ± 0.43 de

11.94 ± 0.96 de

21.28 ± 1.70 ef

C3 Control (with Cd 100 ppm) 48.98 ± 4.41 cd

54.00 ± 4.86 c

44.32 ± 3.99 bcde

69.12 ± 6.22 bcd

4.58 ± 0.41 e

3.91 ± 0.35 e

8.49 ± 0.76 e

15.63 ± 1.41 f


48.00 ± 3.84 cd

39.00 ± 3.12 d

44.97 ± 3.60 bcde

62.00 ± 4.96 d

7.89 ± 0.63 d

6.00 ± 0.48 cde

13.89 ± 1.11 d

28.45 ± 2.28 cde


52.00 ± 5.72 bcd

48.00 ± 5.28 cd

52.00 ± 5.72 abc

68.23 ± 7.51 bcd

8.18 ± 0.90 d

7.06 ± 0.78 cd

15.24 ± 1.68 d

30.20 ± 3.32 cd


64.07 ± 5.12 ab

49.23 ± 3.94 cd

55.00 ± 4.40 ab

76.35 ± 6.11 abcd

8.52 ± 0.68 cd

7.19 ± 0.57 cd

15.71 ± 1.26 d

34.20 ± 2.74 bc


54.00 ± 4.32 abcd

45.23 ± 3.62 cd

38.41 ± 3.07 def

62.33 ± 4.99 d

7.12 ± 0.57 de

6.08 ± 0.49 cd

13.20 ± 1.06 de

24.30 ± 1.94 de


65.00 ± 7.80 ab

43.00 ± 5.16 cd

40.71 ± 4.89 cde

63.71 ± 7.65 cd

7.45 ± 0.67 de

6.51 ± 0.59 cd

13.96 ± 1.26 d

26.03 ± 2.34 de


67.00 ± 6.03 a

40.21 ± 3.22 d

34.73 ± 2.78 ef

61.41 ± 4.91 d

7.38 ± 0.59 de

6.15 ± 0.49 cd

13.53 ± 1.08 de

24.60 ± 1.97 de


47.00 ± 3.76 cd

55.00 ± 4.40 bc

48.24 ± 3.86 abcd

81.88 ± 6.55 abc

8.97 ± 0.72 cd

7.56 ± 0.60 c

16.53 ± 1.32 cd

36.25 ± 2.90 abc


60.00 ± 6.00 abc

67.00 ± 5.36 ab

59.92 ± 4.79 a

91.77 ± 7.34 a

12.15 ± 0.97 b

10.37 ± 0.83 b

22.52 ± 1.80 b

41.48 ± 3.32 ab


67.70 ± 5.20 ab

67.98 ± 5.44 a

57.00 ± 4.56 a

86.23 ± 6.90 ab

11.45 ± 0.92 bc

10.00 ± 0.80 b

21.45 ± 1.72 bc

44.00 ± 3.52 a

CHAPTER # 3 RESULTS

60

Table 3. 6: Role of different Mo treatments on free proline, total phenolic compounds and photosynthetic pigments in Ricinus communis plant grown in

50 ppm Cd contaminated soil. Note: ‗SD‘ denote ‗standard deviation‘ and the different letter in superscript present significant difference among the

values within a column.

Treatments Free proline (ppm) Total Phenolics (ppm) Chlorophyll contents (ppm) Carotenoids (ppm)

Roots Leaves Roots leaves a b total chlorophyll

C2 Control (with Cd 50 ppm) 43.54 ± 4.79 ab

40.86 ± 4.49 e

39.00 ± 4.29 b

67.74 ± 7.45 d

6.62 ± 0.53 b

5.32 ± 0.43 d

11.94 ± 0.96 b

21.28 ± 1.70 d

T10 Cd 50 ppm+ Mo 0.5 ppm (Seed soaking) 45.23 ± 4.07 ab

48.65 ± 4.38 de

41.23 ± 3.71 b

84.15 ± 7.57 cd

7.08 ± 0.64 ab

5.68 ± 0.51 bcd

12.76 ± 1.15 ab

23.98 ± 1.92 cd

T11 Cd 50 ppm+ Mo 1.0 ppm (Seed soaking) 54.32 ± 5.98 ab

59.87 ± 6.59 abcd

46.54 ± 5.12 ab

92.61 ± 10.19 abcd

8.00 ± 0.64 ab

6.88 ± 0.55 abc

14.88 ± 1.19 ab

28.02 ± 2.24 bc

T12 Cd 50 ppm+ Mo 2.0 ppm (Seed soaking) 57.43 ± 4.59 a

61.41 ± 4.91 abcd

51.01 ± 4.08 ab

112.00 ± 8.96 abc

8.42 ± 0.67 a

7.33 ± 0.59 a

15.75 ± 1.26 a

33.10 ± 2.65 ab

T13 Cd 50 ppm+ Mo 0.5 ppm (Soil addition) 55.00 ± 6.05 ab

56.42 ± 6.21 bcde

50.12 ± 5.51 ab

92.00 ± 10.12 abcd

6.39 ± 0.51 b

5.49 ± 0.44 bcd

11.88 ± 0.95 b

22.05 ± 1.54 d


53.63 ± 4.29 bcde

44.54 ± 3.56 ab

88.23 ± 7.06 bcd

6.69 ± 0.53 ab

5.75 ± 0.46 bcd

12.43 ± 0.99 b

22.35 ± 1.34 cd


52.00 ± 5.72 cde

41.24 ± 4.54 b

83.35 ± 9.17 cd

7.36 ± 0.59 ab

6.40 ± 0.51 abcd

13.76 ± 1.10 ab

24.00 ± 1.92 cd

T16 Cd 50 ppm+ Mo 0.5 ppm (Foliar spray) 42.17 ± 3.37 b

68.60 ± 5.49 abc

45.38 ± 3.63 ab

111.14 ± 8.89 abc

7.49 ± 0.60 ab

7.00 ± 0.56 ab

14.49 ± 1.16 ab

35.00 ± 2.45 a

T17 Cd 50 ppm+ Mo 1.0ppm (Foliar spray) 46.23 ± 5.09 ab

70.00 ± 7.70 ab

49.35 ± 5.43 ab

116.00 ± 12.76 ab

7.29 ± 0.58 ab

6.42 ± 0.51 abcd

13.71 ± 1.10 ab

28.00 ± 2.24 bc

T18 Cd 50 ppm+ Mo 2.0 ppm (Foliar spray) 49.23 ± 5.91 ab

75.00 ± 9.00 a

55.68 ± 6.68 a

120.00 ± 14.40 a

6.73 ± 0.81 ab

5.45 ± 0.65 cd

12.18 ± 1.46 b

24.32 ± 1.70 cd

CHAPTER # 3 RESULTS

61

Table 3. 7: Effect of Mo treatments on free proline, total phenolics and photosynthetic pigments in Ricinus communis plant grown in 100 ppm Cd

contaminated soil. Note: ‗SD‘ denote ‗standard deviation‘ and the different letter in superscript present significant difference among the values within a

column.

Treatments Free proline (ppm) Total Phenolics (ppm) Chlorophyll contents (ppm) Carotenoids (ppm)

Roots Leaves Roots leaves a b total chlorophyll

C3 Control (with Cd 100 ppm) 35.00 ± 3.85 c

30.25 ± 3.33 d

33.25 ± 3.66 c

67.74 ± 7.45 cd

27.30 ± 2.18 abc

20.22 ± 1.62 ab

47.52 ± 3.80 abc

42.20 ± 3.38 d

T19 Cd 100 ppm+ Mo 0.5 ppm (Seed soaking) 54.94 ± 4.94 abc

34.16 ± 3.07 cd

57.00 ± 5.13 ab

85.00 ± 7.65 abcd

28.46 ± 2.56 ab

23.22 ± 2.09 a

51.68 ± 4.65 ab

45.12 ± 3.61 cd

T20 Cd 100 ppm+ Mo 1.0 ppm (Seed soaking) 71.25 ± 7.84 a

49.43 ± 5.44 ab

63.00 ± 6.93 ab

91.05 ± 10.02 ab

25.37 ± 2.03 bc

21.20 ± 1.70 ab

46.57 ± 3.73 abc

47.02 ± 3.76 cd

T21 Cd 100 ppm+ Mo 2 ppm (Seed soaking) 67.00 ± 5.36 ab

36.24 ± 2.90 cd

61.25 ± 4.90 ab

75.00 ± 6.00 bcd

20.84 ± 1.67 c

16.82 ± 1.35 b

37.66 ± 3.01 c

50.23 ± 4.02 cd

T22 Cd 100 ppm+ Mo 0.5 ppm (Soil addition) 47.49 ± 5.22 bc

43.59 ± 4.79 abc

47.57 ± 5.23 bc

70.35 ± 7.74 bcd

24.75 ± 1.98 bc

18.06 ± 1.44 ab

42.81 ± 3.42 bc

46.31 ± 3.24 cd


44.53 ± 3.56 abc

51.73 ± 4.14 ab

73.00 ± 5.84 bcd

30.11 ± 2.41 ab

21.17 ± 1.69 ab

51.28 ± 4.10 ab

53.02 ± 3.18 bcd

T24 Cd 100 ppm+ Mo 2 ppm (Soil addition) 67.00 ± 7.37 ab

39.97 ± 4.40 bcd

65.00 ± 7.15 a

64.48 ± 7.09 d

30.88 ± 2.47 ab

21.71 ± 1.74 ab

52.59 ± 4.21 ab

67.42 ± 5.39 bc

T25 Cd 100 ppm+ Mo 0.5 ppm (Foliar spray) 51.22 ± 4.10 abc

45.14 ± 3.61 abc

57.00 ± 4.56 ab

101.85 ± 8.15 a

28.09 ± 2.25 abc

19.88 ± 1.59 ab

47.97 ± 3.84 abc

58.03 ± 4.06 b

T26 Cd 100 ppm+ Mo 1.0ppm (Foliar spray) 57.99 ± 6.38 ab

51.24 ± 5.64 a

64.00 ± 7.04 a

96.36 ± 10.60 a

31.71 ± 2.54 a

22.30 ± 1.78 a

54.01 ± 4.32 a

81.89 ± 6.55 a

T27 Cd 100 ppm+ Mo 2.0 ppm (Foliar spray) 69.00 ± 8.28 ab

40.56 ± 4.87 abc

66.00 ± 7.92 a

85.00 ± 10.20 abc

33.52 ± 4.02 a

23.57 ± 2.83 a

57.09 ± 6.85 a

88.61 ± 6.20 a

CHAPTER # 3 RESULTS

62

Figure 3. 2: Overall effect of the molybdenum treatments on total phenolic and free proline

concentration in plants grown under different concentration of cadmium in soil.

b

c ab

bc

a

a

ab

ab

0

10

20

30

40

50

60

70

80

90

Free Proline Total phenolics

Co

nce

ntr

ario

n (

pp

m)

control with only Cd

25 ppm Cd in soil + Mo



CHAPTER # 3 RESULTS

63

3.3.1.3 Cadmium concentration and accumulation in different plant parts under various

treatments of molybdenum.

Variation in concentration, accumulation, translocation and bioconcentration of Cd in

different parts of Ricinus communis plant is given in tables (3.8, 3.9 and 3.10). Table 3.8

demonstrate the effect of different concentration of cadmium in soil on uptake and

accumulation of cadmium in plant tissues. A gradual increase was noted in plant Cd

concentration with increasing concentration of Cd in soils. The table 3.8 also shows the effect

of molybdenum treatments (T1-T9) on plant Cd uptake from 25 ppm Cd contaminated soil as

compare to C1 (25 ppm Cd, without Mo). The treatment T8 (1 ppm Mo foliar spray) most

significantly increased Cd concentration in roots. Stem and leaves of the plant demonstrated

highest significant increase in Cd concentration with 2 ppm Mo foliar spray (T9) as given in

table 3.8. It was found that 1.00 and 2.00 ppm Mo (seed soaking and foliar spray)

significantly increased Cd accumulation in the plant tissues. The treatment T9 showed

highest significant Cd accumulation in root, leaf and entire plant while the stem demonstrated

highest Cd accumulation in the treatment T8 (1 ppm Mo foliar spray) as shown in table 3.8.

The Mo treated plants (T1 –T9) showed increase in Cd bioconcentration as compared to C1.

Effect of Mo treatments in combination with 50 ppm Cd in soil (T10-18) on Cd uptake in

Ricinus communis is presented in table 3.9. Cadmium concentration in different parts of the

plant increased significantly in treatments T13 (0.5 ppm Mo added to soil) and T18 (2.00

ppm Mo foliar spray) as compared to C2 (50 ppm Cd in soil, without Mo treatments). Roots

accumulated Cd most significantly high in plants sprayed with 0.5 ppm Mo (T16) while stem

and leaves showed highly significant accumulated Cd in plants treated with 2 ppm Mo foliar

spray (T18). Cadmium translocation into leaves increased significantly with 0.5 ppm Mo as

seed soaking (T10). Bioconcentration of Cd was significantly increased by the treatments

T13 (0.5 ppm Mo into soil) and T18 (2 ppm Mo foliar spray) as compared to C2.

Variations in Cd uptake in plant tissues with Mo treatments (T19-T27) under 100 ppm Cd in

soil are given in table 3.10. Application of 0.5 ppm Mo (seed soaking and foliar spray)

significantly increased Cd concentration in roots of the plant. The same concentration (0.5

ppm) of Mo as soil addition significantly increased Cd concentration in stem (Table 3.10).

CHAPTER # 3 RESULTS

64

Foliar spray of 2.00 ppm Mo highly increased Cd concentration in leaves of the plant.

Highest significant accumulation of Cd in different parts of the plant were recorded in the

treatment T27 (2.00 ppm Mo foliar spray). Translocation and bioconcentration of Cd were

highly significant in plants sprayed with 2.00 ppm Mo (T27) as given in table 3.10.

Figure 3.3 present the overall effect of Mo treatments on Cd accumulation and

bioconcentration in Ricinus communis plant under varied Cd concentration in soil. The Mo

treatment showed an overall increase in plant Cd accumulation while decrease was recorded

in Cd bioconcentration with the increasing Cd concentration in soil.

CHAPTER # 3 RESULTS

65

Table 3. 8: Role of different treatments of Mo on Cd contents in Ricinus communis plant grown in 25 ppm Cd contaminated soil. Note: ―R-S‖ denote ―

Roots into Stem‖, ―R-L‖ denote ―Roots into Leaves‖, ―SD‖ represent ―Standard Deviation‖, while different letter in superscript represent significant

difference among the values in a column.

Treatments Cd concentration (ppm) Cd accumulation (mg/DW) % Cd accumulation Cd translocation

factor

Cd-Bioconcentration factor

Roots Stem Leaves Roots Stem Leaves Entire plant Roots Stem Leaves R-S R-L


137.00 ± 16.44 b

58.07 ± 6.38 c

78.00 ± 10.14 b

0.11 ± 0.02 e

0.10 ± 0.02 d

0.15 ± 0.03 c

0.35 ± 0.07 e

30.80 27.19 42.01 0.42 ± 0.05 a

0.57 ± 0.01 a

3.25 ± 0.41 c


219.05 ± 26.28 a

79.00 ± 8.69 b

87.00 ± 11.31 b

0.14 ± 0.03 de

0.09 ± 0.02 d

0.14 ± 0.05 c

0.38 ± 0.08 e

38.41 25.07 36.52 0.36 ± 0.03 c

0.40 ± 0.03 e

2.19 ± 0.25 c


268.70 ± 32.16 a

114.00 ± 12.54 a

125.00 ± 16.25 a

0.15 ± 0.05 de

0.12 ± 0.02 d

0.13 ± 0.01 c

0.40 ± 0.08 de

37.36 29.45 33.19 0.43 ± 0.02 a

0.47 ± 0.02 b

1.51 ± 0.11 d


195.60 ± 23.40 ab

65.23 ± 7.18 b

85.23 ± 8.52 b

0.25 ± 0.02 bcde

0.14 ± 0.03 cd

0.23 ± 0.04 abc

0.62 ± 0.12 bcde

39.80 22.52 37.69 0.35 ± 0.04 d

0.44 ± 0.01 d

4.04 ± 0.48 bc


204.09 ± 24.48 ab

69.00 ± 7.59 b

89.28 ± 8.92 b

0.31 ± 0.06 bcd

0.19 ± 0.04 abcd

0.31 ± 0.06 ab

0.81 0.16 abcd

38.42 23.16 38.42 0.34 ± 0.05 d

0.44 ± 0.01 d

4.19 ± 0.47 bc


214.54 ± 25.68 ab

81.10 ± 9.03 b

98.87 ± 9.89 ab

0.35 ± 0.07 abc

0.23 ± 0.04 abc

0.32 ± 0.05 ab

0.90 0.17 abc

39.02 25.31 35.67 0.38 ± 0.03 b

0.46 ± 0.01 bc

4.70 ± 0.52 abc


208.68 ± 24.96 ab

70.00 ± 7.70 b

91.00 ± 8.19 b

0.21 ± 0.04 cde

0.13 ± 0.03 cd

0.20 ± 0.03 bc

0.53 0.10 cde

38.24 25.11 36.64 0.34 ± 0.02 d

0.44 ± 0.03 cd

4.23 ± 0.47 bc


200.35 ± 24.00 ab

67.00 ± 7.37 b

84.56 ± 7.61 b

0.25 ± 0.05 bcde

0.14 ± 0.03 bcd

0.21 ± 0.02 bc

0.60 0.11 bcde

41.62 24.16 34.22 0.33 ± 0.03 d

0.42 ± 0.02 d

4.11 ± 0.42 bc


196.63 ± 23.52 ab

69.42 ± 7.64 b

83.24 ± 7.49 b

0.29 ± 0.06 bcd

0.14 ± 0.03 cd

0.24 ± 0.04 abc

0.67 0.12 abcde

43.32 20.69 36.00 0.35 ± 0.06 c

0.43 ± 0.01 d

4.21 ± 0.45 bc


245.00 ± 29.40 a

67.89 ± 7.47 b

82.43 ± 10.03 b

0.32 ± 0.08 abcd

0.17 ± 0.03 abcd

0.25 ± 0.05 abc

0.73 0.15 abcde

43.48 22.80 33.72 0.28 ± 0.04 f

0.34 ± 0.02 f

4.33 ± 0.51 bc


267.86 ± 32.04 a

70.45 ± 7.75 b

100.20 ± 12.22 ab

0.39 ± 0.08 ab

0.24 ± 0.05 a

0.34 ± 0.07 ab

0.97 0.19 ab

40.19 25.05 34.76 0.26 ± 0.03 g

0.38 ± 0.03 e

4.69 ± 0.57 abc


256.00 ± 30.72 a

82.23 ± 8.94 b

102.12 ± 12.46 a

0.49 ± 0.11 a

0.24 ± 0.05 ab

0.35 ± 0.08 a

1.08 0.24 a

45.42 22.24 32.34 0.32 ± 0.02 e

0.40 ± 0.02 e

5.21 ± 0.62 ab

CHAPTER # 3 RESULTS

66

Table 3. 9: Effect of Mo treatments on Cd contents in Ricinus communis plant grown in 50 ppm Cd contaminated soil. Note: ―R-S‖ denote ― Roots into

Stem‖, ―R-L‖ denote ―Roots into Leaves‖, ―SD‖ represent ―Standard Deviation‖, while different letter in superscript represent significant difference among

the values in a column.


factor

Cd-Bioconcent

ration factor Roots Stem Leaves Roots Stem Leaves Entire plant Roots Stem Leaves R-S R-L


219.07 ± 19.71 b

79.00 ± 9.48 b

87.00 ± 11.31 d

0.14 ± 0.03 b

0.09 ± 0.02 b

0.14 ± 0.03 b

0.38 ± 0.09 b

38.46 25.04 36.49 0.36 ± 0.05 cde

0.40 ± 0.02 e

2.19 ± 0.25 b


228.47 ± 20.52 ab

88.35 ± 10.60 b

137.07 ± 17.81 abc

0.26 ± 0.05 ab

0.17 ± 0.04 ab

0.29 ± 0.06 ab

0.72 ± 0.15 ab

35.91 23.44 40.66 0.39 ± 0.03 abc

0.60 ± 0.03 a

2.78 ± 0.31 ab


232.00 ± 20.88 ab

89.23 ± 10.71 ab

125.25 ± 16.25 abcd

0.29 ± 0.06 ab

0.23 ± 0.05 ab

0.36 ± 0.09 a

0.88 ± 0.20 ab

33.13 25.83 41.04 0.38 ± 0.04 bcd

0.54 ± 0.04 bc

2.63 ± 0.30 ab


238.75 ± 21.42 ab

95.00 ± 11.40 ab

98.45 ± 12.74 bcd

0.33 ± 0.08 a

0.22 ± 0.06 ab

0.31 ± 0.08 ab

0.87 ± 0.22 ab

38.41 25.72 35.88 0.40 ± 0.06 ab

0.41 ± 0.05 e

2.50 ± 0.28 ab


289.05 ± 26.01 a

121.00 ± 14.52 a

142.35 ± 18.46 ab

0.25 ± 0.05 ab

0.15 ± 0.03 ab

0.24 ± 0.06 ab

0.64 ± 0.15 ab

39.56 22.79 37.65 0.42 ± 0.03 a

0.49 ± 0.02 cd

3.39 ± 0.38 a


246.63 ± 22.14 ab

86.92 ± 10.43 b

128.12 ± 16.64 abcd

0.27 ± 0.06 ab

0.17 ± 0.04 ab

0.26 ± 0.07 ab

0.71 ± 0.17 ab

39.02 23.83 37.15 0.35 ± 0.02 de

0.52 ± 0.02 c

2.77 ± 0.31 ab


229.08 ± 20.61 ab

83.25 ± 9.99 b

92.20 ± 11.99 cd

0.28 ± 0.06 ab

0.18 ± 0.04 ab

0.29 ± 0.07 ab

0.75 ± 0.17 ab

37.62 23.45 38.93 0.36 ± 0.05 cd

0.40 ± 0.03 e

2.30 ± 0.26 b


248.12 ± 22.32 ab

79.23 ± 9.51 b

112.00 ± 14.56 bcd

0.36 ± 0.08 a

0.19 ± 0.05 ab

0.30 ± 0.08 ab

0.84 ± 0.21 ab

42.93 20.94 36.13 0.32 ± 0.06 f

0.45 ± 0.04 de

2.63 ± 0.29 ab


257.58 ± 23.13 ab

85.00 ± 10.20 b

123.98 ± 16.12 abcd

0.34 ± 0.08 a

0.21 ± 0.06 ab

0.31 ± 0.09 ab

0.86 ± 0.23 ab

39.63 24.63 35.74 0.33 ± 0.03 ef

0.48 ± 0.03 cd

2.73 ± 0.30 ab


287.21 ± 25.83 a

108.00 ± 12.96 ab

168.00 ± 21.84 a

0.32 ± 0.07 ab

0.27 ± 0.07 a

0.36 ± 0.08 a

0.93 ± 0.22 a

34.81 29.29 35.91 0.38 ± 0.01 bcd

0.58 ± 0.02 ab

3.30 ± 0.37 a

CHAPTER # 3 RESULTS

67

Table 3. 10: Effect of Mo treatments on Cd contents in Ricinus communis plant grown in 100 ppm Cd contaminated soil. Note: ―R-S‖ denote ― Roots into

Stem‖, ―R-L‖ denote ―Roots into Leaves‖, ―SD‖ represent ―Standard Deviation‖, while different letter in superscript represent significant difference among

the values in a column.


factor

Cd-Bioconcentra

tion factor

Roots Stem Leaves Roots Leaves Entire plant Roots Stem Leaves R-S R-L


268.70 ± 32.16 c

114.00 ± 12.54 c

125.00 ± 16.25 e

0.15 ± 0.05 b

0.12 ± 0.02 c

0.13 ± 0.01 b

0.40 ± 0.08 b

37.36 29.45 33.19 0.43 ± 0.02 d

0.47 ± 0.02 g

1.51 ± 0.11 c


331.05 ± 19.86 a

142.90 ± 10.00 ab

190.75 ± 16.21 bcd

0.27 ± 0.04 ab

0.23 ± 0.04 abc

0.34 ± 0.06 ab

0.83 ± 0.14 ab

31.96 27.29 40.75 0.43 ± 0.02 d

0.58 ± 0.02 ef

2.00 ± 0.15 ab


302.45 ± 18.12 abc

144.33 ± 10.10 ab

172.89 ± 14.70 bcd

0.30 ± 0.05 ab

0.31 ± 0.06 ab

0.44 ± 0.09 a

1.05 ± 0.20 a

28.92 29.09 41.99 0.48 ± 0.01 c

0.57 ± 0.01 ef

1.85 ± 0.14 abc


274.51 ± 16.44 c

153.66 ± 10.76 ab

167.86 ± 14.27 cde

0.36 ± 0.07 a

0.30 ± 0.06 ab

0.47 ± 0.10 a

1.12 ± 0.24 a

31.94 26.71 41.35 0.56 ± 0.03 a

0.61 ± 0.02 e

1.86 ± 0.14 abc


326.36 ± 19.56 ab

164.98 ± 11.55 a

215.63 ± 18.33 ab

0.24 ± 0.04 ab

0.18 ± 0.03 bc

0.29 ± 0.06 ab

0.71 ± 0.14 ab

33.65 25.99 40.36 0.51 ± 0.01 b

0.66 ± 0.04 d

2.23 ± 0.16 a


295.28 ± 17.70 abc

152.50 ± 10.68 ab

210.29 ± 17.87 abc

0.27 ± 0.05 ab

0.25 ± 0.05 abc

0.36 ± 0.08 ab

0.88 ± 0.18 ab

31.23 27.95 40.81 0.52 ± 0.02 b

0.71 ± 0.02 bc

2.07 ± 0.15 ab


287.58 ± 17.25 abc

134.65 ± 9.43 bc

152.93 ± 13.00 de

0.32 ± 0.06 a

0.24 ± 0.04 abc

0.41 ± 0.08 a

0.94 ± 0.11 a

33.12 24.50 42.38 0.47 ± 0.01 c

0.53 ± 0.03 f

1.74 ± 0.13 bc


275.00 ± 16.50 c

128.15 ± 8.97 bc

185.77 ± 15.79 bcd

0.33 ± 0.07 a

0.23 ± 0.05 abc

0.42 ± 0.10 a

0.98 ± 0.22 a

33.63 23.96 42.41 0.47 ± 0.03 c

0.67 ± 0.02 cd

1.86 ± 0.14 abc


279.72 ± 16.74 bc

133.26 ± 9.33 bc

205.64 ± 17.48 abc

0.31 ± 0.07 ab

0.27 ± 0.06 ab

0.41 ± 0.10 a

0.99 ± 0.23 a

30.87 27.75 41.38 0.48 ± 0.03 c

0.74 ± 0.04 b

1.92 ± 0.14 ab


290.43 ± 17.40 abc

162.81 ± 10.42 a

242.17 ± 20.58 a

0.32 ± 0.05 a

0.37 ± 0.06 a

0.51 ± 0.07 a

1.20 ± 0.18 a

26.67 30.08 42.50 0.51 ± 0.02 b

0.83 ± 0.03 a

2.25 ± 0.15 a

CHAPTER # 3 RESULTS

68

Figure 3. 3: Overall effect of the molybdenum treatments on Cd accumulation and bioconcentration

in Ricinus communis plant grown in soil containing different concentrations of Cd (25, 50 and 100

ppm).

0.37

4.5

0.77

4.41

1.50

2.78

1.74

1.96

0

1

2

3

4

5

6

Cd accumulation (mg Cd/ dry biomass of plant) Bioconcentraion

control with only Cd




CHAPTER # 3 RESULTS

69

3.3.1.4 Correlation among different parameters in Ricinus communis plant

Tables 3.11 - 3.19 present correlations among different parameters in roots, stem and leaves

of Ricinus communis plant grown in 25, 50 and 100 ppm Cd contaminated soil, under various

treatments of Mo (0.5, 1.00 and 2.00 ppm). Total phenolics concentration showed

significantly positive correlations with Cd accumulation in plant roots (table 3.11, 3.12 and

3.13) and leaves (table 3.17, 3.18 and 3.19). Proline concentrations in roots (tables 3.11, 3.12)

and leaves (tables 3.17, 3.18) also demonstrated significantly positive correlations with Cd

accumulation in plants grown in 25 and 50 ppm Cd contaminated soil respectively. Proline

concentration showed strong positive correlation with Cd accumulation in roots in 25, 50 and

100 ppm Cd contaminated soil (Tables 3.11, 3.12 and 3.13). Photosynthetic pigments

(chlorophyll and carotenoids) showed strong correlation with total phenolics concentration

within leaves of the plant at all the Cd concentrations (25, 50 and 100 ppm in soil) as shown

in table 3.17, 3.18 and 3.19. It was found that dry weight in roots, stem and leaves

demonstrated significantly positive correlation with Cd accumulation (tables 3.11 – 3.19).

CHAPTER # 3 RESULTS

70

Table 3. 11: Correlations among different parameter in roots of Ricinus communis plant grown in 25 ppm Cd contaminated soil.

Le

ng

th

Fre

sh

we

igh

t

Dry

we

igh

t

To

tal

wa

ter

co

nte

nt

Cd

co

nc

en

tra

tion

Cd

ac

cu

mu

lat

ion

Fre

e

Pro

line

To

tal

Ph

en

olic

s

Length (cm) Pearson Correlation

1 0.758** 0.731

** 0.699

** -0.117 0.646

* 0.340 0.596

*

Sig. (1-tailed) 0.002 0.003 0.006 0.358 0.012 0.140 0.021

Fresh weight (g) Pearson Correlation

0.758** 1 0.896

** 0.964

** -0.114 0.777

** 0.637

* 0.640

*

Sig. (1-tailed) 0.002 0.000 0.000 0.362 0.001 0.013 0.013

Dry weight (g) Pearson Correlation

0.731** 0.896

** 1 0.744

** 0.056 0.939

** 0.721

** 0.644

*

Sig. (1-tailed) 0.003 0.000 0.003 0.431 0.000 0.004 0.012

Total water content

(g)

Pearson Correlation 0.699

** 0.964

** 0.744

** 1 - 0.205 0.602

* 0.624

* 0.694

*

Sig. (1-tailed) 0.006 0.000 0.003 0.262 0.019 0.020 0.016

Cd concentration

(ppm)

Pearson Correlation -0.117 -0.114 0.056 -0.205 1 0.377 0.668

** 0.793

**

Sig. (1-tailed) 0.358 0.362 0.431 0.262 0.113 0.009 0.001

Cd accumulation

(mg/DW)


* 0.777

** 0.939

** 0.602

* 0.377 1 0.668

** 0.793

**

Sig. (1-tailed) 0.012 0.001 0.000 0.019 0.113 0.009 0.001

Free Proline (ppm) Pearson Correlation

0.340 0.637* 0.721

** 0.624

* 0.093 0.668

** 1 0.342

Sig. (1-tailed) 0.140 0.013 0.004 0.020 0.387 0.009 0.138

Total Phenolics

(ppm)


* 0.640

* 0.644

* 0.574

* 0.619

* 0.793

** 0.342 1

Sig. (1-tailed) 0.021 0.013 0.012 0.026 0.016 0.001 0.138


CHAPTER # 3 RESULTS

71


Le

ng

th

Fre

sh

we

igh

t

Dry

we

igh

t

To

tal

wa

ter

co

nte

nt

Cd

co

nc

en

t-

ratio

n

Cd

ac

cu

mu

l-

atio

n

Fre

e

Pro

line

To

tal

Ph

en

olic

s

Length Pearson Correlation 1 0.695* 0.581

* 0.661

* -0.064 0.508 0.124 0.271

Sig. (1-tailed) 0.013 0.039 0.019 0.430 0.067 0.367 0.224

Fresh weight Pearson Correlation 0.695* 1 0.717

** 0.982

** -0.021 0.632

* 0.610

* 0.410

Sig. (1-tailed) 0.013 0.010 0.000 0.477 0.025 0.030 0.119

Dry weight Pearson Correlation 0.581* 0.717

** 1 0.573

* -0.026 0.915

** 0.094 0.322

Sig. (1-tailed) 0.039 0.010 0.042 0.472 0.000 0.398 0.182

Total water content Pearson Correlation 0.661* 0.982

** 0.573

* 1 -0.018 0.497 0.692

* 0.396

Sig. (1-tailed) 0.019 0.000 0.042 0.480 0.072 0.013 0.129

Cd concentration Pearson Correlation -0.064 -0.021 -0.026 -0.018 1 0.373 0.289 0.809**

Sig. (1-tailed) 0.430 0.477 0.472 0.480 0.144 0.209 0.002

Cd accumulation Pearson Correlation 0.508 0.632* 0.915

** 0.497 0.373 1 0.694* 0.807*

*

Sig. (1-tailed) 0.067 0.025 0.000 0.072 0.144 0.013 0.026

Free Proline Pearson Correlation 0.124 0.610* 0.094 0.692

* 0.289 0.694* 1 0.522

Sig. (1-tailed) 0.367 0.030 0.398 0.013 0.209 0.013 0.061

Total Phenolics Pearson Correlation 0.271 0.410 0.322 0.396 0.809** 0.807

** 0.522 1

Sig. (1-tailed) 0.224 0.119 0.182 0.129 0.002 0.026 0.061


CHAPTER # 3 RESULTS

72


Le

ng

th

Fre

sh

we

igh

t

Dry

we

igh

t

To

tal

wa

ter

co

nte

nt

Cd

co

nc

en

tratio

n

Cd

ac

cu

mu

lat

ion

Fre

e

Pro

line

To

tal

ph

en

olic

s

Length Pearson Correlation 1 0.799** 0.794** 0.756** -0.354 0.751** 0.444 0.246

Sig. (1-tailed) 0.003 0.003 0.006 0.158 0.006 0.099 0.356

Fresh weight Pearson Correlation 0.799** 1 0.824** 0.989** -0.113 0.665* 0.634* 0.201

Sig. (1-tailed) 0.003 0.002 0.000 0.378 0.019 0.025 0.289

Dry weight Pearson Correlation 0.794** 0.824** 1 0.733** -0.375 0.763* 0.325 0.203

Sig. (1-tailed) 0.003 0.002 0.008 0.143 0.012 0.180 0.287

Total water content Pearson Correlation 0.756** 0.989** 0.733** 1 -0.040 0.792** 0.677* 0.189

Sig. (1-tailed) 0.006 0.000 0.008 0.457 0.003 0.016 0.300

Cd concentration Pearson Correlation -0.354 -0.113 -0.375 -0.040 1 -0.115 0.315 0.450

Sig. (1-tailed) 0.158 0.378 0.143 0.457 0.376 0.188 0.096

Cd accumulation Pearson Correlation 0.751** 0.665* 0.763* 0.792** -0.115 1 0.673* 0.339

Sig. (1-tailed) 0.006 0.019 0.012 0.003 0.376 0.021 0.739*

Free Proline Pearson Correlation 0.444 0.634* 0.325 0.677* 0.315 0.673* 1 0.010

Sig. (1-tailed) 0.099 0.025 0.180 0.016 0.188 0.021 0.164

Total phenolics Pearson Correlation 0.246 0.201 0.203 0.189 0.650* 0.739* 0.346 1

Sig. (1-tailed) 0.356 0.289 0.287 0.300 0.016 0.010 0.164


CHAPTER # 3 RESULTS

73

Table 3. 14: Correlations among different parameter in stem of Ricinus communis plant grown in 25 ppm Cd contaminated soil.

Le

ng

th

Fre

sh

we

igh

t

Dry

w

eig

ht

To

tal

wa

ter

co

nte

nt

Cd

co

nc

en

t-

ratio

n

Cd a

ccu

mu

-

latio

n

Length (cm) Pearson Correlation

1 0.954** 0.920** 0.916** -0.527* 0.753**

Sig. (1-tailed) 0.000 0.000 0.000 0.039 0.002

Fresh weight (g) Pearson Correlation

0.954** 1 0.957** 0.967** -0.418 0.862**

Sig. (1-tailed) 0.000 0.000 0.001 0.088 0.000

Dry weight (g) Pearson Correlation

0.920** 0.957** 1 0.851** -0.342 0.926**

Sig. (1-tailed) 0.000 0.000 0.000 0.138 0.000

Total water content (g) Pearson Correlation

0.916** 0.967** 0.851** 1 -0.454 0.747**

Sig. (1-tailed) 0.000 0.000 0.000 0.069 0.003

Cd concentration

(ppm)

Pearson Correlation -0.527* -0.418 -0.342 -0.454 1 0.018

Sig. (1-tailed) 0.039 0.088 0.138 0.069 0.478

Cd accumulation

(mg/DW)

Pearson Correlation 0.753** 0.862** 0.926** 0.747** 0.018 1

Sig. (1-tailed) 0.002 0.001 0.001 0.003 0.478


CHAPTER # 3 RESULTS

74


Length Fresh weight

Dry weight Total water content

Cd concent-ration

Cd accumul-ation

Length (cm) Pearson Correlation 1 0.348 0.522 0.172 -0.215 0.442

Sig. (1-tailed) 0.163 0.061 0.318 0.276 0.101

Fresh Pearson Correlation 0.348 1 0.912** 0.949

** -0.033 0.879

**

Sig. (1-tailed) 0.163 0.000 0.000 0.464 0.000

Dry weight Pearson Correlation 0.522 0.912** 1 0.735

** -0.069 0.964

**

Sig. (1-tailed) 0.061 0.000 0.008 0.425 0.000

Total water content Pearson Correlation 0.172 0.949** 0.735

** 1 0.000 0.708

*

Sig. (1-tailed) 0.318 0.000 0.008 0.499 0.011

Cd concentration Pearson Correlation -0.215 -0.033 -0.069 0.000 1 0.187

Sig. (1-tailed) 0.276 0.464 0.425 0.499 0.303

Cd accumulation Pearson Correlation 0.442 0.879** 0.964

** 0.708

* 0.187 1

Sig. (1-tailed) 0.101 0.000 0.000 0.011 0.303


CHAPTER # 3 RESULTS

75


Len

gth

Fre

sh

we

igh

t

Dry

we

igh

t

To

tal

wa

ter

co

nte

nt

Cd

co

ncen

t

-ratio

n

Cd

acc

um

ul

-atio

n

Length Pearson Correlation 1 0.541 0.773** 0.270 -0.361 0.686*

Sig. (1-tailed) 0.053 0.004 0.225 0.153 0.014

Fresh weight Pearson Correlation 0.541 1 0.829** 0.920** -0.304 0.759**

Sig. (1-tailed) 0.053 0.001 0.000 0.197 0.005

Dry weight Pearson Correlation 0.773** 0.829** 1 0.543 -0.270 0.953**

Sig. (1-tailed) 0.004 0.001 0.052 0.225 0.000

Total water content Pearson Correlation 0.270 0.920** 0.543 1 -0.266 0.471

Sig. (1-tailed) 0.225 0.000 0.052 0.229 0.085

Cd concentration Pearson Correlation -0.361 -0.304 -0.270 -0.266 1 0.027

Sig. (1-tailed) 0.153 0.197 0.225 0.229 0.471

Cd accumulation

(mg/DW)

Pearson Correlation 0.686* 0.759** 0.953** 0.471 0.027 1

Sig. (1-tailed) 0.014 0.005 0.000 0.085 0.471


CHAPTER # 3 RESULTS

76

Table 3. 17: Correlations among different parameter in Leaves of Ricinus communis plant grown in 25 ppm Cd contaminated soil.

Length Fresh weight

Dry weight

Total water

content

Cd concentration

Cd accumulation

Free Proline

Total phenolics

Chlorophyll

a

Chlorophyll

b

Carotenoids

Length (cm) Pearson Correlation 1 0.932

** 0.981

** -0.095 0.947

** 0.532

* 0.600

* 0.870

** 0.877

** 0.875

** 0.836

**

Sig. (1-tailed) 0.000 0.000 0.385 0.000 0.037 0.020 0.000 0.000 0.000 0.000

Fresh weight (g)

Pearson Correlation 0.932** 1 0.843

** -0.219 0.949

** 0.433 0.531

* 0.817

** 0.810

** 0.816

** 0.823

**

Sig. (1-tailed) 0.000 0.000 0.247 0.000 0.080 0.038 0.001 0.001 0.001 0.001

Dry weight (g) Pearson Correlation 0.981

** 0.843

** 1 -0.023 0.893

** 0.556

* 0.603

* 0.849

** 0.864

** 0.858

** 0.796

**

Sig. (1-tailed) 0.000 0.000 0.472 0.000 0.030 0.019 0.000 0.000 0.000 0.001

Total water content (g)

Pearson Correlation -0.095 -0.219 -0.023 1 0.077 0.582* 0.393 -0.064 -0.013 -0.040 -0.103

Sig. (1-tailed) 0.385 0.247 0.472 0.406 0.023 0.103 0.422 0.484 0.451 0.375

Cd concentration

(ppm)

Pearson Correlation 0.947** 0.949

** 0.893

** 0.077 1 0.637

* 0.687

** 0.855

** 0.863

** 0.861

** 0.845

**

Sig. (1-tailed) 0.000 0.000 0.000 0.406 0.013 0.007 0.000 0.000 0.000 0.000

Cd accumulation

(mg/DW)

Pearson Correlation 0.532* 0.433 0.556

* 0.582

* 0.637

* 1 0.831

** 0.707

** 0.753

** 0.730

** 0.670

**

Sig. (1-tailed) 0.037 0.080 0.030 0.023 0.013 0.001 0.005 0.002 0.004 0.009

Free Proline (ppm)

Pearson Correlation 0.600* 0.531

* 0.603

* 0.393 0.687

** 0.831

** 1 0.794

** 0.813

** 0.805

** 0.768

**

Sig. (1-tailed) 0.020 0.038 0.019 0.103 0.007 0.001 0.001 0.001 0.001 0.002

Total phenolics

(ppm)


** 0.849

** -0.064 0.855

** 0.707

** 0.794

** 1 0.991

** 0.998

** 0.955

**

Sig. (1-tailed) 0.000 0.001 0.000 0.422 0.000 0.005 0.001 0.000 0.000 0.000

Chlorophyll a (ppm)


** 0.864

** -0.013 0.863

** 0.753

** 0.813

** 0.991

** 1 0.998

** 0.949

**

Sig. (1-tailed) 0.000 0.001 0.000 0.484 0.000 0.002 0.001 0.000 0.000 0.000

Chlorophyll b (ppm)


** 0.858

** -0.040 0.861

** 0.730

** 0.805

** 0.998

** 0.998

** 1 0.954

**

Sig. (1-tailed) 0.000 0.001 0.000 0.451 0.000 0.004 0.001 0.000 0.000 0.000

Carotenoids (ppm)


** 0.796

** -0.103 0.845

** 0.670

** 0.768

** 0.955

** 0.949

** 0.954

** 1

Sig. (1-tailed) 0.000 0.001 0.001 0.375 0.000 0.009 0.002 0.000 0.000 0.000


CHAPTER # 3 RESULTS

77

Table 3. 18: Correlations among different parameter in Leaves of Ricinus communis plant grown in 50 ppm Cd contaminated soil.

Length (cm)

Fresh weight (g)

Dry weight (g)


Cd concentration

(ppm)

Cd accumulation

(mg/DW)

Free Proline (ppm)

Total phenolics

(ppm)

Chlorophyll a (ppm)

Chlorophyll b (ppm)

Carotenoids (ppm)


* 0.848

** -0.027 0.620

* 0.350 0.421 0.783

** 0.722

** 0.764

** 0.603

*

Sig. (1-tailed) 0.032 0.001 0.471 0.028 0.161 0.113 0.004 0.009 0.005 0.032 Fresh weight

(g) Pearson Correlation 0.606* 1 0.092 -0.400 0.783

** 0.276 0.324 0.892

** 0.900

** 0.912

** 0.683

*

Sig. (1-tailed) 0.032 0.400 0.126 0.004 0.220 0.181 0.000 0.000 0.000 0.015 Dry weight

(g) Pearson Correlation 0.848** 0.092 1 0.233 0.255 0.254 0.311 0.386 0.304 0.348 0.300

Sig. (1-tailed) 0.001 0.400 0.259 0.239 0.240 0.191 0.135 0.197 0.162 0.200 Total water content (g) Pearson Correlation -0.027 -0.400 0.233 1 0.247 0.627

* 0.578

* -0.430 -0.358 -0.398 -0.132

Sig. (1-tailed) 0.471 0.126 0.259 0.246 0.026 0.040 0.107 0.155 0.127 0.358 Cd

concentration (ppm)


** 0.255 0.247 1 0.743

** 0.763

** 0.680

* 0.738

** 0.723

** 0.684

*

Sig. (1-tailed) 0.028 0.004 0.239 0.246 0.007 0.005 0.015 0.007 0.009 0.015 Cd

accumulation (mg/DW)

Pearson Correlation 0.350 0.276 0.254 0.627* 0.743

** 1 0.964

** 0.721* 0.369 0.320 0.562

*

Sig. (1-tailed) 0.161 0.220 0.240 0.026 0.007 0.000 0.011 0.147 0.184 0.045 Free Proline

(ppm) Pearson Correlation 0.421 0.324 0.311 0.578* 0.763

** 0.964

** 1 0.349 0.438 0.403 0.642

*

Sig. (1-tailed) 0.113 0.181 0.191 0.040 0.005 0.000 0.161 0.103 0.124 0.023 Total

phenolics (ppm)


** 0.386 -0.430 0.680

* 0.621* 0.349 1 0.932

** 0.981

** 0.787

**

Sig. (1-tailed) 0.004 0.000 0.135 0.107 0.015 0.011 0.161 0.000 0.000 0.003 Chlorophyll a

(ppm) Pearson Correlation 0.722** 0.900

** 0.304 -0.358 0.738

** 0.369 0.638 0.932

** 1 0.985

** 0.890

**

Sig. (1-tailed) 0.009 0.000 0.197 0.155 0.007 0.147 0.010 0.000 0.000 0.000 Chlorophyll

b (ppm) Pearson Correlation 0.764** 0.912

** 0.348 -0.398 0.723

** 0.320 0.563 0.981

** 0.985

** 1 0.856

**

Sig. (1-tailed) 0.005 0.000 0.162 0.127 0.009 0.184 0.012 0.000 0.000 0.001 Carotenoids

(ppm) Pearson Correlation 0.603* 0.683

* 0.300 -0.132 0.684

* 0.562

* 0.642

* 0.787

** 0.890

** 0.856

** 1

Sig. (1-tailed) 0.032 0.015 0.200 0.358 0.015 0.045 0.023 0.003 0.000 0.001

**. Correlation is significant at the 0.01 level (1-tailed).

*. Correlation is significant at the 0.05 level (1-tailed).

CHAPTER # 3 RESULTS

78

Table 3. 19: Correlations among different parameter in leaves of Ricinus communis plant grown in 100 ppm Cd contaminated soil.

Length (cm)

Fresh weight

(g)

Dry weight

(g)


Cd concentra

tion (ppm)

Cd accumulation (mg/DW)

Free Proline (ppm)

Total phenolics

(ppm)

Chlorophyll a (ppm)

Chlorophyll b (ppm)

Carotenoids (ppm)


** 0.863

** -0.468 0.702

* 0.068 0.179 0.607

* 0.593

* 0.064 0.456

Sig. (1-tailed) 0.009 0.001 0.086 0.012 0.426 0.310 0.031 0.035 0.430 0.093

Fresh weight (g)


1 0.271 -0.717**

0.905**

0.147 0.241 0.791**

0.775**

-0.035 0.575*

Sig. (1-tailed) 0.009 0.224 0.010 0.000 0.343 0.251 0.003 0.004 0.461 0.041

Dry weight (g) Pearson Correlation 0.863

** 0.271 1 -0.127 0.314 -0.012 0.073 0.266 0.258 0.115 0.214

Sig. (1-tailed) 0.001 0.224 0.363 0.188 0.486 0.420 0.228 0.236 0.376 0.277


Pearson Correlation -0.468 -0.717**

-0.127 1 -0.365 0.423 0.427 -0.265 -0.336 -0.311 -0.165

Sig. (1-tailed) 0.086 0.010 0.363 0.150 0.112 0.109 0.229 0.171 0.191 0.325

Cd concentration

(ppm)


** 0.314 -0.365 1 0.456 0.585

* 0.924

** 0.869

** -0.166 0.714

*

Sig. (1-tailed) 0.012 0.000 0.188 0.150 0.092 0.038 0.000 0.001 0.324 0.010

Cd accumulation

(mg/DW)

Pearson Correlation 0.068 0.147 -0.012 0.423 0.456 1 0.930**

0.866* 0.646

* -0.335 0.721

**

Sig. (1-tailed) 0.426 0.343 0.486 0.112 0.092 0.000 0.008 0.022 0.172 0.009

Free Proline (ppm)

Pearson Correlation 0.179 0.241 0.073 0.627 0.585* 0.930

** 1 0.723

** 0.661

* -0.309 0.702

*

Sig. (1-tailed) 0.310 0.251 0.420 0.010 0.038 0.000 0.009 0.019 0.193 0.012

/Total phenolics

(ppm)


** 0.266 -0.265 0.924

** 0.866

* 0.723

** 1 0.959

** 0.773* 0.896

**

Sig. (1-tailed) 0.031 0.003 0.228 0.229 0.000 0.008 0.009 0.000 0.017 0.000

Chlorophyll a (ppm)


** 0.258 -0.336 0.869

** 0.646

* 0.661

* 0.959

** 1 0.037 0.946

**

Sig. (1-tailed) 0.035 0.004 0.236 0.171 0.001 0.019 0.018 0.000 0.459 0.000

Chlorophyll b (ppm)

Pearson Correlation 0.064 -0.035 0.115 -0.311 -0.166 -0.335 -0.609 0.773* 0.037 1 0.013

Sig. (1-tailed) 0.430 0.461 0.376 0.191 0.324 0.172 0.019 0.017 0.459 0.486

Carotenoids (ppm)

Pearson Correlation 0.456 0.575* 0.214 -0.165 0.714

* 0.721

** 0.702

* 0.896

** 0.946

** 0.013 1

Sig. (1-tailed) 0.093 0.041 0.277 0.325 0.010 0.009 0.012 0.000 0.000 0.486


CHAPTER # 3 RESULTS

79

3.3.2 Cannabis sativa plant

3.3.2.1 Effects of molybdenum on plant growth parameters (length, biomass and water

contents) under cadmium stress

The effect of molybdenum on plant length, biomass and water content in Cannabis sativa

grown in 25, 50 and 100 ppm cadmium contaminated soil are given in Tables 3.20, 3.21 and

3.22. In table 3.20 and figure 3.4 X, the control C (without Cd and Mo) is compared with C1

(25 ppm Cd), C2 (50 ppm Cd) and C3 (100 ppm) for the effect of Cd on plant growth and

biomass. The table also compare C1 with treatments T1 – T9 for the molybdenum effect on

plant growth under 25 ppm cadmium contaminated soil. It was found that application of Mo

in the form of foliar spray and seed soaking significantly increased plant length, biomass and

water content in Cannabis sativa plant (Table 3.20). Highest significant increase in roots

length and biomass was demonstrated by 1.0 ppm Mo seed soaking treatment (T2) as

compared to C1. Stem and leaves showed highest significant increase in length and biomass

with 1.00 ppm (T8) and 2.00 ppm (T9) Mo foliar spray respectively (Table 3.20).

The effect of molybdenum treatments (T10-T18) on growth parameters of Cannabis sativa

plants in 50 ppm Cd contaminated soil is given in Table 3.21 and figure 3.4Y. Foliar spray of

1 and 2 ppm molybdenum significantly increased plant growth parameters, especially

biomasses (fresh and dry) in all parts of the plant as compared to C2 (50 ppm Cd into soil,

without Mo treatment). Biomass (fresh and dry) in stem, leaves and entire plant increased

most significantly in the treatment T18 (2.00 ppm Mo foliar spray) as given in table 2.21.

The table 3.22 shows the effect of molybdenum treatments on growth parameter of Ricinus

communis plants grown in 100 ppm Cd contaminated soil. Roots and stem lengths were

increased most significantly with 2.00 ppm Mo foliar spray (T27) as compared to C3 (100

ppm Cd added to soil, without Mo treatment) as given in table 3.22 and figure 3.4Z. Highest

biomass (fresh and dry) in roots were recorded with the treatment T21 (2.00 ppm Mo seed

soaking) while the stem and leaves showed highest biomass with the treatment T27 (2 ppm

Mo foliar spray) as shown in table 3.22.

CHAPTER # 3 RESULTS

80

Table 3. 20: Effect of molybdenum (Mo) treatments on length, biomass and water content in different parts of Cannabis sativa plant in soil contaminated with 25

ppm Cd. C1 (25 ppm Cd only) was used as control for the treatments (T1 – T9). Table also compares C1 (25 ppm Cd), C2 (50 ppm Cd) and C3 (100 ppm Cd) with

each other and also with control C (without Cd and Mo). Note: ‗SD‘ stands for ‗standard deviation‘ and the different alphabets in superscript represent significant

difference between the values within a single column.

Treatments Length (cm) ±

SD

FW (g) ± SD DW (g) ± SD TWC (g) ± SD

Root Stem Root Stem Leaves Entire

Plant

Root Stem Leaves Entire

Plant


Plant

C Control (without Cd

and Mo)

12.50 ±

1.00 bc

31.25 ±

3.13 abc

4.75 ±

0.38 ab

8.74 ±

0.79 cde

9.77 ±

0.78 bc

23.26 ±

1.86 bcd

1.83 ±

0.15 a

2.34 ±

0.19 cde

2.93 ±

0.23 cd

7.10 ±

0.57 bcde

2.93 ±

0.23 ab

6.40 ±

0.51 cd

6.84 ±

0.55 ab

16.16 ±

1.29 bcd

C1 Control (with Cd 25

ppm)

10.00 ±

0.80 cde

24.32 ±

2.43 cde

3.12 ±

0.28 fg

6.12 ±

0.49 fg

6.99 ±

0.63 de

16.24 ±

1.46 fgh

1.22 ±

0.11 def

1.64 ±

0.15 fg

1.89 ±

0.15 fgh

4.75 ±

0.38 gh

1.90 ±

0.15 de

4.48 ±

0.36 ef

5.10 ±

0.41 cde

11.49 ±

0.92 fgh


ppm)

8.12 ±

0.65 de

20.30 ±

2.03 de

2.65 ±

0.21 gh

5.20 ±

0.52 g

5.84 ±

0.58 e

13.69 ±

1.09 gh

1.02 ±

0.08 fg

1.60 ±

0.14 fg

1.58 ±

0.13 gh

4.19 ±

0.34 gh

1.64 ±

0.13 ef

3.60 ±

0.29 f

4.26 ±

0.34 de

9.50 ±

0.76 gh


ppm)

6.81 ±

0.54 e

17.02 ±

1.70 e

2.04 ±

0.14 h

4.34 ±

0.35 g

4.89 ±

0.39 e

11.28 ±

1.13 h

0.85 ±

0.09 g

1.23 ±

0.10 g

1.32 ±

0.11 h

3.40 ±

0.27 h

1.19 ±

0.10 f

3.11 ±

0.25 f

3.57 ±

0.29 e

7.87 ±

0.63 h

T1 Cd 25 ppm+ Mo 0.5

ppm (Seed soaking)

12.25 ±

1.23 bc

27.23 ±

2.45 cd

3.68 ±

0.40 cdef

9.87 ±

1.09 cd

8.71 ±

0.78 cd

22.26 ±

1.78 cde

1.36 ±

0.14 cdef

3.12 ±

0.25 ab

2.70 ±

0.22 de

7.18 ±

0.57 bcd

2.31 ±

0.19 cd

6.75 ±

0.61 cd

6.01 ±

0.54 bc

15.08 ±

1.21 cdef


ppm (Seed soaking)

17.50 ±

1.75 a

35.00 ±

3.15 ab

5.00 ±

0.45 a

10.45 ±

0.84 bc

11.20 ±

0.78 ab

26.65 ±

3.20 abc

1.75 ±

0.12 ab

2.71 ±

0.30 bc

3.47 ±

0.28 bc

7.93 ±

0.63 abc

3.25 ±

0.26 a

7.74 ±

0.62 bc

7.73 ±

0.62 a

18.72 ±

1.50 abc


ppm (Seed soaking)

11.20 ±

1.12 bcd

28.00 ±

2.52 bc

4.35 ±

0.44 abcd

7.99 ±

0.80 def

8.96 ±

0.99 bcd

21.30 ±

1.92 cdef

1.57 ±

0.13 abcd

2.42 ±

0.19 cd

3.50 ±

0.28 bc

7.49 ±

0.60 abc

2.78 ±

0.22 abc

5.57 ±

0.45 de

5.46 ±

0.44 bcd

13.81 ±

1.10 def


ppm (Soil addition)

10.52 ±

1.05 cd

26.31 ±

2.37 cd

3.87 ±

0.31 bcdef

5.00 ±

0.45 g

8.22 ±

0.74 cd

17.09 ±

1.20 def

1.60 ±

0.14 abc

1.67 ±

0.13 fg

2.14 ±

0.17 efg

5.41 ±

0.43 fg

2.27 ±

0.18 cd

3.33 ±

0.27 f

6.08 ±

0.49 bc

11.68 ±

0.93 efg


ppm (Soil addition)

11.20 ±

1.01 bcd

29.45 ±

2.36 abc

3.52 ±

0.28 defg

6.00 ±

0.36 fg

9.20 ±

0.74 bcd

18.72 ±

1.50 defg

1.40 ±

0.11 bcde

1.72 ±

0.15 efg

2.39 ±

0.22 def

5.51 ±

0.50 efg

2.12 ±

0.19 de

4.28 ±

0.34 ef

6.81 ±

0.68 ab

13.21 ±

1.06 def


ppm (Soil addition)

11.50 ±

1.04 bc

31.00 ±

2.48 abc

3.44 ±

0.31 defg

6.53 ±

0.72 efg

8.54 ±

0.68 cd

18.51 ±

1.48 defg

1.09 ±

0.09 efg

1.98 ±

0.16 def

2.65 ±

0.21 de

5.72 ±

0.46 defg

2.35 ±

0.19 bcd

4.55 ±

0.36 ef

5.89 ±

0.47 bc

12.79 ±

1.02 defg


ppm (Foliar spray)

10.90 ±

0.98 bcd

25.94 ±

2.16 cd

3.31 ±

0.23 efg

9.68 ±

0.77 cd

8.97 ±

0.90 bcd

21.96 ±

1.98 cde

1.25 ±

0.11 cdef

2.42 ±

0.22 cd

2.96 ±

0.27 bcd

6.62 ±

0.60 cdef

2.06 ±

0.19 de

7.26 ±

0.65 bc

6.01 ±

0.54 bc

15.33 ±

1.38 cde


ppm (Foliar spray)

13.20 ±

1.19 bc

36.00 ±

2.88 a

4.26 ±

0.38 abcde

12.25 ±

1.10 ab

12.15 ±

1.09 a

28.66 ±

2.58 ab

1.40 ±

0.13 bcde

3.42 ±

0.27 a

4.25 ±

0.34 a

9.07 ±

0.73 a

2.86 ±

0.23 abc

8.83 ±

0.71 b

7.90 ±

0.63 a

19.59 ±

1.57 ab


ppm (Foliar spray)

14.00 ±

1.26 b

36.59 ±

2.93 a

4.56 ±

0.36 abc

14.24 ±

1.14 a

10.46 ±

0.84 abc

29.26 ±

2.34 a

1.43 ±

0.14 bcde

3.56 ±

0.36 a

3.66 ±

0.37 ab

8.65 ±

0.86 ab

3.13 ±

0.31 a

10.68 ±

1.07 a

6.80 ±

0.68 ab

20.61 ±

2.06 a

CHAPTER # 3 RESULTS

81

Table 3. 21: Effects of various Mo concentrations (0.5, 1.0 and 2.0 ppm applied in different ways) on length, biomass and water content within Cannabis sativa

plant grown in 50 ppm Cd contaminated soil. Note: ‗SD‘ stands for ‗standard deviation‘ and the different alphabets in superscript represent significant difference

between the values within a single column.


SD


Root Stem Root Stem Leaves Entire

Plant


Plant

Root Stem Leaves Entire Plant


ppm) 8.12 ±

0.97 c

20.30 ±

2.44 d

2.65 ±

0.32 d

5.20 ±

0.62 d

5.84 ±

0.70 b

13.69 ±

1.64 c

1.02 ±

0.12 b

1.60 ±

0.19 c

1.58 ±

0.19 e

4.19 ±

0.50 d

1.64 ±

0.20 c

3.60 ±

0.43 e

4.26 ±

0.51 cd

9.50 ±

1.14 c

T10 Cd 50 ppm+ Mo 0.5

ppm (Seed soaking) 10.20 ±

0.92 bc

24.00 ±

2.16 bcd

2.75 ±

0.25 cd

9.08 ±

0.82 bc

8.00 ±

0.72 ab

18.20 ±

1.64 abc

1.12 ±

0.10 b

2.87 ±

0.26 ab

2.54 ±

0.23 bcd

6.53 ±

0.59 abc

1.63 ±

0.15 c

6.21 ±

0.56 bcd

5.46 ±

0.49 ab

13.30 ±

1.20 abc

T11 Cd 50 ppm+ Mo 1.0


2.06 a

27.00 ±

3.24 abcd

4.60 ±

0.55 a

8.56 ±

1.03 bc

7.25 ±

0.87 ab

17.26 ±

2.07 bc

1.45 ±

0.17 ab

2.00 ±

0.24 c

2.78 ±

0.33 abc

6.23 ±

0.75 bcd

3.15 ±

0.38 a

6.56 ±

0.79 bcd

4.47 ±

0.54 cd

14.18 ±

1.70 abc

T12 Cd 50 ppm+ Mo 0.5

ppm (Soil addition) 15.20 ±

1.99 a

22.00 ±

3.08 cd

4.75 ±

0.67 a

6.24 ±

0.87 cd

6.20 ±

0.87 b

13.85 ±

1.94 c

1.41 ±

0.20 ab

1.78 ±

0.25 c

3.01 ±

0.42 ab

6.20 ±

0.87 bcd

3.34 ±

0.47 a

4.46 ±

0.62 cde

3.19 ±

0.45 d

10.99 ±

1.54 c

T13 Cd 50 ppm+ Mo 0.5


1.02 c

24.21 ±

2.90 bcd

3.14 ±

0.38 cd

4.75 ±

0.57 d

7.00 ±

0.84 ab

13.00 ±

1.56 c

1.25 ±

0.15 b

1.54 ±

0.18 c

1.67 ±

0.20 de

4.46 ±

0.53 cd

1.89 ±

0.23 bc

3.21 ±

0.39 e

5.33 ±

0.64 ab

10.43 ±

1.25 c

T14 Cd 50 ppm+ Mo 1.0


1.38 bc

27.09 ±

3.52 abcd

3.48 ±

0.45 abcd

5.00 ±

0.65 d

7.91 ±

1.03 ab

14.29 ±

1.86 c

1.38 ±

0.18 b

1.58 ±

0.21 c

1.58 ±

0.21 e

4.54 ±

0.59 cd

2.10 ±

0.27 bc

3.42 ±

0.44 e

6.33 ±

0.82 a

11.85 ±

1.54 bc

T15 Cd 50 ppm+ Mo 2


1.11 c

32.00 ±

3.84 abc

3.32 ±

0.40 bcd

6.12 ±

0.73 cd

7.34 ±

0.88 ab

14.58 ±

1.75 bc

1.12 ±

0.13 b

1.82 ±

0.22 c

2.65 ±

0.32 abc

5.59 ±

0.67 bcd

2.20 ±

0.26 bc

4.30 ±

0.52 de

4.69 ±

0.56 abc

11.19 ±

1.34 bc

T16 Cd 50 ppm+ Mo 0.5

ppm (Foliar spray) 10.36 ±

1.55 bc

24.79 ±

3.72 bcd

2.85 ±

0.43 cd

8.91 ±

1.34 bc

6.40 ±

0.96 b

16.42 ±

2.46 bc

1.11 ±

0.17 b

2.21 ±

0.33 bc

2.02 ±

0.30 cde

5.34 ±

0.80 bcd

1.74 ±

0.26 c

6.70 ±

1.00 bc

4.38 ±

0.66 cd

12.82 ±

1.92 bc

T17 Cd 50 ppm+ Mo

1.0ppm (Foliar spray) 12.54 ±

2.01 bc

33.12 ±

5.30 ab

4.05 ±

0.65 abc

11.27 ±

1.80 ab

8.20 ±

1.31 ab

20.75 ±

3.32 ab

1.28 ±

0.20 b

3.15 ±

0.50 a

3.02 ±

0.48 ab

7.45 ±

1.19 ab

2.77 ±

0.44 ab

8.12 ±

1.30 ab

5.18 ±

0.83 ab

16.07 ±

2.57 ab

T18 Cd 50 ppm+ Mo 2.0


1.71 ab

37.20 ±

4.46 a

4.10 ±

0.49 abc

13.10 ±

1.57 a

9.23 ±

1.11 a

24.20 ±

2.90 a

1.87 ±

0.22 a

3.28 ±

0.39 a

3.54 ±

0.42 a

8.69 ±

1.04 a

2.23 ±

0.27 bc

9.83 ±

1.18 a

5.69 ±

0.68 ab

17.74 ±

2.13 a

CHAPTER # 3 RESULTS

82

Table 3. 22: Effect of Mo (0.5, 1.0 and 2.0 ppm) on growth parameters (length, biomass and water content) in Cannabis sativa plant grown in 100 ppm Cd

contaminated soil Note: ‗SD‘ stands for ‗standard deviation‘ and the different alphabets in superscript represent significant difference between the values within a

single column.


SD


Stem Root Stem Leaves Entire

Plant


Plant


Plant

Entire

Plant


ppm) 6.81 ±

0.82 b

17.02 ±

2.04 b

2.04 ±

0.25 d

4.34 ±

0.52 cd

4.89 ±

0.59 cd

11.28 ±

1.35 c

0.85 ±

0.10 b

1.23 ±

0.15 d

1.32 ±

0.16 c

3.40 ±

0.41 d

1.19 ±

0.14 c

3.11 ±

0.37 bc

3.57 ±

0.43 bcd

7.87 ±

0.94 b

T19 Cd 100 ppm+ Mo 0.5


0.89 ab

18.54 ±

1.67 b

2.97 ±

0.27 bcd

5.45 ±

0.49 abcd

6.21 ±

0.56 bcd

14.63 ±

1.32 abc

1.10 ±

0.10 ab

1.61 ±

0.14 abcd

1.78 ±

0.16 bc

4.49 ±

0.40 abcd

1.87 ±

0.17 bc

3.84 ±

0.35 abc

4.43 ±

0.40 abc

10.14 ±

0.91 ab

T20 Cd 100 ppm+ Mo 1.0


1.32 ab

20.24 ±

2.43 ab

4.04 ±

0.48 ab

6.75 ±

0.81 a

6.87 ±

0.82 abc

17.66 ±

2.12 ab

1.25 ±

0.15 ab

1.87 ±

0.22 abcd

2.24 ±

0.27 ab

5.36 ±

0.64 abc

2.79 ±

0.33 a

4.88 ±

0.59 a

4.63 ±

0.56 ab

12.30 ±

1.48 a

T21 Cd 100 ppm+ Mo 2


1.80 a

22.14 ±

3.10 ab

4.32 ±

0.60 a

6.34 ±

0.89 abc

5.23 ±

0.73 bcd

15.89 ±

2.22 abc

1.38 ±

0.19 a

2.02 ±

0.28 ab

2.26 ±

0.32 ab

5.66 ±

0.79 ab

2.94 ±

0.41 a

4.32 ±

0.60 ab

2.97 ±

0.42 d

10.23 ±

1.43 ab

T22 Cd 100 ppm+ Mo 0.5


0.87 b

17.23 ±

2.07 b

3.13 ±

0.38 abcd

3.77 ±

0.45 d

4.45 ±

0.53 d

11.35 ±

1.36 c

0.95 ±

0.11 ab

1.26 ±

0.15 cd

1.45 ±

0.17 c

3.66 ±

0.44 cd

2.18 ±

0.26 ab

2.51 ±

0.30 c

3.00 ±

0.36 d

7.69 ±

0.92 b

T23 Cd 100 ppm+ Mo 1.0


1.11 ab

22.22 ±

2.89 ab

2.84 ±

0.37 bcd

4.53 ±

0.59 bcd

5.94 ±

0.77 bcd

13.30 ±

1.73 bc

1.08 ±

0.14 ab

1.42 ±

0.18 bcd

1.48 ±

0.19 bc

3.98 ±

0.52 bcd

1.76 ±

0.23 bc

3.11 ±

0.40 bc

4.46 ±

0.58 abc

9.32 ±

1.21 ab

T24 Cd 100 ppm+ Mo 2


1.11 ab

20.00 ±

3.24 ab

2.78 ±

0.33 bcd

5.45 ±

0.65 abcd

5.51 ±

0.66 bcd

13.74 ±

1.65 abc

1.10 ±

0.13 ab

1.49 ±

0.18 abcd

1.71 ±

0.21 bc

4.30 ±

0.52 abcd

1.68 ±

0.20 bc

3.96 ±

0.47 abc

3.80 ±

0.46 bcd

9.43 ±

1.13 ab

T25 Cd 100 ppm+ Mo 0.5


1.13 b

23.15 ±

3.47 ab

2.67 ±

0.40 cd

7.00 ±

1.05 a

4.87 ±

0.73 cd

14.54 ±

2.18 abc

1.01 ±

0.15 ab

2.10 ±

0.32 a

1.65 ±

0.25 bc

4.76 ±

0.71 abcd

1.66 ±

0.25 bc

4.90 ±

0.74 a

3.22 ±

0.48 cd

9.78 ±

1.47 ab

T26 Cd 100 ppm+ Mo

1.0ppm (Foliar spray) 9.21 ±

1.47 ab

23.12 ±

3.54 ab

3.44 ±

0.55 abc

7.01 ±

0.96 a

7.84 ±

1.25 a

17.29 ±

2.77 a

1.13 ±

0.18 ab

1.78 ±

0.28 abc

2.74 ±

0.44 a

5.65 ±

0.90 a

2.31 ±

0.37 ab

4.23 ±

0.68 ab

5.09 ±

0.82 a

11.63 ±

1.86 a

T27 Cd 100 ppm+ Mo 2.0


1.47 a

27.12 ±

2.41 a

3.68 ±

0.44 abc

5.87 ±

0.70 abcd

6.75 ±

0.81 ab

16.30 ±

1.96 abc

1.25 ±

0.15 ab

1.87 ±

0.22 abcd

2.86 ±

0.34 a

5.98 ±

0.72 ab

2.43 ±

0.29 ab

4.00 ±

0.48 ab

3.89 ±

0.47 abcd

10.32 ±

1.24 ab

CHAPTER # 3 RESULTS

83

Figure 3. 4: Effect of Mo treatments on growth of Cannabis sativa plant under Cd stress. In

figure X, the control C (without Cd and Mo) was compared with C1 (25 ppm Cd), C2 (50

ppm Cd) and C3 (100 ppm Cd) while the treatments T1 –T9 were compared with C2. In

figure Y, the treatments T10 – T18 are compared with C2 while in figure Z, the treatments

T19 –T27 are compared with C3.

X

Y

Z

CHAPTER # 3 RESULTS

84

3.2.2 Effect of different concentrations of Mo on free proline, total phenolics, chlorophyll and

carotenoids contents in Cannabis sativa plant

The effect of Mo treatments on concentration of free proline, total phenolics, chlorophyll and

carotenoids in Cannabis sativa plants grown in 25, 50 and 100 ppm Cd contaminated soil is

given in table 3.23, 3.24 and 3.25. The table 2.23 compare C (without Cd and Mo) with C1

(25 ppm Cd only), C2 (50 ppm Cd only) and C3 (100 ppm Cd only) for the effect on Cd on

free proline, total phenolics and chlorophyll pigments in the plant. In the same table (3.23)

treatments (T1 – T9) are compared with C1 for the effect of molybdenum treatments on the

above biochemical parameters. It was found that free proline concentration in roots and

leaves increased significantly in C1, C2 and C3 as compared to C (Table 2.23). Molybdenum

concentration of 1.00 ppm as seed soaking (T2) and foliar spray (T8) highly increased free

proline in roots and leaves respectively. All the treatments increased phenolics concentration

in roots and leaves of the plant as compared to C1. Highest significant concentration of total

phenolics in roots and leaves were noted in 0.5 ppm Mo application as seed soaking (T1) and

foliar spray (T7) respectively. Chlorophyll and carotenoids concentration decreased in leaves

with increasing concentration of Cd in soil i.e. in the order of C (without Cd) > C1 (25 ppm

Cd in soil) > C2 (50 ppm Cd in soil) > C3 (100 ppm Cd in soil). The photosynthetic pigments

increased in the treatments (T1-T9) as compared to C1 (Table 3.23). Highest significant

increase in chlorophyll and carotenoid contents were recorded in the treatment T9 (25 ppm

Cd + 2.0 ppm Mo foliar spray) as given in Table 3.23.

The effects of Mo treatments (T10-T18) on concentration of free proline, total phenolics and

photosynthetic pigments in Cannabis sativa plants grown in 50 ppm Cd contaminated soil are

presented in Table 3.24. Highest significant increase in free proline and total phenolics

concentrations in roots were found in 1.00 ppm Mo as seed soaking (T11) and foliar spray

(T7) treatments respectively. Total phenolics and free proline concentrations in leaves were

highly increased by foliar application of 0.5 ppm (T16) and 2.00 ppm (T18) Mo respectively

(Table 3.24). Highest increase in concentration chlorophyll a, b and carotenoids was recorded

in the treatment T18 (2.00 ppm Mo foliar spray) as compared to C2 (Table 324).

Effect of molybdenum treatments (T19 – T27) on the concentration of free proline, total

phenolics and photosynthetic pigments in Cannabis sativa plant grown in 100 ppm Cd

contaminated soil is given in Table 3.25. Highest significant increase in concentration of free

proline and total phenolics in roots were recorded in the treatment T20 (2.00 ppm Mo seed

CHAPTER # 3 RESULTS

85

soaking). Concentration of free proline and total phenolics in leaves were highly increased in

with foliar spray of 1.00 ppm (T26) and 2.00 ppm (T27) Mo respectively (Table 3.25). It was

also noted that free proline concentration in roots was higher than leaves of the Cannabis

plant in all the treatments and controls given Tables 3.25. Highest significant increase in

concentration of chlorophyll ‗b‘ was recorded in the treatment T19 (0.5 ppm Mo seed

soaking). The chlorophyll a and total chlorophyll (a + b) concentration were increased

significantly with the treatment T27 (2 ppm Mo foliar spray). Carotenoids concentration in

leaves were significantly increased by application of Mo in the form of soil addition and

foliar spray (i.e. treatments T23 to T27) as compared to the control C3 (Table 3.25).

Overall effect of Mo treatments on free proline and total phenolics in Cannabis sativa under

different concentrations (25, 50 and 100 ppm) of cadmium in soil is given in Figure 3.5. It

was noted that free proline concentration increased in plant as the soil Cd concentration

increased from 25 - 50 ppm and then decreased when soil Cd concentration reached 100 ppm.

Concentration of total phenolics in plant tissues was found to increased rapidly by increasing

Cd concentration in soil from 25 ppm to 50 ppm and beyond 50 ppm Cd (in soil) the increase

in phenolics concentration became very slow (Figure 3.5).

CHAPTER # 3 RESULTS

86

Table 3. 23: Effect of Mo treatments on free proline, phenolic compounds, chlorophyll (a, b) and carotenoids concentration in Cannabis sativa plant grown

in soil contaminated with 25 ppm Cd. C1 (25 ppm Cd only) was used as control for the treatments (T1 – T9). The C1 (25 ppm Cd), C2 (50 ppm Cd) and C3

(100 ppm Cd) are compared with C (without Cd). Note: ‗SD‘ stands for ‗standard deviation‘ and different alphabets in superscript represent significant

difference between the values within a single column.

Treatments Free proline (ppm) ± SD Total Phenolics (ppm) ± SD Chlorophyll contents (ppm) ± SD Carotenoids

(ppm) ± SD Root Leaves Root Leaves A b total

chlorophyll

C Control (without Cd and Mo) 12.65 ±

1.01 g

18.70 ±

1.66 f

10.54 ±

0.84 f

36.80 ±

2.94 e

36.61 ±

2.93 a

26.00 ±

2.08 abc

62.61 ±

5.01 a

48.00 ±

3.84 cde

C1 Control (with Cd 25 ppm) 23.00 ±

2.76 fg

25.00 ±

3.00 ef

25.23 ±

3.03 e

58.54 ±

7.02 de

25.06 ±

3.01 cd

21.00 ±

2.52 bcde

46.06 ±

5.53 cde

45.00 ±

5.40 de


2.80 ef

30.25 ±

2.42 cde

33.25 ±

2.66 de

67.74 ±

5.42 bcde

27.30 ±

2.18 bcd

20.22 ±

1.62 cde

47.52 ±

3.80 bcde

42.20 ±

3.38 de


3.62 de

32.12 ±

2.89 bcde

40.22 ±

3.62 cd

75.00 ±

6.75 bcde

22.33 ±

2.01 d

16.54 ±

1.49 e

38.87 ±

3.50 e

36.23 ±

3.26 e

T1 Cd 25 ppm+ Mo 0.5 ppm (Seed

soaking)

52.32 ±

4.19 bcd

28.00 ±

2.24 def

56.55 ±

4.52 a

76.48 ±

6.12 bcde

31.41 ±

2.51 abc

27.00 ±

2.16 ab

58.41 ±

4.67 abc

47.00 ±

3.76 cde


soaking)

67.86 ±

7.46 a

40.52 ±

4.46 ab

45.67 ±

5.02 abc

86.71 ±

9.54 abc

28.00 ±

3.08 bcd

24.65 ±

2.71 abcd

52.65 ±

5.79 abcde

48.98 ±

5.39 cde

T3 Cd 25 ppm+ Mo 2 ppm (Seed

soaking)

56.00 ±

4.48 abc

32.00 ±

2.56 bcde

50.23 ±

4.02 abc

79.01 ±

6.32 abcd

23.00 ±

1.84 d

19.56 ±

1.56 de

42.56 ±

3.40 de

52.32 ±

4.19 cd

T4 Cd 25 ppm+ Mo 0.5 ppm (Soil

addition)

45.23 ±

3.62 cde

35.73 ±

2.86 abcd

45.30 ±

3.62 abc

67.00 ±

5.36 cde

27.32 ±

2.19 bcd

21.00 ±

1.68 bcde

48.32 ±

3.87 bcde

48.24 ±

3.86 cde


addition)

54.36 ±

6.52 abc

34.23 ±

4.11 abcd

49.27 ±

5.91 abc

63.71 ±

7.65 de

33.23 ±

2.99 ab

24.61 ±

2.22 abcd

57.84 ±

5.21 abc

55.23 ±

4.97 cd

T6 Cd 25 ppm+ Mo 2 ppm (Soil

addition)

59.12 ±

5.32 ab

32.76 ±

2.62 bcde

52.23 ±

4.18 ab

61.41 ±

4.91 de

34.08 ±

2.73 ab

25.24 ±

2.02 abcd

59.33 ±

4.75 abc

70.23 ±

5.62 b

T7 Cd 25 ppm+ Mo 0.5 ppm (Foliar

spray)

48.78 ±

3.90 bcd

37.00 ±

2.96 abc

54.30 ±

4.34 ab

97.00 ±

7.76 a

31.00 ±

2.48 abc

23.12 ±

1.85 abcd

54.12 ±

4.33 abcd

60.45 ±

4.84 bc

T8 Cd 25 ppm+ Mo 1.0ppm (Foliar

spray)

55.23 ±

5.52 abc

42.00 ±

3.36 a

44.24 ±

3.54 bcd

91.77 ±

7.34 ab

35.00 ±

2.80 ab

25.93 ±

2.07 abc

60.93 ±

4.87 ab

85.30 ±

6.82 a


spray)

57.45 ±

4.60 abc

27.18 ±

2.17 def

40.14 ±

3.21 cd

79.01 ±

6.32 abcd

37.00 ±

2.96 a

27.41 ±

2.19 a

64.41 ±

5.15 a

92.30 ±

7.38 a

CHAPTER # 3 RESULTS

87


in soil contaminated with 50 ppm Cd. Note: ‗SD‘ stands for ‗standard deviation‘ and different alphabets in superscript represent significant difference


Treatments Free proline (ppm) ± SD Total Phenolics (ppm) ±

SD


Root Leaves Root Leaves a B total

chlorophyll

C2 Cd 50 ppm 35.00 ± 3.85 d

30.25 ± 3.33 c

33.25 ± 3.66 c

69.35 ± 7.45 cd

27.30 ± 2.18 ab

20.22 ± 1.62 abc

47.52 ± 3.80 abc

42.20 ± 3.38 d

T10 Mo 0.5 ppm (S.S) 54.94 ± 4.94 abc

34.16 ± 3.07 c

57.00 ± 5.13 ab

85.00 ± 7.65 abcd

28.46 ± 2.56 a

23.22 ± 2.09 ab

51.68 ± 4.65 ab

45.12 ± 3.61 cd

T11 Mo 1.0 ppm (S.S) 71.25 ± 7.84 a

49.43 ± 5.44 a

63.00 ± 6.93 ab

91.05 ± 10.02 abc

25.37 ± 2.03 ab

21.20 ± 1.70 abc

46.57 ± 3.73 abc

47.02 ± 3.76 cd

T12 Mo 2.0 ppm (S.S) 67.00 ± 5.36 ab

36.24 ± 2.90 bc

61.25 ± 4.90 ab

75.00 ± 6.00 bcd

20.84 ± 1.67 b

16.82 ± 1.35 c

37.66 ± 3.01 c

50.23 ± 4.02 cd

T13 Mo 0.5 ppm (A.S) 47.49 ± 5.22 cd

43.59 ± 4.79 ab

47.57 ± 5.23 bc

70.35 ± 7.74 cd

24.75 ± 1.98 ab

18.06 ± 1.44 bc

42.81 ± 3.42 bc

46.31 ± 3.24 cd

T14 Mo 1.0 ppm (A.S) 57.08 ± 4.57 abc

44.53 ± 3.56 ab

51.73 ± 4.14 ab

73.00 ± 5.84 bcd

30.11 ± 2.41 a

21.17 ± 1.69 abc

51.28 ± 4.10 ab

53.02 ± 3.18 cd

T15 Mo 2.0 ppm (A.S) 67.00 ± 7.37 ab

39.97 ± 4.40 abc

65.00 ± 7.15 a

64.48 ± 7.09 d

30.88 ± 2.47 a

21.71 ± 1.74 abc

52.59 ± 4.21 ab

67.42 ± 5.39 b

T16 Mo 0.5 ppm (F.S) 51.22 ± 4.10 bcd

45.14 ± 3.61 ab

57.00 ± 4.56 ab

101.85 ± 8.15 a

28.09 ± 2.25 a

19.88 ± 1.59 abc

47.97 ± 3.84 abc

58.03 ± 4.06 bc

T17 Mo 1.0 ppm (F.S) 57.99 ± 6.38 abc

51.24 ± 5.64 a

64.00 ± 7.04 ab

96.36 ± 10.60 ab

31.71 ± 2.54 a

22.30 ± 1.78 ab

54.01 ± 4.32 ab

81.89 ± 6.55 a

T18 Mo 2.0 ppm (F.S) 69.00 ± 8.28 a

40.56 ± 4.87 abc

66.00 ± 7.92 a

85.00 ± 10.20 abcd

33.52 ± 4.02 a

23.57 ± 2.83 a

57.09 ± 6.85 a

88.61 ± 6.20 a

CHAPTER # 3 RESULTS

88


in soil contaminated with 50 ppm Cd. Note: ‗SD‘ stands for ‗standard deviation‘ and different alphabets in superscript represent significant difference


Treatments Free proline (ppm) ± SD Total Phenolics (ppm)

± SD


R S R S a B total

chlorophyll

C3 Cd 100 ppm 40.20 ±

2.41 c

32.12 ±

1.93 d

40.22 ±

2.41 c

75.00 ±

4.50 bcd

22.33 ±

1.34 c

16.54 ±

0.99 c

38.87 ± 2.33 d

36.23 ± 2.90 d

T19 Mo 0.5 ppm (S.S) 46.70 ±

4.20 c

35.53 ±

3.20 cd

53.94 ±

5.66 ab

83.51 ±

7.52 bcd

29.32 ±

2.64 abc

25.45 ±

2.29 a

54.77 ± 4.93 ab

40.12 ± 3.61 cd

T20 Mo 1.0 ppm (S.S) 60.57 ±

6.66 ab

51.41 ±

5.66 ab

50.83 ±

5.59 abc

94.69 ±

10.42 ab

25.65 ±

2.05 bc

23.21 ±

1.86 ab

48.86 ± 3.91 abcd

45.02 ± 3.76 cd

T21 Mo 2.0 ppm (S.S) 68.23 ±

5.46 a

40.60 ±

3.25 bcd

65.91 ±

4.47 a

86.27 ±

6.90 abcd

27.00 ±

2.16 abc

16.82 ±

1.35 c

43.82 ± 3.51 bcd

48.23 ± 4.02 cd

T22 Mo 0.5 ppm (A.S) 40.37 ±

4.44 c

45.33 ±

4.99 abc

50.42 ±

5.55 abc

73.16 ±

8.05 bcd

25.23 ±

2.02 bc

15.87 ±

1.27 c

41.10 ± 3.29 cd

44.31 ± 3.24 cd

T23 Mo 1.0 ppm (A.S) 48.52 ±

3.88 bc

43.43 ±

3.47 abcd

54.83 ±

4.39 ab

69.57 ±

5.57 cd

26.87 ±

2.15 abc

17.15 ±

1.37 c

44.02 ± 3.52 bcd

53.02 ± 3.18 bc

T24 Mo 2.0 ppm (A.S) 52.76 ±

5.80 bc

41.57 ±

4.57 bcd

58.13 ±

6.39 b

67.06 ±

7.38 d

30.88 ±

2.47 ab

20.30 ±

1.62 bc

51.18 ± 4.09 abc

67.42 ± 5.39 b

T25 Mo 0.5 ppm (F.S) 43.54 ±

3.48 c

41.12 ±

3.29 bcd

60.44 ±

4.83 ab

100.92 ±

8.47 ab

25.00 ±

2.00 bc

15.23 ±

1.22 c

40.23 ± 3.22 cd

60.03 ± 4.06 bc

T26 Mo 1.0 ppm (F.S) 46.00 ±

5.06 bc

53.29 ±

5.86 a

49.24 ±

5.42 abc

105.21 ±

11.02 a

32.56 ±

2.60 a

22.10 ±

1.77 ab

54.66 ± 4.37 a

84.89 ± 6.55 a

T27 Mo 2.0 ppm (F.S) 41.23 ±

4.95 c

34.49 ±

4.14 bcd

44.68 ±

5.36 bc

113.27

±10.35 a

33.52 ±

4.02 a

23.14 ±

2.78 ab

56.66 ± 6.80 a

88.61 ± 6.20 a

CHAPTER # 3 RESULTS

89

Figure 3. 5: Overall effect of the molybdenum on concentration of total phenolic and free

proline Cannabis sativa plant grown in soil containing different concentrations of Cd (25, 50

and 100 ppm).

24.43

38.68

54.26 59.92

55.23

73.59

51.55

70.81

0.00

10.00

20.00

30.00

40.00

50.00

60.00

70.00

80.00

90.00

Free Proline Total phenolics

Co

nce

ntr

ario

n (

pp

m)

controls without Mo25 ppm Cd in soil + Mo50 ppm Cd in soil + Mo100 ppm Cd in soil + Mo

CHAPTER # 3 RESULTS

90

3.3.2.3 Effect of different treatments of molybdenum on cadmium uptake in Cannabis sativa

plant.

The effect of Mo on Cd concentration, accumulation, translocation and bioconcentration in

Cannabis sativa plant is given in Tables (3.26, 3.27 and 3.28). The table 3.26 shows the

effect of different concentrations of Cd in soil i.e. C1 (25 ppm Cd), C2 (50 ppm Cd) and C3

(100 ppm Cd) on uptake and accumulation of Cd in the plants. It was found that Cd

concentration and accumulation in the plants increased in the order of C1< C2< C3. In the

same table effect of molybdenum treatments (T1-T9) on Cd uptake by the plant from 25 ppm

Cd contaminated soil as compare to C1 (25 ppm Cd, without Mo) is given. Molybdenum

treatments increased cadmium contents in the plant as compared to C1. Concentration of Cd

in roots and stem of the plant were highly increased by 2 ppm Mo in the form of soil addition

(T6) and seed soaking (T3) respectively. Application of 2 ppm Mo foliar spray (T9) most

significantly increased Cd concentration in leaves as compared to C1 (Table 3.26). The

treatments (T1 – T9) increased accumulation of Cd in different parts of the plant. Highest Cd

accumulation in roots, stem and leaves of the plant were recorded in the treatments T5 (1

ppm Mo added to soil), T9 (2 ppm Mo foliar spray) and T8 (1 ppm Mo foliar spray)

respectively. Cadmium translocation from roots into aerial parts of the plant was highly

increased by the treatment T2 (1.00 ppm Mo seed soaking). The treatments of Mo

demonstrated increase in Cd-bioconcentration as compared to C1. Highest increase in Cd

bioconcentration was noted in the treatment T5 (1.00 ppm Mo into soil) as given in table

3.26.

The effect of molybdenum treatments on cadmium concentration, accumulation, translocation

and bioconcentration in Cannabis sativa plant grown in soil contaminated with 50 ppm Cd is

given in table 3.27. The treatments of molybdenum demonstrated increase in Cd

concentration in different parts of the plant as compared to C2 (50 ppm Cd added to soil).

Highest significant Cd concentrations in roots, stem and leaves were found in the treatments

T15 (2.00 ppm Mo into soil), T12 (2.00 ppm Mo as seed soaking) and T16 (0.5 ppm Mo as

foliar spray) respectively (Table 3.27). Foliar application of 2.00 ppm Mo (T18) most

significantly increased Cd accumulation in roots and stem of the plant. Treatment of 2.00

ppm Mo as seed soaking (T12) demonstrated highly significant Cd accumulation in leaves of

the plant (Table 3.27). Cadmium translocation from roots into stem and leaves were

significantly increased by the treatment T12 (2.00 ppm Mo as seed soaking). Treatments of

CHAPTER # 3 RESULTS

91

Mo (T10 – T18) have increased the Cd bioconcentration as compared to C2 while most

significantly higher bioconcentration of Cd was recorded in the treatments T12 (Table 3.27).

The effect of Mo treatments (T19 –T27) on Cd uptake in Cannabis sativa plant grown in 100

ppm Cd contaminated soil is given in table 3.28. The treatments of Mo increased

concentration of Cd in all parts of the plant as compared to C3 (100 ppm Cd in soil, without

Mo). Highest increase in roots Cd concentration was shown by 2 ppm Mo added to soil

(T24). Stem and leaves of the plant showed highest concentration of Cd in 2 ppm (T27) and

0.5 ppm Mo foliar spray (T25) respectively. Accumulation of Cd in roots of the plant was

highly increased by 2 ppm Mo added to soil (T24). Stem and leaves demonstrated highest

significant Cd accumulation with foliar sprayed of 0.5 ppm Mo (T25) and 2 ppm Mo (T27)

respectively. The treatment T27 significantly increased Cd accumulation in the entire plant as

compared C3 (100 ppm Cd in soil, without Mo), given in table 3.28.

Overall effect of Mo treatments on Cd accumulation and bioconcentration in Cannabis sativa

plant under different concentrations of Cd in soil is given in Figure 3.6. The figure showed

that Cd accumulation increased with increase in concentration of cadmium in soil while Cd

bioaccumulation decreased with increasing Cd in soil.

CHAPTER # 3 RESULTS

92

Table 3. 26: Concentration, accumulation, translocation and bioconcentration of Cd in Cannabis sativa plant under different treatments of Mo and grown in

soil polluted with 25 ppm Cd. C1 (25 ppm Cd only) was used as control for the treatments. The table also draw comparison between controls having different

concentrations of Cd i.e. C1 (25 ppm Cd), C2 (50 ppm Cd) and C3 (100 ppm Cd) and with control C (without Cd). Note: ―TF‖ represent ―Translocation

Factor‖, ―BCF‖ denote ―Bio-concentration Factor‖, ―R-S‖ denote ―Roots into Stem‖, ―R-L‖ denote ―Roots into Leaves‖, ‗SD‘ stands for ‗standard deviation‘

and different alphabets in superscript represent significant difference between the values within a single column.

Treatments Cd concentration (ppm) ± SD Cd accumulation (mg/DW) ± SD % Cd accumulation Cadmium TF ± SD Cadmium

BCF ± SD Roots Stem Leaves Roots Stem Leaves Entire

plant

Roots Stem Leaves R-S R-L


11.86 d

47.23 ±

5.20 d

76.23 ±

9.91 ab

0.12 ±

0.03 c

0.08 ±

0.02 d

0.14 ±

0.03 b

0.34 ±

0.07 d

35.18 22.71 42.11 0.48 ±

0.01 b

0.77 ±

0.01 a

2.88 ±

0.37 b


18.12 cd

75.23 ±

8.28 abc

95.00 ±

12.35 ab

0.15 ±

0.03 bc

0.12 ±

0.02 cd

0.15 ±

0.03 b

0.43 ±

0.09 cd

36.21 28.45 35.34 0.50 ±

0.01 a

0.63 ±

0.01 bc

4.04 ±

0.50 ab


24.14 abc

94.29 ±

10.37 a

121.20 ±

15.76 a

0.17 ±

0.04 bc

0.12 ±

0.02 cd

0.16 ±

0.03 b

0.45 ±

0.09 bcd

38.25 25.96 35.79 0.47 ±

0.02 b

0.60 ±

0.03 c

5.26 ±

0.68 a

T1 Cd 25 ppm+ Mo 0.5 ppm

(Seed soaking)

206.43 ±

24.77 abc

58.57 ±

6.44 bcd

90.83 ±

9.08 ab

0.28 ±

0.06 ab

0.18 ±

0.03 abc

0.25 ±

0.04 ab

0.71 ±

0.14 abcd

39.58 25.78 34.64 0.28 ±

0.01 f

0.44 ±

0.01 g

3.95 ±

0.47 ab


(Seed soaking)

160.00 ±

19.20 cd

74.42 ±

8.19 abc

103.56 ±

10.36 ab

0.28 ±

0.05 ab

0.20 ±

0.04 abc

0.36 ±

0.06 a

0.85 ±

0.16 ab

33.27 23.95 42.77 0.47 ±

0.04 b

0.65 ±

0.02 b

4.24 ±

0.48 ab

T3 Cd 25 ppm+ Mo 2 ppm

(Seed soaking)

173.57 ±

20.83 bc

80.00 ±

8.80 ab

96.67 ±

9.67 ab

0.27 ±

0.05 abc

0.19 ±

0.04 abc

0.34 ±

0.06 a

0.81 ±

0.15 abc

33.82 24.08 42.10 0.46 ±

0.02 b

0.56 ±

0.05 d

4.30 ±

0.47 ab


(Soil addition)

189.29 ±

22.71 abc

67.86 ±

7.46 bcd

118.33 ±

10.65 a

0.31 ±

0.06 ab

0.11 ±

0.02 cd

0.25 ±

0.04 ab

0.67 ±

0.13 abcd

45.20 16.94 37.86 0.36 ±

0.03 e

0.63 ±

0.02 bc

4.95 ±

0.55 a


(Soil addition)

235.71 ±

28.29 ab

62.14 ±

6.84 bcd

105.83 ±

9.53 ab

0.33 ±

0.07 a

0.11 ±

0.02 cd

0.25 ±

0.05 ab

0.69 ±

0.13 abcd

47.82 15.47 36.71 0.26 ±

0.02 g

0.45 ±

0.01 g

5.01 ±

0.51 a


addition)

246.43 ±

29.57 a

57.14 ±

6.29 cd

98.33 ±

8.85 ab

0.27 ±

0.05 abc

0.11 ±

0.02 cd

0.26 ±

0.04 ab

0.65 ±

0.12 abcd

41.78 17.61 40.61 0.23 ±

0.01 h

0.40 ±

0.02 h

4.49 ±

0.48 a


(Foliar spray)

232.14 ±

27.86 ab

64.29 ±

7.07 bcd

115.00 ±

14.03 a

0.29 ±

0.06 ab

0.16 ±

0.03 bcd

0.34 ±

0.07 a

0.79 ±

0.16 abc

36.84 19.84 43.32 0.28 ±

0.03 fg

0.50 ±

0.04 f

4.74 ±

0.56 a

T8 Cd 25 ppm+ Mo 1.0ppm

(Foliar spray)

181.43 ±

21.77 abc

69.45 ±

7.64 bcd

95.83 ±

11.69 ab

0.26 ±

0.05 abc

0.24 ±

0.05 ab

0.41 ±

0.08 a

0.90 ±

0.18 a

28.24 26.44 45.32 0.38 ±

0.03 d

0.53 ±

0.03 e

3.96 ±

0.48 ab


(Foliar spray)

172.14 ±

20.66 bc

72.86 ±

8.01 abc

120.50 ±

11.90 a

0.25 ±

0.05 abc

0.26 ±

0.05 a

0.36 ±

0.08 a

0.87 ±

0.19 a

28.51 30.10 41.39 0.42 ±

0.01 c

0.57 ±

0.02 d

3.99 ±

0.47 ab

CHAPTER # 3 RESULTS

93

Table 3. 27: Effect of Mo treatments on Cd contents in Cannabis sativa plant grown in 50 ppm Cd polluted soil. Note: ―TF‖ represent ―Translocation



Treatments Cd concentration (ppm) ± SD Cd accumulation (mg/DW) ± SD % Cd accumulation Cd TF ± SD Cd BCF

± SD Roots Stem Leaves Roots Stem Leaves Entire

plant



13.59 d

75.23 ±

9.03 b

95.00 ±

12.35 b

0.15 ±

0.03 b

0.12 ±

0.03 b

0.15 ±

0.04 b

0.43 ±

0.10 b

36.27 28.42 35.31 0.50 ±

0.02 a

0.63 ±

0.03 a

2.02 ±

0.23 b


(Seed soaking)

227.07 ±

20.44 abc

76.49 ±

9.18 b

113.13 ±

14.71 ab

0.26 ±

0.05 ab

0.22 ±

0.05 ab

0.29 ±

0.06 ab

0.77 ±

0.16 ab

33.47 28.83 37.70 0.34 ±

0.03 f

0.50 ±

0.05 de

2.33 ±

0.27 ab


(Seed soaking)

206.43 ±

18.58 abcd

85.82 ±

10.30 ab

126.62 ±

16.46 ab

0.30 ±

0.06 ab

0.17 ±

0.04 ab

0.36 ±

0.09 ab

0.83 ±

0.19 ab

36.44 20.85 42.72 0.41 ±

0.05 d

0.61 ±

0.06 ab

2.64 ±

0.30 ab

T12 Cd 50 ppm+ Mo 2 ppm

(Seed soaking)

217.86 ±

19.61 abc

108.00 ±

12.96 a

132.58 ±

17.23 ab

0.31 ±

0.07 ab

0.19 ±

0.05 ab

0.40 ±

0.11 a

0.91 ±

0.23 ab

34.25 21.39 44.36 0.49 ±

0.03 ab

0.62 ±

0.07 ab

3.02 ±

0.33 a


(Soil addition)

203.57 ±

18.32 bcd

88.62 ±

10.63 ab

125.00 ±

16.25 ab

0.26 ±

0.05 ab

0.14 ±

0.03 b

0.21 ±

0.05 ab

0.60 ±

0.14 ab

42.52 22.70 34.78 0.43 ±

0.05 cd

0.61 ±

0.02 ab

2.69 ±

0.30 ab


(Soil addition)

244.29 ±

21.99 abc

76.12 ±

9.13 b

131.81 ±

17.14 ab

0.34 ±

0.07 a

0.12 ±

0.03 b

0.21 ±

0.05 ab

0.67 ±

0.16 ab

50.70 18.07 31.23 0.31 ±

0.06 fg

0.54 ±

0.06 cd

2.90 ±

0.22 a


addition)

260.71 ±

23.46 a

72.39 ±

8.69 b

118.18 ±

15.36 ab

0.29 ±

0.06 ab

0.13 ±

0.03 b

0.32 ±

0.08 ab

0.74 ±

0.17 ab

39.68 17.88 42.43 0.28 ±

0.08 g

0.45 ±

0.03 e

2.64 ±

0.30 ab


(Foliar spray)

255.36 ±

22.98 ab

73.88 ±

8.87 b

143.23 ±

18.62 a

0.29 ±

0.07 ab

0.17 ±

0.04 b

0.29 ±

0.08 ab

0.74 ±

0.19 ab

38.57 22.17 39.25 0.29 ±

0.07 g

0.56 ±

0.05 bcd

2.76 ±

0.31 ab

T17 Cd 50 ppm+ Mo 1.0ppm

(Foliar spray)

210.71 ±

18.96 abc

78.36 ±

9.40 b

119.36 ±

15.52 ab

0.27 ±

0.07 ab

0.25 ±

0.07 ab

0.37 ±

0.10 ab

0.89 ±

0.24 ab

30.82 28.11 41.07 0.37 ±

0.03 e

0.57 ±

0.08 abc

2.35 ±

0.27 ab


(Foliar spray)

196.43 ±

17.68 cd

91.00 ±

10.92 ab

106.06 ±

13.79 ab

0.37 ±

0.08 a

0.30 ±

0.07 a

0.38 ±

0.09 ab

1.05 ±

0.24 a

35.35 28.62 36.03 0.46 ±

0.07 bc

0.54 ±

0.07 cd

2.40 ±

0.27 ab

CHAPTER # 3 RESULTS

94

Table 3. 28: Effect of Mo treatments on Cd contents in Cannabis sativa plant grown in 100 ppm Cd polluted soil. Note: ―TF‖ represent ―Translocation



Treatments Cd concentration (ppm) Cd accumulation (mg/DW) % Cd accumulation Cd TF BCF

Roots Stem Leaves Roots Leaves Entire

plant



24.12 c

94.29 ±

11.31 b

121.20 ±

15.76 b

0.23 ±

0.05 b

0.12 ±

0.03 b

0.16 ±

0.04 b

0.51 ±

0.12 b

45.30 22.98 31.71 0.35 ±

0.05 ef

0.45 ±

0.05 f

1.48 ±

0.16 b


soaking)

287.00 ±

25.83 c

167.35 ±

20.08 a

171.73 ±

22.32 ab

0.32 ±

0.06 ab

0.27 ±

0.06 ab

0.31 ±

0.07 ab

0.90 ±

0.18 ab

35.49 30.23 34.28 0.58 ±

0.02 a

0.60 ±

0.02 bcd

1.98 ±

0.22 ab


soaking)

293.00 ±

26.37 c

138.56 ±

16.63 ab

192.21 ±

24.99 a

0.37 ±

0.08 ab

0.26 ±

0.06 ab

0.43 ±

0.14 ab

1.07 ±

0.20 ab

34.75 24.53 40.72 0.47 ±

0.05 c

0.65 ±

0.03 ab

1.97 ±

0.22 ab

T21 Cd 100 ppm+ Mo 2 ppm (Seed

soaking)

324.00 ±

29.16 bc

134.21 ±

16.11 ab

182.76 ±

23.76 ab

0.45 ±

0.10 ab

0.27 ±

0.07 ab

0.42 ±

0.11 ab

1.14 ±

0.24 ab

39.61 23.96 36.44 0.41 ±

0.06 d

0.56 ±

0.04 cde

2.00 ±

0.22 ab


addition)

326.48 ±

29.38 bc

168.32 ±

20.68 a

230.23 ±

29.93 a

0.31 ±

0.07 ab

0.22 ±

0.05 ab

0.34 ±

0.09 ab

0.87 ±

0.20 ab

36.08 25.20 38.72 0.53 ±

0.04 b

0.70 ±

0.03 a

2.35 ±

0.27 a


addition)

400.40 ±

36.04 ab

136.32 ±

16.36 ab

200.09 ±

26.01 a

0.44 ±

0.10 ab

0.20 ±

0.05 ab

0.30 ±

0.08 ab

0.93 ±

0.22 ab

46.96 20.98 32.06 0.34 ±

0.07 ef

0.50 ±

0.02 ef

2.32 ±

0.25 a


addition)

425.04 ±

38.25 a

135.00 ±

16.20 ab

185.91 ±

24.17 ab

0.47 ±

0.10 a

0.20 ±

0.05 ab

0.32 ±

0.06 ab

1.00 ±

0.23 ab

47.44 20.42 32.15 0.32 ±

0.08 f

0.44 ±

0.06 f

2.29 ±

0.25 a


spray)

400.40 ±

36.04 ab

145.20 ±

17.42 ab

235.42 ±

28.26 a

0.41 ±

0.10 ab

0.31 ±

0.08 a

0.36 ±

0.10 ab

1.08 ±

0.28 ab

37.84 28.57 33.59 0.36 ±

0.06 e

0.54 ±

0.02 de

2.24 ±

0.25 a

T26 Cd 100 ppm+ Mo 1.0ppm (Foliar

spray)

312.93 ±

28.16 bc

164.08 ±

19.69 a

165.00 ±

21.45 ab

0.36 ±

0.09 ab

0.30 ±

0.08 ab

0.46 ±

0.15 a

1.11 ±

0.30 ab

32.27 26.58 41.15 0.52 ±

0.02 b

0.53 ±

0.03 e

1.94 ±

0.22 ab


spray)

296.91 ±

26.72 c

171.45 ±

19.97 a

184.33 ±

23.96 ab

0.37 ±

0.08 ab

0.31 ±

0.07 a

0.53 ±

0.13 a

1.22 ±

0.25 a

30.73 25.72 43.55 0.56 ±

0.02 ab

0.62 ±

0.02 bc

2.02 ±

0.23 ab

CHAPTER # 3 RESULTS

95

Figure 3. 6: Overall effect of the molybdenum on Cd accumulation and Cd-bioconcentration in Cannabis

sativa plant grown in soil containing different concentrations of Cd (25, 50 and 100 ppm).

0.41

4.06

0.77

4.40

0.80

2.67

1.05

2.17

0.00

0.50

1.00

1.50

2.00

2.50

3.00

3.50

4.00

4.50

5.00

Cd accumulation (mg Cd/ dry biomass of plant) Bioconcentraion

controls without Mo

25 ppm Cd in soil

50 ppm Cd in soil

100 ppm Cd in soil

CHAPTER # 3 RESULTS

96

3.3.2.4 Correlations among different parameters

The correlations among plant growth parameters, free proline, total phenolics, photosynthetic

pigments and cadmium contents in different parts (roots, stem and leaves) of Cannabis sativa are

presented in Tables (3.29 to3.37). Cadmium accumulation in roots demonstrated strong positive

correlation with free proline and total phenolics as given in Tables 3.29, 3.30 and 3.31. The same

tables also showed positive correlation of dry weight with the concentration of phenolics and proline

in roots of the plant. Positive correlations were noted between dry weight and Cd accumulation in

stem of Cannabis sativa plant (Tables 3.32, 3.33 and 3.34). Total phenolics in plant leaves

demonstrated strong positive correlation with chlorophylls and carotenoids concentrations (Tables

3.35, 3.36 and 3.37). It was noted that free proline concentration showed positive correlation with Cd

accumulation in leaves of the plant. Dry weight in leaves demonstrated strong positive correlation

with total phenolics and chlorophyll a concentrations (Tables 3.35, 3.36 and 3.37).

CHAPTER # 3 RESULTS

97

Table 3. 29: Correlations between different parameters studied in roots of Cannabis sativa plant grown in 25 ppm Cd contaminated soil and under various

treatments of Mo.

Length (cm) Fresh weight

(g)

Dry weight

(g)

Total water

content (g)

Cd concentration (ppm) Cd

accumulation

(mg/DW)

Free Proline

(ppm)

Total Phenolics

(ppm)

Length (cm) Pearson Correlation 1 0.915**

0.781**

0.921**

-0.037 0.504* 0.753

** 0.302

Sig. (1-tailed) 0.000 0.001 0.000 0.455 0.047 0.002 0.170

Fresh weight (g) Pearson Correlation 0.915**

1 0.907**

0.984**

-0.096 0.561* 0.724

** 0.300

Sig. (1-tailed) 0.001 0.000 0.000 0.384 0.029 0.004 0.172

Dry weight (g) Pearson Correlation 0.781**

0.907**

1 0.816**

-0.129 0.621* 0.567

* 0.292

Sig. (1-tailed) 0.001 0.001 0.001 0.345 0.016 0.027 0.179

Total water

content (g)


0.984**

0.816**

1 -0.076 0.504* 0.751

** 0.286

Sig. (1-tailed) 0.000 0.000 0.001 0.407 0.048 0.002 0.184

Cd concentration

(ppm)

Pearson Correlation -0.037 -0.096 -0.129 -0.076 1 0.692**

0.511* 0.823

**

Sig. (1-tailed) 0.455 0.384 0.345 0.407 0.006 0.045 0.001

Cd accumulation

(mg/DW)


* 0.621

* 0.504

* 0.692

** 1 0.773

** 0.850

**

Sig. (1-tailed) 0.047 0.029 0.016 0.048 0.006 0.002 0.000

Free Proline

(ppm)


0.724**

0.567* 0.751

** 0.511

* 0.773

** 1 0.713

**

Sig. (1-tailed) 0.002 0.004 0.027 0.002 0.045 0.002 0.005

Total Phenolics

(ppm)

Pearson Correlation 0.302 0.300 0.292 0.286 0.823**

0.850**

0.713**

1

Sig. (1-tailed) 0.170 0.172 0.179 0.184 0.001 0.000 0.005


CHAPTER # 3 RESULTS

98


treatments of Mo.

Length (cm) Fresh

weight (g)

Dry weight

(g)

Total water

content (g)

Cd

concentration

(ppm)

Cd accumulation

(mg/DW)

Free Proline

(ppm)

Total phenolics

(ppm)


0.703* 0.816

** -0.044 0.574

* 0.773

** 0.644

*

Sig. (1-tailed) 0.000 0.012 0.002 0.452 0.041 0.004 0.022


1 0.695* 0.958

** -0.029 0.588

* 0.783

** .626

*

Sig. (1-tailed) 0.000 0.013 0.000 0.468 0.037 0.004 0.027

Dry weight (g) Pearson Correlation 0.703* 0.695

* 1 0.458 -0.117 0.780

** 0.653

* 0.619*

Sig. (1-tailed) 0.012 0.013 0.091 0.374 0.004 0.020 0.012

Total water

content (g)


0.958**

0.458 1 0.011 0.414 0.706* 0.565

*

Sig. (1-tailed) 0.002 0.000 0.091 0.488 0.117 0.011 0.044

Cd

concentration

(ppm)

Pearson Correlation -0.044 -0.029 -0.117 0.011 1 0.526 0.420 0.548

Sig. (1-tailed) 0.452 0.468 0.374 0.488 0.059 0.114 0.051

Cd

accumulation

(mg/DW)


* 0.780

** 0.414 0.526 1 0.814

** 0.764

**

Sig. (1-tailed) 0.041 0.037 0.004 0.117 0.059 0.002 0.005

Free Proline

(ppm)


0.783**

0.653* 0.706

* 0.420 0.814

** 1 0.907

**

Sig. (1-tailed) 0.004 0.004 0.020 0.011 0.114 0.002 0.000

Total phenolics

(ppm)


* 0.619* 0.565

* 0.548 0.764

** 0.907

** 1

Sig. (1-tailed) 0.022 0.027 0.012 0.044 0.051 0.005 0.000



CHAPTER # 3 RESULTS

99


treatments of Mo.

Length (cm) Fresh

weight (g)

Dry weight

(g)

Total

water

content (g)

Cd

concentration

(ppm)

Cd

accumulation

(mg/DW)

Free Proline

(ppm)

Total phenolics

(ppm)

Length (cm) Pearson Correlation 1 0.860** 0.965** 0.804** -0.227 0.449 0.662* 0.086 Sig. (1-tailed) 0.001 0.000 0.003 0.264 0.097 0.018 0.407

Fresh weight (g)

Pearson Correlation 0.860** 1 0.914** 0.993** -0.229 0.403 0.692* 0.056 Sig. (1-tailed) 0.001 0.000 0.000 0.262 0.124 0.013 0.439

Dry weight (g) Pearson Correlation 0.965** 0.914** 1 0.862** -0.066 0.599* 0.763** 0.540* Sig. (1-tailed) 0.000 0.000 0.001 0.428 0.014 0.005 0.019


Pearson Correlation 0.804** 0.993** 0.862** 1 -0.268 0.335 0.650* 0.013 Sig. (1-tailed) 0.003 0.000 0.001 0.227 0.172 0.021 0.486

Cd concentration (ppm)

Pearson Correlation -0.227 -0.229 -0.066 -0.268 1 0.758** 0.084 0.549 Sig. (1-tailed) 0.264 0.262 0.428 0.227 0.006 0.409 0.050

Cd accumulation (mg/DW)

Pearson Correlation 0.449 0.403 0.599* 0.335 0.758** 1 0.582* 0.555* Sig. (1-tailed) 0.097 0.124 0.014 0.172 0.006 0.039 0.048

Free Proline (ppm)

Pearson Correlation 0.662* 0.692* 0.763** 0.650* 0.084 0.582* 1 0.332 Sig. (1-tailed) 0.018 0.013 0.005 0.021 0.409 0.039 0.174

Total phenolics (ppm)

Pearson Correlation 0.086 0.056 0.540* 0.013 0.549 0.555* 0.332 1 Sig. (1-tailed) 0.407 0.439 0.019 0.486 0.050 0.048 0.174



CHAPTER # 3 RESULTS

100

Table 3. 32: Correlations between different parameters studied in stem of Cannabis sativa plant grown in 25 ppm Cd contaminated soil and under various treatments

of Mo.

Length (cm) Fresh weight (g) Dry weight (g) Total water

content (g)

Cd concentration

(ppm)

Cd accumulation

(mg/DW)


0.797**

0.800**

-0.249 0.713**

Sig. (1-tailed) 0.001 0.001 0.001 0.217 0.005


1 0.969**

0.997**

-0.053 0.918**

Sig. (1-tailed) 0.001 0.000 0.000 0.435 0.000

Dry weight (g) Pearson Correlation 0.797**

0.969**

1 0.946**

-0.085 0.925**

Sig. (1-tailed) 0.001 0.000 0.000 0.397 0.000

Total water content

(g)


0.997**

0.946**

1 -0.042 0.904**

Sig. (1-tailed) 0.001 0.000 0.000 0.449 0.000

Cd concentration

(ppm)

Pearson Correlation -0.249 -0.053 -0.085 -0.042 1 0.283

Sig. (1-tailed) 0.217 0.435 0.397 0.449 0.187

Cd accumulation

(mg/DW)


0.918**

0.925**

0.904**

0.283 1

Sig. (1-tailed) 0.005 0.000 0.000 0.000 0.187


CHAPTER # 3 RESULTS

101

Table 3. 33: Correlations between different parameters studied in stem of Cannabis sativa plant grown in 50 ppm Cd contaminated soil and under various

treatments of Mo.


(g)

Dry weight (g) Total water

content (g)

Cd concentration

(ppm)

Cd accumulation

(mg/DW)

Length (cm) Pearson Correlation 1 0.687* 0.644

* 0.687

* -0.217 0.601

*

Sig. (1-tailed) 0.014 0.022 0.014 0.273 0.033

Fresh weight (g) Pearson Correlation 0.687* 1 0.951

** 0.996

** -0.088 0.935

**

Sig. (1-tailed) 0.014 0.000 0.000 0.405 0.000

Dry weight (g) Pearson Correlation 0.644* 0.951

** 1 0.919

** -0.153 0.956

**

Sig. (1-tailed) 0.022 0.000 0.000 0.336 0.000

Total water content (g) Pearson Correlation 0.687* 0.996

** 0.919

** 1 -0.066 0.910

**

Sig. (1-tailed) 0.014 0.000 0.000 0.428 0.000

Cd concentration (ppm) Pearson Correlation -0.217 -0.088 -0.153 -0.066 1 0.139

Sig. (1-tailed) 0.273 0.405 0.336 0.428 0.351

Cd accumulation

(mg/DW)


** 0.956

** 0.910

** 0.139 1

Sig. (1-tailed) 0.033 0.000 0.000 0.000 0.351



CHAPTER # 3 RESULTS

102

Table 3. 34: Correlations between different parameters studied in stem of Cannabis sativa plant grown in 100 ppm Cd contaminated soil and under various

treatments of Mo.


(g)

Dry weight

(g)

Total water

content (g)

Cd

concentration

(ppm)

Cd accumulation

(mg/DW)

Length (cm) Pearson Correlation 1 0.295 0.364 0.258 0.303 0.411

Sig. (1-tailed) 0.204 0.150 0.236 0.197 0.119

Fresh weight (g) Pearson Correlation 0.295 1 0.946** 0.992** 0.089 0.732**

Sig. (1-tailed) 0.204 0.000 0.000 0.403 0.008

Dry weight (g) Pearson Correlation 0.364 0.946** 1 0.897** 0.232 0.845**

Sig. (1-tailed) 0.150 0.000 0.000 0.260 0.001

Total water content

(g)

Pearson Correlation 0.258 0.992** 0.897** 1 0.030 0.666*

Sig. (1-tailed) 0.236 0.000 0.000 0.467 0.018

Cd concentration

(ppm)

Pearson Correlation 0.303 0.089 0.232 0.030 1 0.707*

Sig. (1-tailed) 0.197 0.403 0.260 0.467 0.011

Cd accumulation

(mg/DW)

Pearson Correlation 0.411 0.732** 0.845** 0.666* 0.707* 1

Sig. (1-tailed) 0.119 0.008 0.001 0.018 0.011



CHAPTER # 3 RESULTS

103

Table 3. 35: Correlations between different parameters studied in leaves of Cannabis sativa plant grown in 25 ppm Cd contaminated soil and under various

treatments of Mo.

Length

(cm)

Fresh

weight (g)

Dry weight

(g)

Total

water

content (g)

Cd

concentration

(ppm)

Cd

accumulation

(mg/DW)

Free

Proline

(ppm)

Total

phenolics

(ppm)

Chlorophyll

a (ppm)

Chlorophyll

b (ppm)

Carotenoids

(ppm)


** 0.969

** -0.131 0.915

** 0.546

* 0.540

* 0.644

* 0.764

** 0.714

** 0.714

**

Sig. (1-tailed) 0.000 0.000 0.342 0.000 0.033 0.035 0.012 0.002 0.005 0.005

Fresh

weight (g)


1 0.822**

-0.172 0.956**

0.456 0.647* 0.541

* 0.649

* 0.752

** 0.752

**

Sig. (1-tailed) 0.000 0.001 0.297 0.000 0.068 0.011 0.035 0.011 0.002 0.002

Dry weight

(g)


0.822**

1 0.093 0.817**

0.568* 0.424 0.669

** 0.787

** 0.633

* 0.633

*

Sig. (1-tailed) 0.000 0.001 0.387 0.001 0.027 0.085 0.009 0.001 0.014 0.014

Total water

content (g)

Pearson Correlation -0.131 -0.172 0.093 1 0.114 0.494 0.295 -0.124 -0.317 -0.145 - 0.145

Sig. (1-tailed) 0.342 0.297 0.387 0.362 0.051 0.176 0.350 0.158 0.326 0.326 Cd

concentration

(ppm)


0.956**

0.817**

0.114 1 0.610* 0.742

** 0.517

* 0.574

* 0.710

** 0.710

**

Sig. (1-tailed) 0.000 0.000 0.001 0.362 0.018 0.003 0.042 0.025 0.005 0.005 Cd

accumulation

(mg/DW)

Pearson Correlation 0.546* 0.456 0.568

* 0.494 0.610

* 1 0.592

* 0.157 0.117 0.186 0.186

Sig. (1-tailed) 0.033 0.068 0.027 0.051 0.018 0.021 0.313 0.359 0.281 0.281

Free Proline

(ppm)


* 0.424 0.295 0.742

** 0.592

* 1 0.181 0.225 0.336 0.336

Sig. (1-tailed) 0.035 0.011 0.085 0.176 0.003 0.021 0.287 0.241 0.143 0.143 Total

phenolics

(ppm)


* 0.669

** -0.124 0.517

* 0.157 0.181 1 0.899

** 0.830

** 0.830

**

Sig. (1-tailed) 0.012 0.035 0.009 0.350 0.042 0.313 0.287 0.000 0.000 0.000

Chlorophyll a

(ppm)


0.649* 0.787

** -0.317 0.574

* 0.117 0.225 0.899

** 1 0.698

** 0.698

**

Sig. (1-tailed) 0.002 0.011 0.001 0.158 0.025 0.359 0.241 0.000 0.006 0.006

Chlorophyll b

(ppm)


0.752**

0.633* -0.145 0.710

** 0.186 0.336 0.830

** 0.698

** 1 0.940

**

Sig. (1-tailed) 0.005 0.002 0.014 0.326 0.005 0.281 0.143 0.000 0.006 0.000

Carotenoids

(ppm)


0.752**

0.633* - 0.145 0.710

** 0.186 0.336 0.830

** 0.698

** 0.940

** 1

Sig. (1-tailed) 0.005 0.002 0.014 0.326 0.005 0.281 0.143 0.000 0.006 0.000


CHAPTER # 3 RESULTS

104


treatments of Mo.

Length

(cm)

Fresh

weight (g)

Dry

weight (g)

Total water

content (g)

Cd

concentration

(ppm)

Cd

accumulation

(mg/DW)

Free

Proline

(ppm)

Total

phenolics

(ppm)

Chlorophyll

a (ppm)

Chlorophyll

b (ppm)

Carotenoids

(ppm)

Length (cm) Pearson Correlation 1 0.532 0.752** -0.195 0.366 0.349 0.241 0.734** 0.775** 0.694* 0.694*

Sig. (1-tailed) 0.057 0.006 0.294 0.149 0.162 0.251 0.008 0.004 0.013 0.013

Fresh weight

(g)

Pearson Correlation 0.532 1 -0.158 -0.109 0.929** 0.175 0.353 0.221 0.367 0.666* 0.666*

Sig. (1-tailed) 0.057 0.332 0.382 0.000 0.314 0.158 0.269 0.148 0.018 0.018

Dry weight (g) Pearson Correlation 0.752** -0.158 1 -0.143 -0.296 0.270 0.006 0.684* 0.619* 0.291 0.291

Sig. (1-tailed) 0.006 0.332 0.347 0.203 0.225 0.493 0.015 0.028 0.207 0.207

Total water

content (g)

Pearson Correlation -0.195 -0.109 -0.143 1 0.252 0.548 0.371 -0.356 -0.450 -0.129 -0.129

Sig. (1-tailed) 0.294 0.382 0.347 0.242 0.050 0.146 0.156 0.096 0.361 0.361

Cd

concentration

(ppm)

Pearson Correlation 0.366 0.929** -0.296 0.252 1 0.341 0.462 0.014 0.137 0.511 0.521

Sig. (1-tailed) 0.149 0.000 0.203 0.242 0.168 0.089 0.484 0.353 0.050 0.050

Cd

accumulation

(mg/DW)

Pearson Correlation 0.349 0.175 0.270 0.548 0.341 1 0.567* 0.213 0.121 0.390 0.390

Sig. (1-tailed) 0.162 0.314 0.225 0.050 0.168 0.044 0.277 0.370 0.133 0.133

Free Proline

(ppm)

Pearson Correlation 0.241 0.353 0.006 0.371 0.462 0.567* 1 0.183 0.290 0.319 0.319

Sig. (1-tailed) 0.251 0.158 0.493 0.146 0.089 0.044 0.306 0.208 0.184 0.184

Total phenolics

(ppm)

Pearson Correlation 0.734** 0.221 0.684* -0.356 0.014 0.213 0.183 1 0.857** 0.737** 0.737**

Sig. (1-tailed) 0.008 0.269 0.015 0.156 0.484 0.277 0.306 0.001 0.008 0.008

Chlorophyll a

(ppm)

Pearson Correlation 0.775** 0.367 0.619* -0.450 0.137 0.121 0.290 0.857** 1 0.525 0.525

Sig. (1-tailed) 0.004 0.148 0.028 0.096 0.353 0.370 0.208 0.001 0.060 0.060

Chlorophyll b

(ppm)

Pearson Correlation 0.694* 0.666* 0.291 -0.129 0.511 0.390 0.319 0.737** 0.525 1 0.900**

Sig. (1-tailed) 0.013 0.018 0.207 0.361 0.050 0.133 0.184 0.008 0.060 .000

Carotenoids

(ppm)

Pearson Correlation 0.694* 0.666* 0.291 -0.129 0.521 0.390 0.319 0.737** 0.525 0.900** 1

Sig. (1-tailed) 0.013 0.018 0.207 0.361 0.050 0.133 0.184 0.008 0.060 0.000

*. Correlation is significant at the 0.05 level (1-tailed). **. Correlation is significant at the 0.01 level (1-tailed).

CHAPTER # 3 RESULTS

105


treatments of Mo.

Length

(cm)

Fresh

weight (g)

Dry

weight (g)

Total water

content (g)

Cd

concentration

(ppm)

Cd

accumulation

(mg/DW)

Free

Proline

(ppm)

Total

phenolics

(ppm)

Chlorophyll

a (ppm)

Chlorophyll

b (ppm)

Carotenoids

(ppm)

Length (cm) Pearson Correlation 1 0.777** 0.882** -0.242 0.604* 0.449 0.398 0.687* 0.567* 0.599* 0.599*

Sig. (1-tailed)

0.004 0.000 0.250 0.032 0.097 0.127 0.014 0.044 0.034 0.034

Fresh weight

(g) Pearson Correlation 0.777** 1 0.389 -0.084 0.900** 0.287 0.528 0.739** 0.411 0.759** 0.759**

Sig. (1-tailed) 0.004

0.134 0.409 0.000 0.210 0.058 0.007 0.119 0.005 0.005

Dry weight

(g) Pearson Correlation 0.882** 0.389 1 -0.291 0.210 0.441 0.188 0.553* 0.523* 0.309 0.309

Sig. (1-tailed) 0.000 0.134

0.207 0.280 0.101 0.302 0.094 0.060 0.192 0.192

Total water

content (g) Pearson Correlation -0.242 -0.084 -0.291 1 0.352 0.383 0.089 -0.002 -0.109 0.089 0.089

Sig. (1-tailed) 0.250 0.409 0.207

0.159 0.137 0.404 0.498 0.382 0.403 0.403

Cd

concentration

(ppm)

Pearson Correlation 0.604* 0.900** 0.210 0.352 1 0.404 0.537 0.667* 0.351 0.737** 0.737**

Sig. (1-tailed) 0.032 0.000 0.280 0.159

0.124 0.055 0.018 0.160 0.008 0.008

Cd

accumulation

(mg/DW)

Pearson Correlation 0.449 0.287 0.441 0.383 0.404 1 0.351 0.121 -0.183 0.186 0.186

Sig. (1-tailed) 0.097 0.210 0.101 0.137 0.124

0.160 0.370 0.306 0.304 0.304

Free Proline

(ppm) Pearson Correlation 0.398 0.528 0.188 0.089 0.537 0.351 1 0.097 -0.036 0.282 0.282

Sig. (1-tailed) 0.127 0.058 0.302 0.404 0.055 0.160

0.395 0.461 0.215 0.215

Total

phenolics

(ppm)

Pearson Correlation 0.687* 0.739** 0.553* -0.002 0.667* 0.121 0.097 1 0.644* 0.876** 0.876**

Sig. (1-tailed) 0.014 0.007 0.094 0.498 0.018 0.370 0.395

0.022 0.000 0.000

Chlorophyll a

(ppm) Pearson Correlation 0.567* 0.411 0.523 -0.109 0.351 -0.183 -0.036 0.644* 1 0.407 0.407

Sig. (1-tailed) 0.044 0.119 0.060 0.382 0.160 0.306 0.461 0.022

0.121 0.121

Chlorophyll b

(ppm) Pearson Correlation 0.599* 0.759** 0.309 0.089 0.737** 0.186 0.282 0.876** 0.407 1 0.950**

Sig. (1-tailed) 0.034 0.005 0.192 0.403 0.008 0.304 0.215 0.000 0.121

0.000

Carotenoids

(ppm) Pearson Correlation 0.599* 0.759** 0.309 0.089 0.737** 0.186 0.282 0.876** 0.407 0.950** 1

Sig. (1-tailed) 0.034 0.005 0.192 0.403 0.008 0.304 0.215 0.000 0.121 0.000




106

3.4 DISCUSSION

The effect of molybdenum on phytoextraction potential of Ricinus communis and Cannabis

sativa plants were evaluated in the present work. Molybdenum effect was also studied on

concentration of free proline, total phenolics and photosynthetic pigments in plant tissues

under varied Cd stress.

Presence of toxic heavy metals in soil significantly reduces growth and biomass of a plant

(Hadi et al 2010; John et al 2009; Hadi and Bano 2009). In present research, Ricinus

communis and Cannabis sativa plants demonstrated significant reduction in growth and

biomass when subjected to varied concentration of Cd in soil. Heavy metals have been

reported to disturb the function of some key enzymes involved in metabolism and

consequently reduce plant growth and biomass (John et al 2009). It has been found that

nitrate absorption into roots and its translocation and assimilation in plant tissues is

negatively affected by presence of Cd in the soil and in plant tissues (Gouia et al 2000;

Hernandez et al 1996). Toxic effect of Cd on growth and biomass have been reported in other

plants i.e. Parthenium hysterophorus, Lycopersicon esculentum, Pisum sativum and Brassica

juncea (Hadi el al., 2014; Haouari et al 2012; Bavi et al 2011; John et al 2009). Our results

showed that different treatments of Mo restored the plant growth and biomass in both the

plants under cadmium stress. Seed soaking and foliar treatments of Mo most significantly

increased biomass and growth of Ricinus communis plant. Molybdenum might counter

balance the negative effect of Cd on plant growth and biomass due to its key role on nitrate

assimilation; a major nitrogen source for proteins synthesis and biomass production in plants

(Hristozkova et al 2006). Vargas and Ramirez (1989) reported that Mo application

significantly increased dry weight in cowpea and soybean pods. Acidity in soil decrease bio-

availability of Mo and consequently its uptake by plant roots, which might be the reason of

Mo deficiency in many plant species like herbs, crops and trees grown in acidic soils (Kaiser

et al 2005; Saco et al 1995). Molybdenum application through foliar sprays can effectively

supplement internal molybdenum deficiencies and rescue the activity of molybdoenzymes

(Kaiser et al 2005). Our results showed that foliar spray of Mo significantly increased

biomass in plants as compared to the treatments of molybdenum addition into soil.

Plants under stress conditions such as heavy metals, salinity and high/low temperatures

produce and accumulate high concentration of free proline in their tissues (Hadi et al 2015;

Ahmad et al 2015; Khatamipour et al 2011; Ahmad et al 2008; Sun et al 2007; Ahmad and

Jhon, 2005). Free proline accumulation in plants act as indicator for environmental stress in

http://link.springer.com/search?facet-creator=%22L.+E.+Hern%C3%A1ndez%22


107

many plant species (Khatamipour et al 2011). Several plants species such as Cannabis,

tomato, sunflower, cowpea and wheat have been found to increase concentration of free

proline in their tissues under heavy metals stress (Hadi et al 2014; Zengin and Munzuroglu,

2006; De and Mukherjee, 1998; Lalk and Dorfling, 1985). Present results demonstrated high

concentration of free proline in roots and leaves of Ricinus communis and Cannabis sativa

plants under Cd stress. Treatments of Mo further increased free proline concentration in the

plant tissues. Seed soaking with Mo increase free proline in roots while foliar spray

treatments demonstrated highly increased in free proline in leaves of the plants. Free proline

concentration in roots of both plants were found higher than leaves in all the treatments and

controls. High concentration of free proline in roots as compared to leaves in Cannabis plant

has previously been reported by Ahmad et al, (2015).

Toxic heavy metals results in the production of reactive oxygen species that in turn cause

oxidative stress in plants. To combat oxidative stress plants synthesize a variety of phenolic

compounds. These compounds possess antioxidant potential and protect cellular components

from oxidative damage caused by reactive oxygen species (Diaz et al 2001). In previous

literature, increase in concentration of phenolic compounds in plants have been reported

under Cd stress (Hadi et al 2015; Michalak, 2006; Uraguchi et al 2006). Present results

demonstrated high concentration of phenolic compounds in roots and leaves of Ricinus

communis and Cannabis sativa plants under Cd stress. It was found that treatments of Mo

further increased total phenolics in roots and leaves of plants grown in Cd contaminated soil.

Foliar application of Mo was most significant in terms of total phenolics concentration in

roots and leaves of Ricinus communis plant. Both the plants showed high concentration of

phenolic compounds in leaves as compared to roots which is in complete agreement with the

work of Ahmad et al (2015) on Cannabis sativa, Ali and Hadi, (2015) on Parthenium

hysterophorus and Uraguchi et al (2006) on Crotalaria juncea.

Due to high bioavalibility of cadmium, it can easily be absorbed by plant roots and

translocate into its aerial parts. Our results showed highest concentration of Cd in roots of

both the plant while the lowest Cd concentration was present in stem. Which means that

translocation rate of Cd from roots into leaves was higher than into stem of the plants.

Similarly, higher concentration of Cd were reported by Ahmad et al (2015), Linger et al

(2005) and Citterio et al (2003) in roots, followed by leaves and stem respectively. Metals

phytoextraction potential can be enhanced by increasing plant biomass as well as heavy metal

concentration in the biomass (Ahmad et al 2015). Molybdenum has been reported to increase


108

plant dry weight due to its role as cofactor for enzymes involved in nitrate metabolism

(Hristozkova et al 2006; Vargas and Ramirez, 1989; Kaiser et al 2005; Hadi et al 2014).

Nitrate is an important source of plants nitrogen and high availability of nitrogen results in

high biomass production in plants. Foliar spray of Mo increased plant biomass as well as

concentration of Cd within the biomass and both the plants showed high Cd accumulation in

the foliar treatments as compared to the seed soaking and soil addition treatments. Ricinus

communis and Cannabis sativa are considered as hyperaccumulator of cadmium due to its

higher Cd bioconcentration factor (greater than one) in untreated plants (only Cd added to

soil). Concentration of phenolic compounds in roots and leaves of the plants demonstrated

strong positive correlation with Cd accumulation and dry weight of the plant. Strong

correlation conform the significant role of phenolic compounds in protection of plant cells

against the toxic effects of Cd metal (Hadi et al 2015; Ahmad et al 2015; Khatamipour et al

2011; Sun et al 2007).

3.4.5 Conclusions

Ricinus communis and Cannabis sativa are good candidates for toxic metals phytoextraction

due to their high biomass and tolerance to toxic metals. Foliar spray of Mo demonstrated

significant increase in both biomass and Cd accumulation in both the plants. Phenolic

compounds in the plants leaves were highly increased by the Mo treatments, especially foliar

spray treatments. Strong correlation between dry weight, Cd accumulation and total phenolics

under different treatments of Mo was observed in the plants.

CHAPTER # 4

EXPRESSION OF CBF/DREB LIKE GENES IN RICINUS COMMUNIS AND

CANNABIS SATIVA PLANTS UNDER CADMIUM STRESS AND

MOLYBDENUM FOLIAR SPRAY.


109

CHAPTER 4: Expression of CBF/DREB like transcriptional factors genes

in Ricinus communis and Cannabis sativa plants under

cadmium stress and molybdenum foliar spray

ABSTRACT

Transcriptional factors such as CBF/DREB have been studied mostly under various abiotic

stress. Presently we investigated the expression of DREB 1A, DREB 1B, DREB 1F and CBF

like genes in Ricinus communis and Cannabis sativa plants under the cadmium (Cd) stress

and different treatments of molybdenum (Mo). Cadmium (50 ppm) was added into soil while

Mo (0.5, 1.0 and 2.0 ppm) was applied as foliar spray. The DREB 1B, DREB 1F and CBF

like genes showed expression in both the plants due to Cd exposure. Their expression was

further increased by molybdenum foliar sprayed on the plants under Cd stress. It was noted

that molybdenum alone (without Cd) have no effect on the expression of DREB1B and

DREB 1F while the CBF like gene showed expression under Mo only (without Cd). No

expression was observed in DREB 1A gene either under Cd stress or Mo application.

Application of 2.00 ppm Mo in combination with 50 ppm Cd highly increased expression of

CBF like gene in both plants. Positive correlation of DREB 1B, DREB 1F and CBF like

genes expression was found with Cd accumulation, free proline and total phenolics

concentration in both the plants.


110

4.1 INTRODUCTION

Cadmium is toxic heavy metal and its presence in soil offer stress condition to plants,

resulting in reduced growth and development. To combat the stress condition, plants respond

by the activation of several stress related genes. These genes can be categorized into two

groups; one group of genes have the direct impact on stress (i.e. they producing function

proteins such as enzymes involved in detoxification reactions, betaines, membrane

transporters and water channel proteins etc.) while the second group of genes indirectly

combat stress condition (by produce regulatory proteins that control transduction of signals

and expression of stress related genes, including several transcription factors [TF].

Transcriptional factors are important because a single TF protein can activate several related

genes at a time and thus produce a strong response to stress condition (Hadi et al 2011).

Research on TF (involved in different stresses) have received much attention in the past few

decades due to their important role in combating different environmental stresses. CBF (C-

repeat binding factors)/DREB (Dehydration response element binding protein) are important

group of transcription factors (TF) belong to ethylene responsive element binding proteins

(ERF) that follow the ABA-independent signal transduction pathway (Agarwal et al 2006).

These TFs have been studied mostly under dehydration (Thomashow 1999), low temperature

(Kume et al 2005), and salinity (Liu et al 1998; Nakashima et al 2000; Suzuki et al 2001)

stresses and have been found to improve plant tolerance to unfavorable environmental

conditions (Zarka et al 2003; Knight et al 2004). No literature is available on the role of

CBF/DREB genes in plants under heavy metals stress. Kohan and Bagherieh-Najjar (2011)

suggested that CBF/DREB transcription factors might have some role in plant defence

against heavy metals toxicity.

Micronutrients play important role in plant growth and development. Molybdenum (Mo) is a

micronutrient required by plants in a very minute quantity for normal physiology. It mainly

act as a cofactor for some important enzymes, such as Nitrate reductase (NR) and xanthine

dehydrogenase/oxidase (XDH), involved in nitrate metabolism in plants (Agarwala et al

1978; Jones et al 1976). Two different plants (Ricinus communis and Cannabis sativa) were

selected for the experiment. Ricinus communis belongs to the family Euphorbiaceous in plant

kingdom. It is a perennial shrub which reach a height of more than 3 meter and are found

mostly in the tropical regions (Rana et al 2012). Cannabis sativa is an annual herb and belong


111

to Cannabaceae family. These plants were selected due to their fast growth, high biomass and

non-palatable nature; suitable for growing in heavy metal polluted soil.

CHAPTER # 4 AIMS AND OBJECTIVES

112

4.1.1. Aim and objectives

Aim:

The aim of the chapter was to investigate the role of CBF/DREB genes in Ricinus communis

and Cannabis sativa plants for the development of phytoextraction technology.

Objectives:

1. To study the presence of DREB 1A, DREB 1B, DREB 1F and CBF like genes in

Ricinus communis and Cannabis sativa plants.

2. To investigate the effect of cadmium (Cd) on expression of DREB 1A, DREB 1B,

DREB 1F and CBF like genes in the experimental plants.

3. To find out the role of molybdenum (Mo) alone and in combination with Cd on the

expression of the DREB 1A, DREB 1B, DREB 1F and CBF like genes.

4. To study the correlation of DREB 1A, DREB 1B, DREB 1F and CBF like genes

expression with cadmium accumulation in the selected plants.

5. To find out the correlations of free proline and total phenolics with the expression of

DREB 1A, DREB 1B, DREB 1F and CBF like genes.


113


4.2.1 Plant materials and growth conditions

Viable seeds of Ricinus communis and Cannabis sativa were obtained from Herbarium

University of Malakand, Pakistan. Seeds were sown in pots containing soil (collected from

agricultural fields). Water holding capacity (300 ± 3 mL water per kg soil) and pH (6.5) of

soil was measured. Plants were grown under conditions of 30 ± 5 0C (daytime)/25 ± 4

0C

(night) and 85 ± 5 % relative humidity. Cadmium (50 ppm) was added to the respective pots

in the form of cadmium acetate dehydrate solution (CH3COO)2 Cd·2H2O (CAS # 5743-04-4,

Merck, Germany) while molybdenum (0.5, 1.0 and 2.0 ppm) in the form of ammonium

molybdate pentahydrate was applied in the form of foliar spray. Treatments of cadmium (into

soil) and molybdenum (foliar spray) were applied after 4 weeks of germination.

4.2.2 Treatments during the experiment

Three controls (C, C1 and C2) and three treatments (T1, T2 and T3) were used during the

experiment as shown in table 4.1. The control C1 (only Cd) and C2 (only Mo) are compared

with the control C (without Cd and Mo). The treatments (T1, T2 and T3) are compared with

the control C1 to find the effect of different Mo concentrations on gene expression. While the

control C2 is used to know the effect of molybdenum alone (without cadmium).

Table 4. 1: The following treatments were made during the experiment.

S/No. Treatments Symbols

1 Control without Cd and Mo C

2 Control with Cd (50 ppm) only C1

3 Control with Mo (1.00 ppm) only C2

4 Cd (50 ppm) + Mo (0.5 ppm) T1

5 Cd (50 ppm) + Mo (1.0 ppm) T2

6 Cd (50 ppm) + Mo (2.0 ppm) T3

4.2.3 Genomic DNA extraction and amplification of DREB 1A, DREB 1B, DREB 1F

and CBF like genes sequences

Fresh leaves were taken from the plants (Ricinus communis and Cannabis sativa),

immediately frozen in liquid nitrogen and grinded into powdered form using mortar and


114

pestle. Then 100 mg of grinded samples were transferred into 1.5 mL Eppendorf tubes and

Lysis buffer was added immediately to each tube to prevent DNA damage. GeneJET plant

genomic DNA purification mini kit (Cat # K0792, Thermo Scientific, Lithuania) was used for

DNA extraction according to the manufacturer protocol. Four degenerate primers (given in

table 4.2) were used for identification of four genes (DREB-1A, DREB-1B, DREB-1F and

CBF-like gene sequences) in Ricinus communis and Cannabis sativa genomes. The primers

were design using pick primer tool at NCBI website. Polymerase Chain Reactions (PCRs)

were carried out in a thermocycler (Kyratec supercycler, model # SC300, thermal cycler,

Queensland Australia) in 50 μL of solution containing 15 μL of extracted DNA, 5 μL of each

primer (forward and reverse) and 25 μL of PCR master mix (Cat # K0171,

Fermentas,Thermo). The mixture was treated at 95ºC (5 min) and subjected to 35 cycles of

amplification (denaturation at 95ºC for 1 min, primer annealing (temperature for each primer

given in table 4.1) for 1 min and polymerization at 72ºC for 2 min) with a final elongation

cycle of 5 min at 72ºC. The PCR products were run on Agarose (CAS # 9012-36-6, Bio-

Basic, Canada) gel (1 %) for 30 mins at 70 volts potential difference in horizontal Midi-Gel

systems. The DNA bands were detected using Gel documentation system.

Table 4. 2: Primers used during the experiments.

S/N

o. Name of Genes Primers

Am

plified

Pro

duct

size

Annealin

g

Tem

peratu

r

e

1 Ricinus communis Dehydration-

responsive element binding

protein 1A, putative, mRNA

Forward CGCGTGCGTAAGACTGAAAG 233

pb 55

0C Reverse AGCCACAGAGTTGGAAGGTG


responsive element-binding

protein 1B, putative, mRNA

Forward GAAATGGGAAGTGGGTCAGT 458

bp 52

0C Reverse TTTCATGGCTGGTGGAGTAA


responsive element binding

protein 1F, putative, mRNA

Forward GCCACGAGCTATCCGAAGAA 253

bp 55

0C Reverse GCAACCTCCAAGCAGAGTCA

4 Ricinus communis CBF-like

transcription factor

Forward CCAGCAAAGAAGAGGAAAGC 382

bp 51

0C

Reverse TAGAAGACGCAGACGAACAA

5 Actin gene (Housekeeping

gene)

Forward AACAGCCCTTCTTTGGTTTT 423

bp

50 0C

Reverse AGAGAGAGAGACAGAATGGT


115

4.2.4 Total RNA extraction

Total RNA extraction from leaves was done after six hour of treatments (Table 4.1). All the

apparatus and working area were cleaned with RNAase cleaning agent (Sigma Rnase ZAP

cat # R2020) to avoid RNA degradation by RNAases. Fresh leaves were taken from treated

and control plants, immediately frozen in liquid nitrogen and grinded into powdered form.

Then 100 mg of grinded tissue is immediately transferred into RNAse free 1.5 mL micro-

centrifuge (Eppendorf) tubes and 500 ml of lyses buffer was immediately added to the tubes.

Tubes were vortexed for about 30 seconds. GeneJET Plant RNA Purification Mini Kit (Cat#

K0801, Thermo Scientific) was used for total RNA extraction. Extracted RNA was stored at

- 20 0C for a short time.

4.2.5 cDNA synthesis and Identification of DREB 1A, DREB 1B, DREB 1F and CBF

like genes

First strand cDNA was synthesized using total RNA as template via Revert Aid First Strand

cDNA Synthesis Kit (Fermentas, Thermo, Cat # K1622). Reaction mixture was prepared in

PCR tubes by mixing 5 µg Total RNA (5 µL), 1 µL Oligo (dT)18 primer, 4 µL 5X reaction

Buffer, 1 µL RiboLock Rnase Inhibitor (20 U/µL), 10 µL dNTP Mix (10mM) and 1µL

RevertAid M-MuLV RT (200 U/µL). Total volume was raised up to 20 µL by adding

nuclease-free water. During addition the tubes were kept on ice to prevent degradation of

reaction components. Thermal cycler (Kyratec supercycler, model # SC300, thermal cycler,

Queensland Australia) was used for the synthesis of first strand cDNA. The samples were

incubate in the thermal cycler at 25 0C for 5 mins followed by 42

0C for 60 mins and the

reaction was terminated at 70 0C for 5 mins according to the kit manufacturer instructions.

After completing the synthesis of first strand cDNA, the next step was to amplify specific

sequences of the target genes (cDNA) using thermal cycler. For each sample 50 μL of

reaction mixture were prepared containing 15 μL of cDNA, 5 μL of each primer (forward and

reverse) and 25 μL of PCR master mix (Cat # K0171, Fermentas,Thermo). The mixture was

treated at 95ºC (5 min) and subjected to 35 cycles of amplification (denaturation at 95ºC for 1

min, primer annealing [temperatures given in table 4.2] for 1 min and polymerization at 72ºC

for 2 min) with a final elongation cycle of 5 min at 72ºC. Reverse transcriptase [RT] PCR of

Actin gene sequences were performed to test the optimization of experiments. The PCR

products were run on Agarose (CAS # 9012-36-6, Bio-Basic, Canada) gel (1 %) for 30 mins

at 70 volts potential difference. PCR products were visualized under UV light and

photographs were taken using gel documentation system. The DNA bands were compared


116

with a 100 bp DNA ladder (Thermo Scientific cat # SM0323) for size determination.

Intensities of bands were measured semi quantitatively using Quantity one 4.6.3 Bio-Rad

software. The molecular analysis took about eight (8) months.

4.2.6. Sequence analysis

The PCR product for each primer was purified using PCR purification Kit (Cat # K0702,

Thermo scientific). The purified PCR products were then analyzed for nucleotides sequence

using DNA sequencer (Applied Biosystems 3730/3730xl DNA Analyzers, USA). Some of

the purified DNA was stored at 4 0C for a short time and at -20

0C for longer storage.

Multiple sequence alignments of the deduced amino acid sequence sequences were carried

out using BLAST (NCBI).

4.2.7. Data analysis

Nucleotides sequences and deduced amino acid sequences were analyzed for percent

homology with related genes of other plants. Gene expression (semi-quantitatively) was

correlated with Cd accumulation, concentration of free proline and total phenolics using MS

excel and SPSS softwares.


117

4.2.8. Experimental Design

Seed sowing in soil and germination

Treatments of Molybdenum (foliar spray) and Cadmium (addition into soil).

Three controls (C, C1 and C2) and three combination treatments (T1, T2 and T3) were used.

Cadmium was added to soil after three and a half week of germination while molybdenum was applied after four weeks of germination.

Total RNA extraction from fresh leaves after six hours of Mo treatments and then cDNA was synthesis using RT-

PCR

Amplification of CBF/DREB like genes with specific primers from cDNA, using PCR.

Sequencing of CBF/DREB genes fragments, (PCR products)

BLAST analysis of nucleotid sequences and predicted amino acid sequences

Statistical data analysis

CHAPTER # 4 RESULTS

118

4.3. RESULTS

4.3.1 Ricinus communis

4.3.1.1 CBF/DREB Like genes sequences in Ricinus communis genomic DNA

Total genomic DNA extracted from Ricinus communis plant was intact and in good quality as

shown in figure 4.1. The PCR reactions with gene specific primers were carried out to

confirm the presence of DREB 1A, DREB 1B, DREB 1F and CBF like genes sequences in

the Ricinus communis plant. The PCR product confirmed the presence of all four gene

sequences in Ricinus communis plants (Figure 4.2).

Figure 4.1: Total DNA extracted from Ricinus communis.

Figure 4.2: PCR product of four DREB/CBF genes fragments from genomic DNA of Ricinus

communis. PCR product for each gene fragment is given in triplicate. Lane order is (1-3)

represent DREB 1A, (4-6) shows DREB 1B, (7-9) shows DREB 1F and (10-12) represent

CBF like transcription factor. Lane M contain DNA marker.

A M

CHAPTER # 4 RESULTS

119

4.3.1.2 Expression analysis of DREB 1A, DREB 1B, DREB 1F and CBF like genes

Reverse transcriptase (RT) PCR products of the actin (housekeeping) gene in Ricinus

communis (Figure 4.3) confirmed the optimization of RNA extraction kits and protocols used

during the experiments. Variation was found in expression level of DREB 1B (figure 4.4),

DREB 1F and CBF like gene (figure 4.5) in Ricinus communis plants under different

treatments of molybdenum and cadmium. The DREB-1A gene was not expressed under the

treatments (T1, T2 and T3) and controls (C, C1 and C2) as given in figure 4.4A. The DREB

1B gene showed expression in the treatments (T1, T2 and T3) and controls C1 (Cd only).

Combination treatments of Mo and Cd increased DREB 1B gene expression as compared to

the control C1 (only Cd). Highest expression of DREB 1B gene occurred under combination

treatment of 1 ppm Mo foliar spray and 50 ppm Cd added to soil (T3). The DREB 1F gene

showed very low expression in the control C1 while the gene expression was highly increased

in the treatments T2 and T3 as compared to C1 (figure 4.5A). No expression of DREB 1F

gene was observed in the control C2 (Mo only). The CBF like gene expressed in the

treatments and controls C1 and C2 (figure 4.5B). Increase in expression of CBF like gene

was found in the treatments as compared to the controls C1 and C2. It was noted that none of

the gene expressed in the control C (without Cd and Mo).

Figure 4.3: RT-PCR product of Actin gene fragment from Ricinus communis. The symbol

‗M‘ represent DNA marker.

CHAPTER # 4 RESULTS

120

A

B

Figure 4. 4: RT-PCR product of DREB 1A (A) and DREB 1B (B) genes fragments of Ricinus communis under various treatments of cadmium and

molybdenum. Three replicates are given for each gene and designated by numbers 1, 2 and 3. The letter ‗M‘ denote DNA marker, ‗C‘ stands for control

without Cd and Mo, ‗C1‘ represent control having 50 ppm Cd only, ‗C2‘ denote control having 1.00 ppm Mo only, ‗T1, T2 and T3‘ present treatments

containing 50 ppm Cd in combination with 0.5, 1.00 and 2.00 ppm Mo in each treatment respectively and ‗B‘ denote blank without template DNA.

1 2

1 2 3

3

CHAPTER # 4 RESULTS

121

A

B

Figure 4. 5: RT-PCR product of DREB 1F (A) and CBF like factor (B) genes fragments of Ricinus communis plant under various treatments of cadmium and

molybdenum. Three replicates are given for each gene and designated by numbers 1, 2 and 3. The letter ‗M‘ denote DNA marker, ‗C‘ stands for control without Cd and Mo,

‗C1‘ represent control having 50 ppm Cd only, ‗C2‘ denote control having 1.00 ppm Mo only, ‗T1, T2 and T3‘ present treatments containing 50 ppm Cd in combination

with 0.5, 1.00 and 2.00 ppm Mo in each treatment respectively and ‗B‘ denote blank without template DNA.

1 2 3

1 2 3

CHAPTER # 4 RESULTS

122

4.3.1.3 Nucleotides sequence analysis of DREB 1B, DREB 1F and CBF like genes of Ricinus

communis plant

The cDNA sequence of DREB 1B, DREB 1F and CBF like gene fragments in Ricinus

commuis and their sequence alignment with related genes from other plants are presented in

Figures 4.6, 4.7 and 4.8 respectively. Multiple sequence alignment was done using BLAST

tool. The Ricinus communis DREB 1B gene fragment showed 72% nucleotide sequence

similarity with Jatropha curcas DREB 1F gene, 76% with Theobroma cacao DREB 1B gene

(Figure 4.6). The Ricinus communis DREB 1F gene fragment demonstrated 80 % nucleotide

sequence alignments with DREB 1B (of Jatropha curcas) and CBF4 (of Populus deltiodes)

while 81 % sequence homology with CBF2 (of Populus simonii) as shown in figure 4.7. It

was noted that Ricinus communis CBF like gene fragment showed 80%, 83% and 82%

sequence homology with DREB-1A (of Jatropha carcus), DREB1F (of Populus euphratica)

and CBF6 (of Populus balsamifera) gene sequences respectively (Figure 4.8).

CHAPTER # 4 RESULTS

123

Rc DREB 1B 2 AAATGGGAAGTGGGTCAGTGAATTAAGACAACCCTACAATAATAAGTCGAGGATATGGTT 61

Jc DREB 1F 168 AAATGGGAAATGGGTGAGTGAACTGAGAGAAC—-TACAG-ACTAAGTCTCGGATATGGCT 224

Tc DREB 1B ------------------------------------------------------------

Consensus ********* ***** ****** * * * *** **** ******** **********

Rc DREB 1B 62 AGGAACATTTCCATCACCTGACATGGCCGCTAGGGCTTATGACGTAGCAGCTTTTGCATT 121

Jc DREB 1F 225 TGGAACGTTTCCAAACCCTGAAATGGCAGCGAGAGCTTATGATGTAGCTGTTAAAGCACT 284

Tc DREB 1B 336 ---------------------CATGGCTGCTAGGGCTTATGATGCAGCGGCCTTAGCTCT 396

Consensus ***** ** ** ******** * *** ** ** *

Rc DREB 1B 122 ACGAGGAGATTCTGCTTCCTTAAACTTTCCTGAATCAGTTCATTTGTTGCCTCAGGCTAG 181

Jc DREB 1F 285 TCGGGGAAATACGGCGTCATTAAACTTTCCTGAAACAGCGCATTTGTTGCCTCAAGTTGG 344

Tc DREB 1B 397 CAAGGGAGATTCTGCTTCCTTAAACTTTCCTGAGTCAGCTAATGCATTACCACGTGCTAG 456

Consensus *** ** * ** ** ******** ****** *** ** * *** ** ** * *

Rc DREB 1B 182 ATCTACTTCTATAAAGGATATTCAGTATGCAGCTCTGGAAGCTGCTGATCAGAGTGTTAg 241

Jc DREB 1F 345 GTCAACCTCTATAAAGGATATTCAATGCGCCGCATTGGAAGCTGCAG------GTGTTCA 398

Tc DREB 1B 357 GTCATCCTCCATCAGGGATATACAATATGCTGCTATGGAGGCTGCCGA------------

Consensus ** * ** ** * ****** ** * ** ** **** ***** *

Rc DREB 1B 242 tggtggtggtggtggtggtAGTGATGTTGATCATCTGTTTCAATGTTCTTCTTCTTCTCT 301

Jc DREB 1F 399 TGGTGGTGGTGGCGATG---------------------TTCAATGTGCTTCTTCTTC--- 434

Tc DREB 1B ------------------------------------------------------------

Consensus ************ * ** ******** **********

Rc DREB 1B 302 ATCTTTTTGTTCCTCGACTATAGAAGGAAGTGACAATGTTGGGAAAGATTGGAATAAGAA 361

Jc DREB 1F 435 ATCTTCGTTGAAAGCTAGTGTTGAAGAAGGTTATAATAATAATAATAATAATAATAATGA 494

Tc DREB 1B ------------------------------------------------------------

Consensus ***** * * * * * **** * ** * *** * ** ** ***** *

Rc DREB 1B 362 TATGAATATGTTTTTGGATGAAGAGGAGTTGTTTAACATGCCTGCATTACTCGATAGCAT 421

Jc DREB 1F 495 TAATAAAATGTTTCTGGATGAAGAAGAGTTGTTTAATATGCCGGCATTACTTGATAGTAT 554

Tc DREB 1B ------------------------------------------------------------

Consensus ** ** ****** ********** *********** ***** ******** ***** **

Rc DREB 1B 422 GGCAGAAGGGTTAATTCTTACTCCACCAGCCATGAAA 458

Jc DREB 1F 555 GGCAGAAGGGTTGATTCTAACACCACCAGCCATGAAA 591

Tc DREB 1B -------------------------------------

Consensus ************ ***** ** ***************

Figure 4. 6: Nucleotide sequence (Gene fragment) alignment of Ricinus communis DREB-1B with

DREB-1F of Jatropha curcas and DREB-1B of Theobroma cacao. The symbols ‗Rc‘ represent

Ricinus communis, ‗Jc‘ represent Jatropha curcas, ‗Tc‘ represent Theobroma cacao and ‗*‘ indicate

the nucleotide in that column are similar in all the sequences in alignment. The dotted line shows the

missing nucleotides in a sequence. Ricinus communis DREB-1B gene sequences showed 72 % and

76% homology with DREB-1F and DREB-1B genes sequences of Jatropha curcas and Theobroma

cacao respectively.

CHAPTER # 4 RESULTS

124

Rc DREB 1F 1 GCCACGAGCTATCCGAAGAAGCGAGCTGGGCGGAGAGTGTTCAAGGAGACTCGACATCCT 60

Jc DREB 1B 138 GCTACAAGCTACCCGAAGAAGCGAGCAGGGCGGCGAATATTTAAGGAAACACGACATCCC 197

Pd CBF4 139 GCAACAAGTTTTCCGAAAAAACGCGCTGGCCGCAGAATATTCAGGGAGACTCGGCACCCG 198

Ps CBF2 176 GCAACAAGTTTTCCGAAAAAACGCGCTGGCCGCAGAATATTCAGGGAGACTCGGCACCCG 235

Consensus ** ** ** * ****** ** ** ***** ** *** * **** ********* ** **

Rc DREB 1F 61 GTCTTTAGAGGCGTTAGGAATAGGAATAATGACAAGTGGGTCTGCGAGCTACGTGAGCCA 120

Jc DREB 1B 198 ATTTTTCGTGGAGTTCGAAAAAGAAACAATGAAAAATGGGTTTGTGAGCTTAGAGAACCC 257

Pd CBF4 199 GTTTTTAGAGGTGTTCGGAAGAGGAATGGTAACAAATGGGTGTGTGAGATGCGGGAACCA 258

Ps CBF2 236 GTTTTTAGAGGTGTTCGGAAGAGGAATGGTAACAAATGGGTGTGTGAGATGCGGGAACCA 295

Consensus * *** * ** *** * ** ** ** * * ** ***** ** *** * * ** **

Rc DREB 1F 121 AATAAGAAATCAAGAATATGGCTTGGTACTTATCCTACTCCTGAAATGGCAGCTAGAGCA 180

Jc DREB 1B 258 AATAAGAAGACACGTATATGGCTCGGTACATATCCAACACCAGAAATGGCGGCCAGAGCA 317

Pd CBF4 259 AACAAGAAGTCACGAATATGGTTAGGAACATATCCTACACCAGAAATGGCAGCTCGAGCT 318

Ps CBF2 296 AACAAGAAGTCACGAATATGGTTAGGAACATATCCTACACCAGAAATGGCAGCTCGAGCT 355

Consensus ** ***** ** * ****** * ** ** ******** ** *********** ****

Rc DREB 1F 181 CACGACGTTGCTGCATTGGCTCTTAGAGGAAAATCTGCTTGCCTTAACTTTGCTGACTCT 240

Jc DREB 1B 318 CATGACGTGGCTGCATTAGCGCTTAGAGGAAAATCGGCTTGTCTTAACTTTGCAGATTCT 377

Pd CBF4 319 CATGATGTTGCTGCTTTGGCACTTAGAGGCAAATCTGCTTGCCTTAACTTCGCTGATTCT 378

Ps CBF2 356 CATGATGTTGCTGCTTTGGCACTTAGAGGCAAATCTGCTTGCCTTAACTTCGCTGATTCT 415

Consensus ** ** ** ***** ** ** ******** ***** ***** ******** ** ** ***

Rc DREB 1F 241 GCTTGGAGGTTGC 253

Jc DREB 1B 378 TCTTGGAGGTTGC 390

Pd CBF4 379 GCCTGGAGGTTGC 391

Ps CBF2 416 GCCTGGAGGTTGC 428

Consensus * **********

Figure 4. 7: Nucleotide sequence (Gene fragment) alignment of Ricinus communis DREB-1F with

DREB-1B of Jatropha curcas, CBF4 of Popullu deltoids and CBF2 of Populus simonii. Symbols ‗Rc‘

represent Ricinus communis, ‗Jc‘ represent Jatropha curcas, ‗Pd‘ represent Populus deltoids, ‗Ps‘

represent Populus simonii and ‗*‘ indicate the nucleotide in that column are similar in all the

sequences in alignment. The dotted line shows the missing nucleotides in a sequence. Ricinus

communis DREB-1F gene fragment demonstrated 80 % nucleotide sequence homology with DREB-

1B and CBF4 gene sequences of Jatropha curcas and Populus deltoids respectively and 81%

sequence homology with CBF2 gene sequences of Populus simonii plant. Among the four plants 74%

sequence homology was found between the gene sequences.

CHAPTER # 4 RESULTS

125

Rc CBF like factor 7 AAGAAGAGGAAAGCAGGGAGGACCAAGTTCAAGGAGACTAGGCATCCGATTTATAGAGGT 66

Jc DREB 1A 67 AAAAAGAGGAAGGCAGGAAGGACTAAGTTTAAGGAAACCAGACACCCATTTTATAGAGGT 126

Pe DREB 1F 303 AAGAAGAACAAAGCGGGAAGGAAGAAGTTCAAGGAGACGCGGCATCCGGTATATAGGGGG 362

Pb CBF6 124 AAGAAGAACAAAGCGGGAAGGAAGAAGTTCAAGGAGACGAGGCATCCGGTATATAGGGGG 183

Consensus ** **** ** ** ** **** ***** ***** ** * ** ** * ***** **

Rc CBF like factor 67 GTAAGGCGAAGAAATGGGAATAAATGGGTATGTGAAGTTAGAGAACCGAACCTGAAA--- 123

Jc DREB 1A 127 GTTCGACGAAGAAATGGAAATAAATGGGTATGTGAAGTGCGAGAACCAAATAAGAACAAG 186

Pe DREB 1F 363 GTACGGAGAAGAAATGGGAATAAATGGGTATGTGAAGTGCGTGAACCAAACAAGAAATCA 422

Pb CBF6 184 GTAMGGAGAAGAAATGGGAATAAATGGGTATGTGAAGTGCGYGAACCAAACAAGAAATCA 243

Consensus ** * ********** ******************** * ***** ** ***

Rc CBF like factor 124 TCAAGAATATGGCTAGGGACATACCCTACCCCAGAAATGGCAGCTAGGGCACATGATGTT 183

Jc DREB 1A 187 TCAAGGATTTGGTTAGGTACGTTTCCTACTCCAGAAATGGCAGCTAGGGCACACGACATT 246

Pe DREB 1F 423 ---AGAATTTGGGTGGGGACCTTCAAGAGCCCAGAAATGGCAGCTAGGGCACATGATGTT 480

Pb CBF6 244 ---AGAATTTGGGTGGGGACCTTCAAGAGCCCAGAAATGGCAGCTAGGGCACATGATGTT 300

Consensus ** ** *** * ** ** * * * *********************** ** **

Rc CBF like factor 184 GCTGCTTTGGCATTCAGAGGAGAGTTTGCTTCTCTCAACTATCTTGATTCAGCTTGGATA 243

Jc DREB 1A 247 GCTGCTCTGGCATTAAGAGGAGATCTTGCTATACTAAACTTCCCTGATTCTGCTTCGGTA 306

Pe DREB 1F 481 GCTGCTTTAGCACTTAAAGGAGAGTTTGCTGCCTTGAACTTTCTTGATTCAGCTTTGATA 540

Pb CBF6 301 GCTGCTTTAGCACTTAAAGGAGAGCTTGCTGCCTTGAACTTTCTCGATTCAGCTTTGATA 360

Consensus ****** * *** * * ****** ***** * **** * ***** **** * **

Rc CBF like factor 244 CTGCCACGACCAAAGTCGTCTTCTCATGAAGATATTAAAAGGGCTGCTCTTGAAGCTGCT 303

Jc DREB 1A 307 CTTCCAAGACCGAAGTCTTCTTCTGCAAAAGATATTAAAAGGG----------------- 349

Pe DREB 1F 543 CTTCCTCGAGCAAAGTCGTCTTCTGCCGAAGATATACAAAGGGCTGCCCTTGCTGCTACA 602

Pb CBF6 364 CTTCCTCGAGCAAAGTCGTCTTCTGCTAGAGATATACAAAGGGCTGCCCTTGCTGCTACA 425

Consensus ** ** ** * ***** ****** ****** ********** **** *** *

Rc CBF like factor 304 GAGG-CTTT 311

Jc DREB 1A ---------

Pe DREB 1F 603 GAGGTCTTT 611

Pb CBF6 426 GAGG 409

Consensus ****

Figure 4. 8: Nucleotide sequence (Gene fragment) alignment of Ricinus communis CBF like gene

with DREB-1B of Jatropha curcas, DREB-1F of Populus euphratica and CBF6 of Populus

balsamifera. The symbols ‗Rc‘ represent Ricinus communis, ‗Jc‘ represent Jatropha curcas, ‗Pe‘

represent Populus euphratica, ‗Pb‘ represent Populus balsamifera and ‗*‘ indicate the nucleotide in

that column are similar in all the sequences in alignment. The dotted line shows the missing

nucleotides in a sequence. The Ricinus communis CBF like gene fragment showed 80%, 83% and

82% sequence homology with DREB-1B of Jatropha curcas, DREB-1F of Populus euphratica and

CBF6 of Populus balsamifera.

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4.3.1.4 Protein Sequence analysis of DREB 1B, DREB 1F and CBF like genes in Ricinus

communis plant

The predicted amino acid sequence of DREB 1B, DREB 1F and CBF like gene fragments of

Ricinus communis were subjected to multiple sequence alignment with the amino acid

sequences of related proteins from other plant species using BLAST tool (Figure 4.9). Amino

acid sequences of Ricinus communis DREB 1B protein showed 63 % and 60 % homology

with DREB 1F of Jatropha carcus and DREB 1B of Theobroma cocoa (Figure 4.9 A).

Predicted amino acid sequence of Ricinus communis DREB 1F showed 92% sequence

homology with DREB 1B of Jatropha carcus and CBF2 of Populus simonii while 93%

sequence homology with amino acid sequence of Populus deltiodes CBF4 protein (Figure 4.9

B). Amino acid sequence of Ricinus communis CBF like factor showed 75%, 81% and 79%

homology with DREB 1A of Jatropha carcus, DREB 1F of Populus euphratica and CBF6 of

Populus balsamifera amino acid sequences respectively (Figure 4.9 C).

CHAPTER # 4 RESULTS

127

A Rc DREB 1B 3 NGKWVSELRQPYNNKSRIWLGTFPSPDMAARAYDVAAFALRGDSASLNFPESVHLLPQAR 182

Jc DREB 1F 57 NGKWVSELRE-LQTKSRIWLGTFPNPEMAARAYDVAVKALRGNTASLNFPETAHLLPQVG 115

Tc DREB 1B 59 NGKWVSELREPIK-KSRIWLGTFSSPGMAARAYDAAALALKGDSASLNFPESANALPRAR 117

Consensus ********* ********* ** ******* * ** * ******* **

Rc DREB 1B 183 STSIKDIQYAALEAADQsvsggggggsdvdHlfqcsssslsfcssTIEGSDNVGKDWNKN 362

Jc DREB 1F 116 STSIKDIQCAALEAAG----------VHGGGGDVQCASSSSSLKASVEEGYNNNNNNNND 165

Tc DREB 1B 118 SSSIRDIQYAAMEAAEAFGDIAKTPSPSPSLSSSSSLPSPPLPSLE-NSSENVQGSSEK- 175

Consensus * ** *** ****** * *

Rc DREB 1B 363 MNMFLDEEELFNMPALLDSMAEGLILTPPAMK 458

Jc DREB 1F 166 NKMFLDEEELFNMPALLDSMAEGLILTPPAMK 197

Tc DREB 1B 176 --LFLDEEEVFNMPGILDSMAEGLILTPPAMQ 205

Consensus ****** **** ***************

B Rc DREB 1F 1 ATSYPKKRAGRRVFKETRHPVFRGVRNRNNDKWVCELREPNKKSRIWLGTYPTPEMaara 180

Jc DREB 1B 39 ATSYPKKRAGRRIFKETRHPIFRGVRKRNNEKWVCELREPNKKTRIWLGTYPTPEMAARA 98

Pd CBF4 15 ATSRPKKRAGRRIFKETRHPIFRGVRKRNGDKWVCELREPNKKSRIWLGTYPTPEMAARA 74

Ps CBF2 47 ATSFPKKRAGRRIFRETRHPVFRGVRKRNGNKWVCEMREPNKKSRIWLGTYPTPEMAARA 106

Consensus *** ******** * ***** ***** ** ***** ****** ****************

Rc DREB 1F 181 hdvaalalrGKSACLNFADSAWRL 252 Jc DREB 1B 99 HDVAALALRGKSACLNFADSSWRL 122

Pd CBF4 75 HDVAALAFRGKSACLNFADSAWRL 98

Ps CBF2 107 HDVAALALRGKSACLNFADSAWRL 130

Consensus ******************** ***

C Rc CBF 10 KR-KAGRTKFKETRHPIYRGVRRRNGNKWVCEVREPNLKSRIWLGTYPTPEMAARAHDVAA 189

Jc DREB 1A 45 KR-KAGRRVFKETRHPVYRGVRKRNGNKWVCELREPNKKTRIWLGTYPTPEMAARAHDVAA 104

Pe DREB 1F 42 KKNKAGRKKFKETRHPVYRGVRRRNGNKWVCEVREPNKKSRIWVGTFKSPEMAARAHDVAA 104

Pb CBF6 42 KKNKAGRKKFKETRHPVYRGVRRRNGNKWVCEVREPNKKSRIWVGTFKSPEMAARAHDVAA 104

Consensus * **** ******* *************** **** * *** ** ************

Rc CBF 190 LAFRGEFASLNYLDSAWILPRPKSSSHEDIKRAALEAAEAFK 315

Jc DREB 1A 105 LAFRGKSACLNFADSAWRLPVPASRDAKEIRRAASQAAEMFR 146

Pe DREB 1F 105 LALKGEFAALNFLDSALILPRAKSSSAEDIQRAALAATEVF- 143

Pb CBF6 105 LALKGELAALNFLDSALILPRAKSSSARDIQRAALAATEV-- 142

Consensus ** * * ** *** ** * * *** * *

Figure 4. 9: Multiple alignment and comparison of the deduced amino acids sequence of (A) Ricinus

communis DREB-1B with DREB-1F from Jatropha curcas and DREB-1B from Theobroma cacao, (B) Ricinus

communis DREB-1F with DREB-1B from Jatropha curcas, CBF4 from Populus deltoids and CBF2 from

Populus simonii and (C) Ricinus communis CBF like factor with DREB-1B from Jatropha curcas, DREB-1F

from Populus euphratica and CBF6 from Populus balsamifera. The symbols ‗Rc‘ represent Ricinus communis,

‗Jc‘ represent Jatropha curcas, ‗Tc‘ represent Theobroma cacao , ‗Pd‘ represent Populus deltoids, ‗Ps‘

represent Populus simonii, ‗Pe‘ represent Populus euphratica and ‗*‘ indicate the nucleotide in that column are

similar in all the sequences in alignment. The dotted line shows the missing amino acids in a sequence. Ricinus

communis DREB-1B like amino acid sequences showed 63% and 60% homology with DREb-1F and DREB-1B

amino acids sequences of Jatropha curcas and Theobroma cacao respectively. DREB-1F like amino acid

sequences in Ricinus communis demonstrated 92%, 93% and 92% homology with amino acid sequences of

DREB-B, CBF4 and CBF2 proteins in Jatropha curcas, Populus deltoids, and Populus simonii respectively.

Amino acids sequence of CBF like factor in Ricinus communis possessed 75%, 81% and 79% homology with

amino acid sequences of DREB-1A, DREB-1F and CBF6 proteins in Jatropha curcas, Populus euphratica and

Populus balsamifera respectively.

CHAPTER # 4 RESULTS

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4.3.1.5 Correlations of DREB 1B, DREB 1F and CBF like gene with Cd accumulation, free

proline and total phenolics concentration in Ricinus communis plant

Correlations of Cd accumulation in Ricinus communis with expression of DREB 1B, DREB

1F and CBF like genes is given in figure 4.10 (A, B, C). Positive correlations were observed

between the Cd accumulation and expression of DREB 1B (figure 4.10 A), DREB 1F (Figure

4.10 B) and CBF like genes (Figure 4.10 C) in Ricinus communis plant. The figure 4.11 (A –

F) present correlations of DREB 1B, DREB 1F and CBF like genes expression with free

proline and total phenolics concentration in Ricinus communis plant. Free proline

concentration demonstrated highly significant positive correlations with DREB 1B (R2 =

0.92), DREB 1F (R2 = 0.93) and CBF like genes (R

2 = 0.88) expression as given in figure

4.11 (A, B and C respectively). Total phenolics concentration showed positive correlations

with the DREB 1B, DREB 1F and CBF like genes in Ricinus communis (Figure 4.11 D, E,

F).

CHAPTER # 4 RESULTS

129

Figure 4. 10: Correlations of cadmium accumulation with expression of DREB-1F (A), DREB-1F

(B) and CBF like factor (C) gene in Ricinus communis plant.

y = 0.0076x + 0.2423 R² = 0.4779

0.00

0.20

0.40

0.60

0.80

1.00

1.20

0.00 20.00 40.00 60.00 80.00 100.00 120.00

Cd

acc

um

ula

ion

(m

g/D

W)

Expression level

DREB 1B gene

y = 0.0134x + 0.3201 R² = 0.5162

0.00

0.20

0.40

0.60

0.80

1.00

1.20

0.00 10.00 20.00 30.00 40.00 50.00

Cd

acc

um

ula

tio

n (

mg/

DW

)

Expression level

DREB 1F gene

y = 0.0076x + 0.3161 R² = 0.5092

0.00

0.20

0.40

0.60

0.80

1.00

1.20

0.00 20.00 40.00 60.00 80.00 100.00

Cd

acc

um

ula

tio

n (

mg/

DW

)

Expression level

CBF like gene

A

B

C

CHAPTER # 4 RESULTS

130

Figure 4. 11: Correlations of DREB 1B, DREB 1F and CBF like genes expression with free

proline (A, B and C respectively) and total phenolics concentration (D, E and F respectively) in

Ricinus communis plant. The symbol ―*‖ shows significance of the correlations.

y = 0.2262x + 32.492 R² = 0.9202 **

0

10

20

30

40

50

60

0.00 50.00 100.00 150.00

Free

pro

line

con

cen

trat

ion

(p

pm

)

DREB-1B gene expression level A

y = 0.4333x + 33.915 R² = 0.9394 **

0

10

20

30

40

50

60

0.00 20.00 40.00 60.00

Free

pro

line

con

cen

trat

ion

(p

pm

)

DREB-1F gene expression level

y = 0.336x + 27.239 R² = 0.8845 **

0

10

20

30

40

50

60

70

0.00 50.00 100.00

Free

pro

line

con

cen

trat

ion

(p

pm

)

CBF like gene expression level

y = 0.7273x + 54.763 R² = 0.7496 *

0

20

40

60

80

100

120

140

0.00 50.00 100.00 150.00

Tota

l ph

eno

lics

con

cen

trat

ion

(p

pm

)

DREB-1B gene expression level

y = 1.3967x + 59.241 R² = 0.7694 *

0

20

40

60

80

100

120

140

0.00 20.00 40.00 60.00

Tota

l ph

eno

lics

con

cen

trat

ion

(p

pm

)

DREB-1F gene expression level

y = 1.0016x + 41.949 R² = 0.6196 *

0

20

40

60

80

100

120

140

0.00 50.00 100.00

Tota

l ph

eno

lics

con

cen

trat

ion

(p

pm

)


B A

C D

E F

CHAPTER # 4 RESULTS

131

4.3.2 Cannabis sativa

4.3.2.1 Identification of CBF/DREB like gene sequences in Cannabis sativa plant

Genomic DNA extracted from Cannabis sativa plant is presented in figure 4.12. The DNA

was found intact and good quality. PCR product from genomic DNA using degenerate

primers showed the presence of DREB 1A, DREB 1B, DREB 1F and CBF like gene

sequences in Cannabis sativa plant (Figure 4.13).

Figure 4. 12: Genomic DNA extracted from Cannabis sativa plant. The lane 1 – 4 present

replicates.

Figure 4. 13: PCR product of DREB 1A (lane 1 – 3), DREB 1B (Lane 4 – 6), DREB 1F

(lane 7 – 9) and CBF like transcription factor (lane 10 -12) gene fragments of Cannabis

sativa plant. DNA marker is present on the left side and represented by ‗M‘.

M

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132

4.3.2.2 Expression analysis of DREB 1A, DREB 1B, DREB 1F and CBF like genes

sequences in Cannabis sativa

Reverse transcriptase [RT] PCR product of Actin gene of Cannabis sativa plant is given in

figure 4.14. Prominent DNA bands (PCR product) of actin gene fragments in the figure 4.14

reflected the optimization of experiments. Expression of DREB 1A and DREB 1B like genes

sequences is presented in the figure 4.15. No expression of DREB 1A like gene was found in

the treatments and controls (figure 4.15 A). The DREB 1B like gene of Cannabis sativa plant

expressed under the treatments (T1, T2 and T3) and the control C1 (only Cd) as shown in

figure 4.15 B. The treatments of Cd and Mo (T1, T2 and T3) induced slight increase in the

expression of DREB 1B like gene as compared to the control C1. Figure 4.16 (A and B)

shows the expression of DREB 1F and CBF like genes in Cannabis sativa plants under

various treatments of Cd and Mo. The DREB 1F like gene expressed in the treated plants (T1,

T2 and T3) and in the control C1 (Cd only) as given in figure 4.16 A. It was found that

expression of CBF like gene occurred in the controls C1 and C2 (Mo only), while its

expression increased in the treatments (T1 – T3) (Figure 4.16 B).

Figure 4. 14: RT-PCR product of Actin gene fragment of Cannabis sativa. The symbol ‗M‘

represent DNA marker. The number 1 and 2 denote replicates.

CHAPTER # 4 RESULTS

133

A

B

Figure 4. 15: RT-PCR product of DREB 1A (A) and DREB 1B (B) like genes of Cannabis sativa plant under various treatments of cadmium and

molybdenum. Three replicates are given for each gene and designated by numbers 1, 2 and 3. The letter ‗M‘ denote DNA marker, ‗C‘ stands for control

without Cd and Mo, ‗C1‘ represent control having 50 ppm Cd only, ‗C2‘ denote control having 1.00 ppm Mo only, ‗T1, T2 and T3‘ present treatments

containing 50 ppm Cd in combination with 0.5, 1.00 and 2.00 ppm Mo in each treatment respectively, ‗B‘ denote blank without template DNA.

1 2 3

1 2 3

CHAPTER # 4 RESULTS

134

A

B

Figure 4. 16: Reverse transcriptase [RT]-PCR product of DREB 1F (A) and CBF like gene (B) fragment of Cannabis sativa plant under various treatments of

cadmium and molybdenum. Three replicates are given for each gene and designated by numbers 1, 2 and 3. The letter ‗M‘ denote DNA marker, ‗C‘ stands for

control without Cd and Mo, ‗C1‘ represent control having 50 ppm Cd only, ‗C2‘ denote control having 1.00 ppm Mo only, ‗T1, T2 and T3‘ present

treatments containing 50 ppm Cd in combination with 0.5, 1.00 and 2.00 ppm Mo in each treatment respectively, ‗B‘ denote blank without template DNA.

1 2 3

1 2 3

CHAPTER # 4 RESULTS

135

4.3.2.3 Nucleotide sequences analysis

Nucleotides sequence alignment of Cannabis sativa DREB 1B, DREB 1F and CBF like gene

fragments with related gene sequences of other plants are presented in figures 4.17, 4.18 and

4.19 respectively. Nucleotides sequence alignment (Figure 4.17) shows 83 % and 81 %

homology of Cannabis sativa DREB 1B like gene sequences with DREB 1B (of Pyrus

bretschneideri) and DREB 1D (of Malus domestica) respectively. It was noted that Cannabis

sativa DREB 1F like nucleotides sequences were 90%, 92% and 88% homologous to DREB

1F (of Prunus mume), DREB 1E (of Malus domestica) and DREB 1E (of Pyrus

bretschneideri) gene sequences respectively (Figure 4.18). Nucleotide sequence alignment of

Cannabis sativa CBF like gene sequences showed 86 %, 83 % and 83 % similarity with CBF

like gene sequences (of Ricinus communis), CBF5 (of Populus trichcarpa) and CBF5 (of

Populus balsamifera) respectively (Figure 4.19).

CHAPTER # 4 RESULTS

136

Cs DREB 1B 5 TGGGAAGTGGGTGTGTGAAATGAGGCAACCGGATCATAACAAGTCGAGGGTATGGCTCGG 64

Pyb DREB 1B 146 TGGGAAGTGGGTGTGTGAGCTGAGGCAACCGGATCACAAGAAATCGCGGATATGGCTCGG 205

Md DREB 1D 146 TGGGAAGTGGGTGTGTGAGCTGAGGCAACCGGATCACAAGAAATCGCGGATATGGCTCGG 205

Consensus ****************** **************** ** ** *** ** **********

Cs DREB 1B 65 GACATTTCCTTA-CCCTGACATGGCTGCTAGGGCTTATGAAGTAGCAGCTTTTGCATTGA 123

Pyb DREB 1B 206 AAC-TTTCACTAGCCCTGACATGGCTGCTAGGGCTTATGATGTTGCAGCCTTGGCTCTCA 264

Md DREB 1D 206 AAC-TTTCACTAGCCCTGACATGGCTGCTAGGGCTTATGATGTTGCAGCCTTGGCTCTCA 264

Consensus ** **** ** *************************** ** ***** ** ** * *

Cs DREB 1B 124 GAGGAGAGTCTGCTTCGCTAAACTTTCCT---GA-GAT-CA-GTTCATTGTTGCCGCAGTTCA 180

Pyb DREB 1B 265 AGGGTGAGTCTGCTTCACTCAACTTTCCTAACGA GAC-AA-GTGC—T-TTGCCGCGCTTCG 321

Md DREB 1D 265 AGGGTGAGTCTGCTTCACTCAACTTTCCT---AACGAGGCAAGTGC-TT-T-GCCGCGCTTCG 321

Consensus ** *********** ** ********* * ** * ** * * * ***** ***

Cs DREB 1B 181 CGTCTAATGCTAACATGGAAAACGACAAAGTAGGTGCGGGGTCAGCAGG-TGCCGAGGCT 239

Pyb DREB 1B 322 AGTCGAATGCTTATACCGTAAAGGACATACAATGTGCTGCCTTAG-AGGCTGCCGAGGCA 380

Md DREB 1D 322 AGTCGAATGCTTATACCGTAAAGGASATACAATGTGCTGCCTTAG-AGGCTGCCGAGGCA 380

Consensus *** ****** * * * *** ** * * * **** * * ** *** *********

Cs DREB 1B 240 GTCTTGGAGGTTAAAGGTAAAGCCTCTTCATCTTCTTCTTTGAAGTTGGAGAAGGTAGAG 299

Pyb DREB 1B 381 TTCTTGGAGGTTAAAGGTAAAGCCTCTTCATCTTCTTCTTTGAAGTTGGAGAAGGTAGAG 440

Md DREB 1D 381 TTCTTGGAGGTTAAAGTTAAAGCCTCTTCCTCTTCTTCTTTGAAGTTGGAGAAGGTAGAA 440

Consensus *************** ************ *****************************

Cs DREB 1B 300 GAAGAAGAGGTGAGAAAAGTTGT 322

Pyb DREB 1B 441 GAAGAAGAGGTGAGAAAAGTTGT 463

Md DREB 1D 441 GAAGAAGAGATGGGAAAAGTTGT 463

Consensus ********* ** **********

Figure 4. 17: Nucleotide sequence alignment of Cannabis sativa DREB 1B like gene

fragment with DREB 1B of Pyrus x bretschneideriand DREB 1D of Malus domestica. The

symbol ‗Cs‘ represent Cannabis sativa, ‗Pyb‘ represent Pyrus x bretschneideri, ‗Md‘

represent Malus domestica and ‗*‘ indicate the nucleotide in that column are similar in all the

sequences in alignment. The dotted line shows the missing nucleotides in a sequence.

Cannabis sativa DREB-1B like gene sequences showed 83 % and 81 % homology with

DREB-1B and DREB-1D gene sequences of Pyrus x bretschneideri and Malus domestica

respectively. Nucleotide sequence homology among the three genes sequences is equal to

80%.

CHAPTER # 4 RESULTS

137

Cs DREB 1F 13 CCGAAGAAGCGAGCGGGGAGGAGAGTGTTCAAGGAGACGAGGCACCCAGTCTATAGAGGA 72

Pm DREB 1F 175 CCGAAGAAGCGAGCCGGGAGGAGGGTTTTCAAGGAGACGAGGCACCCGGTTTATAGGGGT 234

Md DREB 1E 399 ---AAGAAGCGAGCGGGGAGGAGAGTTTTCAAGGAGACGAGGCACCCAGTTTACAGAGGA 455

Pyb DREB 1E 492 ---AAGAAGCGAGCGGGGAGGAGAGTTTTCAAGGAGACGAGGCACCCGGTTTACAGAGGA 548

Consensus *********************** ******************** ** ** *****

Cs DREB 1F 73 GTTAGGAGGAGGAACAATGACAAGTGGGTGTGCGAGATGAGGGAGCC—-A-AAC---AAGAAG 129

Pm DREB 1F 235 GTGAGGAGGAGGAACAATGACAAGTGGGTTTGTGAAATGAGAGAGCCCAACAAG---AAGAAG 294

Md DREB 1E 456 GTTAGGAGGAGGAACAACAACAAGTGGGTGTGCGAAATGAGGGAACC--A-AACAAGAAGAAG 515

Pyb DREB 1E 549 GTGAGGAGGAGGAACAACAACAAGTGGGTGTGCGAAATGAGAGAACC—-A-AACAAGAAGAAG 608

Consensus ***************** **************** ***** ** ** * ** ******

Cs DREB 1F 130 TCGAGGATATGGCTCGGAACTTATCCTACGGCCGAGATGGCTGCTCGAGCGCATGACGTG 189

Pm DREB 1F 295 TCCAGGATATGGCTCGGGACTTATCCGACGGCTGAGATGGCTGCTCGTGCCCATGACGTG 354

Md DREB 1E 516 TCGAGGATATGGCTCGGAACTTATCCGACGGCCGAGATGGCAGCTCGGGCGCATGACGTG 575

Pyb DREB 1E 609 TCGAGGATATGGCTCGGAACTTATCCGACGGCCGAGATGGCAGCTCGGGCGCATGACGTG 668

Consensus ***************** ******** ************** ***** ** *********

Cs DREB 1F 190 GCCGCATTGGCGTTTAGAGGGAAGCCTGCCTGCCTCAACTTTGCTGACTCCGCGTGGAGG 249

Pm DREB 1F 355 GCGGCATTGGCGTTTAGAGGGAAGCTTGCCTGCCTCAACTTTGCTGACTCCGCGTGGAGG 414

Md DREB 1E 576 GCGGCATTGGCCTTTAGAGGGAAGCTTGCCTGCCTCAATTTTGCAGACTCCGCATGG--- 632

Pyb DREB 1E 668 GCGGCATTGGCCTTTAGAGGGAAGCTTGCCTGCCTCAATTTTGCAGACTCCGCATGG--- 725

Consensus ** ******** ************* ************ ***** ******** ***

Cs DREB 1F 250 TTGC 253

Pm DREB 1F 415 CTGC 418

Md DREB 1E ----

Pyb DREB 1E ----

Consensus

Figure 4. 18: Nucleotide sequence alignment of Cannabis sativa DREB 1F like gene

segment with DREB 1F of Prunus mume, DREB 1E of Malus domestica and DREB 1E of

Pyrus x bretschneideri. The symbols ‗Cs‘ represent Cannabis sativa, ‗Pm‘ Prunus mume,

‗Md‘ represent Malus domestica, ‗Pyb‘ represent Pyrus x bretschneideri and ‗*‘ indicate the

nucleotide in that column are similar in all the sequences in alignment. The dotted line shows

the missing nucleotides in a sequence. Cannabis sativa DREB-1F like gene sequences

demonstrated 90%, 92 % and 88 % homology with DREB-1F, DREB-1E and DREB-1E of

Prunus mume, Malus domestica and Pyrus x bretschneideri respectively. Nucleotides

sequence similarity of 80% was found among the above four genes sequences.

CHAPTER # 4 RESULTS

138

Cs CBF 1 CCAGCAAAGAAGAGGAAAGCAGGGAGGAAGAAGTTCAAGGAGACTCGGCACCCGATGTAC 60

Rc CBF 85 CCAGCAAAGAAGAGGAAAGCAGGGAGGACCAAGTTCAAGGAGACTAGGCATCCGATTTAT 144

Pt CBF5 130 ------AAGAAGAAGAAAGCAGGAAGGAAGAAGTTCAAGGAGACTCGGCACCCGGTATAT 189

Pb CBF5 10 ------AAGAAGAAGAAAGCAGGAAGGAAGAAGTTCAAGGAGACTCGGCACCCGGTATAT 69

Consensus ******* ********* **** *************** **** *** * **

Cs CBF 61 AGCGGTGTCCGGCGAAGAAACTCCAGGAAATGGGTTTGCGAGGTGCGAGAACCCAACAAG 120

Rc CBF 145 AGAGGTGTAAGGCGAAGAAATGGGAATAAATGGGTATGTGAAGTTAGAGAACCGAACCTG 204

Pt CBF5 190 AGGGGGGTACGGAAGAGAAATGGGAATAAATGGGTATGTGAAGTGCGAGAACCGAACAAG 249

Pb CBF5 70 AGGGGGGTACGGAAGAGAAATGGGAATAAATGGGTGTGTGAAGTGCGAGAACCGAACAAG 129

Consensus ** ** ** *** ***** * ******** ** ** ** ******* *** *

Cs CBF 121 AAGACCAGGATTTGGCTAGGGACTTTCCCCACCCCCGAAATGGCAGCTAGGGCACATGAC 180

Rc CBF 205 AAATCAAGAATATGGCTAGGGACATACCCTACCCCAGAAATGGCAGCTAGGGCACATGAT 264

Pt CBF5 250 AAATCAAGAATTTGGTTAGGGACCTTCACTAGCCCAGAAATGGCAGCTAGGGCACATGAC 309

Pb CBF5 130 AAATCAAGAATTTGGTTAGGGACCTTCACTAGCCCAGAAATGGCAGCTAGGGCACATGAC 189

Consensus ** * ** ** *** ******* * * * * *** ***********************

Cs CBF 181 GTGGCGGCCTTAGCACTTAGAGGAGAATTTGCTTCTCTCAATTTTCCTGATTCAGCTTGG 240

Rc CBF 265 GTTGCTGCTTTGGCATTCAGAGGAGAGTTTGCTTCTCTCAACTATCTTGATTCAGCTTGG 324

Pt CBF5 310 GTTGCTGCCTTAGCACTGAAGGGAGAAACTGCTACTTTAAATTTTCCTGATTCAGCTTTG 369

Pb CBF5 190 GTTGCTGCCTTAGCACTGAAGGGAGAAACTGCTACTTTAAATTTTCCTGATTCAGCTTTG 249

Consensus ** ** ** ** *** * * ***** **** ** * ** * ** *********** *

Cs CBF 241 ATACTTCCACGACCGAAGTCGTCTTCTGCTGAAGATATAAAAAGAGCTGCGCTTGAAGCT 300

Rc CBF 325 ATACTGCCACGACCAAAGTCGTCTTCTCATGAAGATATTAAAAGGGCTGCTCTTGAAGCT 384

Pt CBF5 370 ATACTTCCTCGAGCGAAGTCGTCTTCTGCTGGAGATATACGAAGAGCTGCGCGTGATGCT 429

Pb CBF5 250 ATACTTCCTCGAGCGAAGTCGTCTTCTGCTRGAGATATACGAAGAGCTGCGCGTGATGCT 309

Consensus ***** ** *** * ************ * ****** *** ***** * *** ***

Cs CBF 301 GCTGAGGCTTTCAAGCCAAGTGCTTCTGATCTATCCTCAACATCACCACCATCGTCTTCT 360

Rc CBF 385 GCTGAGGCTTTCAAGCCAAGTTCTACTGATCTATCCTCAACATCACCACCATCGTCTTCT 444

Pt CBF5 430 GTTGAGGCCTTTATACCTAGTGCTTCT--------------------------------- 457

Pb CBF5 310 GTTGAGGCCTTTATACCTAGTGCTTCT--------------------------------- 337

Consensus * ****** ** * ** *********

Cs CBF 361 TCTTGTTCGTCTGCGTCTTCTA 382

Rc CBF 445 TCTTGTTCGTCTGCGTCTTCTA 466

Pt CBF5 ----------------------

Pb CBF5 ----------------------

Consensus

Figure 4. 19: Nucleotide sequence alignment of Cannabis sativa CBF like gene segment

with CBF like gene of Ricinus communis, CBF5 of Populus trichocarpa and CBF5 of

Populus balsamifera. The symbol ‗Cs‘ Cannabis sativa, ‗Rc‘ represent Ricinus communis,

‗Pt‘ represent Populus trichocarpa, ‗Pb‘ represent Populus balsamifera and ‗*‘ indicate the

nucleotide in that column are similar in all the sequences in alignment. The dotted line shows

the missing nucleotides in a sequence. The CBF like gene sequences of Cannabis sativa

showed 86 %, 83 % and 83% homology with CBF like gene sequences of Ricinus communis,

Populus trichocarpa and Populus balsamifera plants respectively. Nucleotides sequence

homology of 65% was present among the four gene sequences.

CHAPTER # 4 RESULTS

139

4.3.2.4 Protein sequence analysis of DREB 1B, DREB 1F and CBF like genes of Cannabis

sativa plant

Predicted amino acid sequence of Cannabis sativa DREB 1B, DREB 1F and CBF like

protein fragments and their sequence comparison with related proteins from other plant

species is given in figure 4.19 (A, B, C respectively). The Cannabis sativa DREB 1B like

protein fragment showed 73% and 77% amino acid homology with DREB 1B (of Pyrus x

bretschneideri) and DREB 1D (of Malus domestica) proteins (Figure 4.19 A). Amino acid

sequence similarity of 95% was found between Cannabis sativa DREB-1F like protein

fragment and DREB-1F (of Prunus mume) protein (Figure 4.19 B). Similarly DREB-1E (of

Malus domestica) and DREB-1E (of Pyrus x bretschneideri) proteins demonstrated 94%

amino acid sequence similarity with the Cannabis sativa DREB-1F like protein segment

(Figure 4.19 B). The CBF like protein fragment of Cannabis sativa showed 93% amino acid

sequence homology with CBF like protein fragment of Ricinus communis and 84% amino

acid sequence homology with CBF5 of Populus trichocarpa and Populus balsamifera (Figure

4.19 C).

CHAPTER # 4 RESULTS

140

A Cs DREB 1B 6 GKWVCEMRQPDHNKSRVWLGTFPYPDMAARAYEVAAFALRGESASLNFPEISSLLPQFTS 185

Pyb DREB 1B 34 GKWVCELRQPDHKKSRIWLGTFTSPDMAARAYDVAALALKGESASLNFPNETSALPRFES 93

Md DREB 1D 34 GKWVCELRQPDHKKSRIWLGTFTSPDMAARAYDVAALALKGESASLNFPNEASALPRFES 93

Consensus ****** ********* ***** ******** *** ** ********* * ** * *

Cs DREB 1B 186 SNANMEND 206

Pyb DREB 1B 94 -NAYTVKD 100 Md DREB 1D 94 -NA----D 95

** *

B Cs DREB 1F 1 ATSYPKKRAGRRVFKETRHPVYRGVRRRNNDKWVCEMREPNK-KSRIWLGTYPTAEMAAR 177

Pm DREB 1F 40 ASSRPKKRAGRRVFKETRHPVYRGVRRRNNDKWVCEMREPKKTKSRIWLGTYPTAEMAAR 99

Md DREB 1E 42 ASSRPKKRAGRRVFKETRHPVYRGVRRRNNNKWVCEMREPNKKKSRIWLGTYPTAEMAAR 101

Pyb DREB 1E 42 ASSRPKKRAGRRVFKETRHPVYRGVRRRNNNKWVCEMREPNKKKSRIWLGTYPTAEMAAR 101

Consensus * * ************************** *********** *****************

Cs DREB 1F 178 AHDVAALAFRGKPACLNFADSAWRL 252

Pm DREB 1F 100 AHDVAALAFRGKLACLNFADSAWRL 124

Md DREB 1E 102 AHDVAALAFRGKLACLNFADSAWRL 126

Pyb DREB 1E 102 AHDVAALAFRGKLACLNFADSAWRL 126

Consensus *************************

C Cs CBF 40 ETRHPIYRGVRQRNGNKWVCEVREPNKKSRIWLGTYPTPEMaarahdvaalalrGEFASL 219

Rc CBF 42 ETRHPIYRGVRRRNGNKWVCEVREPNLKSRIWLGTYPTPEMAARAHDVAALAFRGEFASL 101

Pt CBF5 55 ETRHPVYRGVRKRNGNKWVCEVREPNKKSRIWLGTFTSPEMAARAHDVAALALKGETATL 114

Pb CBF5 15 ETRHPVYRGVRKRNGNKWVCEVREPNKKSRIWLGTFTSPEMAARAHDVAALALKGETATL 74

Consensus *********** *********************** *************** * * *

Cs CBF 220 NFPDSAWILPRPKSSSAEDIKRAALEAAEAFK 315

Rc CBF 102 NYLDSAWILPRPKSSSHEDIKRAALEAAEAFK 133

Pt CBF5 115 NFPDSALILPRAKSSSAGDIRRAARDAVEAF- 145

Pb CBF5 75 NFPDSALILPRAKSSSARDIRRAAXDAVEAF- 105

Consensus * *** ********* ****** * ***

Figure 4. 20: Multiple alignment and comparison of the deduced amino acids sequence of (A)

Cannabis sativa DREB-1B like protein fragment with DREB-1B of Pyrus x bretschneideri

and DREB-1B of Malus domestica, (B) Cannabis sativa DREB-1F like protein fragment with

DREB-1F of Prunus mume, DREB-1E of Malus domestica and DREB-1E of Pyrus x

bretschneideriand (C) Cannabis sativa CBF like protein fragment with CBF of Ricinus

communis, CBF5 from Populus trichocarpa and CBF5 of Populus balsamifera. The symbols

‗*‘ indicate the nucleotide in that column are similar in all the sequences in alignment, ―Cs‖

represent Cannabis sativa, ―Pyb‖ denote Pyrus x bretschneideri, ―Md‖ represent Malus

domestica, ―Pm‖ denote Prunus mume, ―Rc‖ shows Ricinus communis, ―Pt‖ represent

Populus trichocarpa and ―Pb‖ denote Populus Balsamifera. The dotted line shows the

missing amino acids in a sequence. Cannabis sativa DREB-1B like protein fragment showed

73% and 77% amino acid sequence homology with DREB-1B proteins of Pyrus x

bretschneideri and Malus domestica respectively. Amino acid sequence of DREB-1F protein

fragment of Cannabis sativa showed amino acid sequence similarity of 95%, 94% and 94%

with DREB-1F, DREB1E and DEB1E amino acids sequences of Prunus mume, Malus

domestica and Pyrus x bretschneideri respectively. Amino acid sequence of Cannabis sativa

CBF like protein fragment demonstrated 93%, 84% and 84% homology with amino acid

sequences of CBF like proteins of Ricinus communis, Populus trichocarpa and Populus

balsamifera respectively.

CHAPTER # 4 RESULTS

141

4.3.2.5 Correlations of DREB 1B, DREB 1F and CBF like gene sequences with Cd

accumulation and concentration of proline and phenolics in Cannabis sativa plant

Correlations of cadmium accumulation in Cannabis sativa plants with expression of DREB

1B, DREB 1F and CBF like genes are presented in Figure 4.21 (A, B and C respectively).

Cadmium accumulation demonstrated significantly positive correlation with expression of the

BREB 1B gene (Figure 4.21 A) while the correlation of Cd accumulation with DREB 1F

(Figure 4.21 B) and CBF like gene (Figure 4.21 C) were positive but not statistically

significant. Expression of Cannabis sativa DREB-1B like gene showed strong positive

correlation (R2 = 0.7509) with accumulation of cadmium in Cannabis sativa plant (Figure

4.21 A). Positive correlations were found between DREB 1B, DREB 1F and CBF like genes

expression and the concentration of free proline (Figure 4.22 A, B, C respectively) and total

phenolics (Figure 4.22 D, E, F respectively) in Cannabis sativa plant.

CHAPTER # 4 RESULTS

142

Figure 4. 21: Correlation of cadmium accumulation in Cannabis sativa with expression of

CsDREB-1B (A), CsDREB-1F (B) and CsCBF like gene (C). The symbol ―*‖ denote the

significance of the correlation.

y = 0.0216x - 0.1604 R² = 0.7509 *

0.00

0.20

0.40

0.60

0.80

1.00

1.20

0.00 10.00 20.00 30.00 40.00 50.00 60.00

Cd

acc

um

ula

tio

n (

mg/

DW

)

Expression level

DREB 1B gene

A

y = 0.0224x - 0.051 R² = 0.551

0.00

0.20

0.40

0.60

0.80

1.00

1.20

0.00 10.00 20.00 30.00 40.00 50.00 60.00

Cd

acc

um

ula

tio

n (

mg/

DW

)

Expression level

DREB 1F Gene

B

y = 0.0178x + 0.1562 R² = 0.5629

0.00

0.20

0.40

0.60

0.80

1.00

1.20

0.00 10.00 20.00 30.00 40.00 50.00 60.00

Cd

acc

um

ula

tio

n (

mg/

DW

)

Expression Level

CBF like gene

C

CHAPTER # 4 RESULTS

143

Figure 4. 22: Correlations of DREB 1B, DREB 1F and CBF like genes expression with free

proline (A, B and C respectively) and total phenolics concentration (D, E and F respectively) in

Cannabis sativa plant.

y = 0.6569x + 59.401 R² = 0.5646

0

20

40

60

80

100

120

0.00 20.00 40.00 60.00

Free

pro

line

con

cen

trat

ion

(p

pm

)

DREB-1B like gene expression level A

y = 0.6702x + 62.433 R² = 0.4162

0

20

40

60

80

100

120

0.00 20.00 40.00 60.00

Free

pro

line

con

cen

trat

ion

(p

pm

)

DREB-F like gene expression level

y = 0.2135x + 31.795 R² = 0.0563

0

10

20

30

40

50

60

0.00 20.00 40.00 60.00

Free

pro

line

con

cen

trat

ion

(p

pm

)

CBF like gene expression level C

y = 0.6569x + 59.401 R² = 0.5646

0

20

40

60

80

100

120

0.00 20.00 40.00 60.00

Tota

l ph

eno

lics

con

cen

trat

ion

(p

pm

)

DREB-1B like gene expression level

y = 0.6702x + 62.433 R² = 0.4162

0

20

40

60

80

100

120

0.00 20.00 40.00 60.00

Tota

l ph

eno

lics

con

cen

trat

ion

(p

pm

)

DREB-F like gene expression level

y = 0.2135x + 31.795 R² = 0.0563

0

10

20

30

40

50

60

0.00 20.00 40.00 60.00

Tota

l ph

eno

lics

con

cen

trat

ion

(p

pm

)


B

D

E F

A

C


144

4.4 DISCUSSION

A variety of transcription factors affect the expression of stress resistant genes and thus play a

crucial role in plant responses to biotic and abiotic stresses (Akhtar et al 2012; Hadi et al

2011; Ito et al 2006; Dubouzet et al 2003). Expression of transcriptional factors like CBF/

DREB have been previously studied under low temperature, drought and salinity stresses

(Hadi et al 2011). In present research we found that DREB 1B, DREB 1F and CBF like genes

in Ricinus communis and Cannabis sativa plants expressed under Cd stress. Extensive cross-

talk between abiotic stress signaling pathways have been reported in previous literature

(Agarwal and Jha 2010; Huang et al 2012). Since the expression of DREB/CBF genes is

known to be influenced by low-temperature, drought or salinity stress, therefore it would not

be surprising that their expression might also be influenced by other abiotic stresses such as

heavy metal exposure. Mechanism by which cadmium induce the expression of DREB/CBF

genes is not known as far as our knowledge is concern. Cadmium has been found to induce

physiological drought by altering water content in plant tissues (Barcelo and Poschenrieder

1990) and this might be one of the reasons for the expression of DREB 1B, DREB 1F and

CBF like genes like genes in Ricinus communis and Cannabis sativa plants under Cd stress.

But a relationship between drought and Cd stresses at the transcription level has not been

reported as far as we know (Oono et al 2014).

In our experiments, the molybdenum foliar spray alone (without Cd) had no effect on the

expression of DREB 1A, DREB 1B and DREB 1F like gene in Ricinus communis and

Cannabis sativa plants. The CBF like genes expressed under the Mo foliar spray (without

Cd) in the plants. Application of molybdenum foliar spray in combination with Cd (added to

soil) increased the expression of DREB 1B, DREB 1F and CBF like genes; suggesting a

possible role of molybdenum in enhancing the expression of these genes under Cd stress.

Molybdenum cofactor (MoCo) is required for the biosynthesis of abscisic acid (ABA) (Xiong

et al 2001). Several investigators reported expression of CBF/ DREB like transcription

factors under high concentration of abscisic acid in plant tissues (Liu et al 1998; Yang et al

2009; Wang et al 2008; Shen et al 2003). It is possible that Mo might enhanced the

expression of DREB 1B, DREB 1F and CBF like genes by increasing biosynthesis of abscisic

acid under cadmium stress. Since we have studied only a small fragments of the DREB 1A,

DREB 1B, DREB 1F and CBF like gene and the full length gene sequences have not been

isolated and investigated due to lack of financial support and time constrain.

Expression of the CBF/ DREB like genes demonstrated positive correlation with the

concentration of free proline in plant tissues, which suggest the possible role of these genes in


145

biosynthesis of proline (Tuteja et al 2012). Increased in concentration of free proline have

been reported under the over-expressed CBF/DREB genes (Gilmour et al 2000; Hadi et al

2011). Proline act as an osmoprotector and thus protect cellular components from toxic

effects of heavy metals. It was noted that total phenolics in plant leaves increased with the

expression of CBF/DREB genes. Phenolic compounds are strong antioxidants and play an

important role in scavenging the reactive oxygen species (ROS) produced by cadmium in the

plants.


146

4.5 Conclusions

DREB/CBF genes have mostly been studied under drought, frost and salinity stresses. In

present research an effort has been made to study the effect of toxic heavy metal (cadmium)

and micronutrient (molybdenum) on the expression of four selected DREB/CBF genes in

Ricinus communis and Cannabis sativa plants. Three of the four genes (DREB-1B, DREB-1F

and CBF like factor) sequences showed expression under cadmium stress. Molybdenum

further increased expression level of the above genes sequences when used in combination

with cadmium. Only the CBF like gene demonstrated expression in plants treated with only

Mo. DREB-1A like gene sequences did not showed expression at all the treatments and

controls. Our results suggested that expression of these genes responsive to drought stress

(also implying a relationship to high-salinity and low temperature stresses) was affected by

Cd exposure, but a relationship between Cd and drought stresses at the transcription level has

not been reported as far as we know. It is further recommended to isolate the whole gene

sequences of the DREB-1B, DREB-1F and CBF like genes to conform that the studied

sequences belongs to these genes. Other DREB/CBF genes should be studied in Ricinus

communis and Cannabis sativa plants for their expression under Cd stress.

CHAPTER # 5

GENERAL DISCUSSION

CHAPTER # 4 GENERAL DISCUSSION

146

CHAPTER 5: General discussion

5.1. Phytoextraction as a promising green technology for heavy metals remediation

Environmental pollution is global problem and number of organizations and agencies are

involved to facilitate the safe restoration of soil and ground water but most of the strategies

adopted for this purpose are highly expensive and laborious. Instead of classical technologies,

phytoextraction is an emerging technology for the safe restoration of toxic metals polluted

soil and water. It is very cheaper, solar driven and environment friendly technology, which is

highly suitable for the developing countries like Pakistan. Many research is going on to find

out the biochemical and molecular mechanisms involved in the uptake and accumulation of

toxic metals in plant. In the present research, we investigated the molecular, biochemical and

physiological aspects of cadmium phytoextraction from contaminated soil using Ricinus

communis, Cannabis sativa and Parthenium hysterophorus plants. All the three plants are

highly suited for the phytoextraction purpose due to their huge biomass, high tolerance to

heavy metals and almost non palatable nature to herbivores to prevent entry of toxic metals

into the food chain.

5.2. CBF/DREB transcriptional factors (CBF regulon) can play role in phytoextraction

of cadmium

Transcriptional factors (TF) play an important role in plant defense against abiotic stress.

Single TF can regulate a number of downstream genes (regulon), their products contribute in

resistance to abiotic stresses. Which make the TF a suitable candidate in research for the

improvement in phytoextraction technology. In present research, we investigated the

expression of well characterized drought, frost and salinity stress-related transcription factors

(i.e. DREB 1A, DREB-1B, DREB-1F and CBF) genes in Ricinus communis and Cannabis

sativa plants under Cd stress. The DREB-1B, DREB-1F and CBF like gene sequences were

identified for the first time in Cannabis sativa plant. It was found that the DREB-1B, DREB-

1F and CBF like genes expressed in Ricinus communis and Cannabis sativa plants under

cadmium stress. The actual mechanism by which Cd induces the expression of DREB/CBF

genes is not known as for as our knowledge is concerned. Extensive cross-talk between

different stress signalling pathways have been reported in plants (Agarwal and Jha 2010;

Huang et al 2012) and it can be expected that these transcription factors might also be

expressed under cadmium stress. Over expression of CBF/DREB genes in plants have been

reported to increase the tolerance level of plants to various abiotic stresses (Hadi et al 2011).

The increase in expression of DREB-B, DREB-1F and CBF like genes and accumulation of


147

cadmium in plants support our hypothesis that the DREB-B, DREB-1F and CBF like genes

might have some important role in increasing plant tolerance and accumulation of cadmium

in plants. The overexpression of CBF/DREB like TF might play two important roles in the

improvement of cadmium phytoextraction; (1) they could increase the phytoextraction

potential of a plant by increasing its tolerance to cadmium and (2) possibly they may provide

an opportunity to use this plants for removal of Cd from soil under harsh conditions such as

salinity, low temperature or drought. The present investigation could open a window for new

research in field of regulon biotechnology (CBF regulon) for development of phytoextraction

technology.

5.3. Proline and phenolic compounds enhance plant defense and cadmium

phytoextraction

Significant increase in concentration of free proline and total phenolics in roots and leaves of

Ricinus communis, Cannabis sativa and Parthenium hysterophorus were noted with increase

in accumulation of cadmium in the plant tissues. Proline and phenolic compounds increase

the tolerance level of a plant by scavenging the reactive oxygen species produced by toxic

heavy metals as well as by shielding the macromolecules and cell organelles from the direct

or indirect toxicity of cadmium (Khatamipour et al 2011; Sun et al 2007). Proline act as an

osmoprotectant and its concentration in plants is highly increased during drought conditions.

Cadmium produce physiological drought in plants which activate the enzyme ‗pyroline-5-

carboxylate‘ (involved in proline bio-synthesis) and thus increase the concentration of free

proline in plant tissues (Ali et al 2001; Delauney and Verma 1993). Phenolic compounds are

produced mainly in response to the increased level of ROS during the heavy metals stress in

plants (Jung et al 2003: Bors et al 1990). The treatments of molybdenum and gibberellic acid

highly increased the concentration of free proline and total phenolics in the plants and thus

amplified the plant defense against cadmium toxicity. A plant with strong biochemical

defense is highly suited for the phytoextraction purposes.

5.4. High biomass and high concentration of toxic metals in the biomass greatly

enhanced the phytoextraction ability of plants

Total amount of metal extracted by plants are determined by concentration of the metals in

biomass and total biomass of the plant and both of these factors are important

(Padmavathiamma and Li 2007). For efficient phytoextraction, a plant must produce high

biomass and should tolerate high concentration of toxic heavy metals in the biomass. Plant

growth parameters such as biomass are highly sensitive to Cd toxicity (Hadi et al 2015;


148

Khatamipour et al 2011; Arun et al 2005). Cadmium disturbs the structure and function of

some important enzymes involved in metabolism and consequently decrease plant growth

and biomass (Houshmandfar and Moraghebi 2011; John et al 2009). To increase the biomass

we used phytohormone gibberellic acid (GA3) in our first experiment on Parthenium

hysterophorus plant and found that biomass of the plant was highly increased by GA3 under

cadmium stress. In the same experiment ethylenediaminetetraacetic acid was used for

increasing Cd bioavalibility in soil. EDTA increased the concentration of Cd in plant but

reduced the biomass which in turn affect the phytoextraction potential of plants. Application

of GA3 in combination with EDTA restored the plant biomass as well as the uptake and

accumulation of Cd in the plant tissues. This might be due to the effect of EDTA on

increasing Cd bio-availability in soil and the role of GA3 on the plant growth and nutrient

uptake (Mamindy-Pajany et al 2014; Hadi et al 2014; Tassi et al 2008; Meers et al 2005;

Thayalakumaran et al 2003; Chen and Cutright 2001).

Ricinus communis and Cannabis sativa plants showed higher biomass with molybdenum

treatments. Molybdenum act as cofactor for enzymes involved in nitrogen metabolism,

synthesis of indole-3-acetic acid (IAA) and abscisic acid ABA (Hesberg et al 2004; Sagi et al

2002; Schwartz et al 1997; Marin and Marion-Poll 1997) that play important role in

increasing plant growth and defense against toxicity of heavy metals. R. communis and C.

sativa with molybdenum (Mo) foliar spray increased the biomass and Cd accumulation in the

plants tissues. All the plants demonstrated highest Cd concentration in their roots which is the

confirmation of results obtained in Nerium oleander (Kadukova et al 2006), Triticum

aestivum and Triticum durum (Kovaeevic et al 1999; Ozturk et al 2003), Pinus sylvestris

(Kim et al 2003) and maize (Ekmekci et al 2008).


149

5.5. Conclusions

The DREB 1A, DREB 1B and CBF like genes expressed in Ricinus communis and Cannabis

sativa plants under cadmium stress. Molybdenum foliar spray further increased the expression of

these genes under Cd stress. The presence of these genes are reported in Cannabis sativa plant

for the first time. Cadmium uptake and accumulation in plants tissues increased with increase in

concentration of free proline and total phenolics. Significantly positive correlations of DREB

1A, DREB 1B and CBF like genes expression with Cd accumulation, free proline and total

phenolics were found in Ricinus communis while such correlations were positive but non-

significant in Cannabis sativa. Foliar spray of Mo was found better in terms of increasing

biomass, Cd accumulation and concentration of proline and phenolics in the plants as compared

to seed soaking and soil addition applications of Mo. Both the plants showed high

bioconcentration of Cd in their tissues and the treatments of Mo further increased the

bioconcentration. The treatments of gibberellic acid and ethylenediaminetetraacetic acid highly

increased Cd uptake, translocation and accumulation in P. hysterophorus plant. Biomass of the

plant was increased by the foliar application of gibberellic acid while uptake and Cd

accumulation was enhanced by ethylenediaminetetraacetic acid.


150

5.6. Recommendations

Since the CBF/DREB genes are investigated for the first time for its role in Cd phytoextraction

and this research will open a new window for investigation of CBF/DREB like genes under

heavy metals stress. We have isolated and studied some parts of the whole genes, we were

interested in further study of whole genes but due to financial and time constraint unable to

perform. Therefore further study is recommended to isolate and investigate the full length

CBF/DREB genes under cadmium stress. Molybdenum have shown good results and it is highly

recommended to study its role in phytoextraction of other toxic metals as well as its effect on

other transcription factors involved in plant abiotic stresses. Mo play important role in nitrate

uptake and assimilation in plants, so it is recommended to test the effect of nitrogen fertilizer in

combination with the foliar spray of Mo on Cd phytoextraction. Further study is recommended to

find the biochemical basis of proline and phenolics synthesis and their role in Cd

phytoextraction. It is highly suggested to investigate the effect of individual phenolics rather

than total phenolics to find out the candidate compound, responsible for metal detoxicificantion

and enhanced phytoextraction. In the present work individual phenolics were not studied due to

financial constraint.

REFERENCES

151

REFERENCES

Adewole MB, Sridhar MKC, Adeoye GO (2010) Removal of Heavy Metals from Soil Polluted

with Effluents from a Paint Industry Using Helianthus annuus L. and Tithonia

diversifolia (Hemsl.) as Influenced by Fertilizer Applications. Bioremediation Journal

14(4), 169–179.

Aery NC, Rana DK (2003) Growth and cadmium uptake in barley under cadmium stress.

Journal of Environmental Biology 24, 117-123.

Agarwal PK, Agarwal P, Reddy MK, Sopory SK (2006) Role of DREB transcription factors in

abiotic and biotic stress tolerance in plants. Plant Cell Reports 25, 1263–1274.

Agarwal PK, Jha B (2010) Transcription factors in plants and ABA dependent and independent

abiotic stress signaling. Biologia Plantarum 54 (2), 201-212.

Agarwala SC, Sharma CP, Farooq S, Chatterjee C (1978) Effect of molybdenum deficiency on

the growth and metabolism of corn plants raised in sand culture. Canadian Journal of

Botany 56, 1905–1909.

Ahmad A, Hadi F, Ali N (2015) Effective Phytoextraction of Cadmium (Cd) with Increasing

Concentration of Total Phenolics and Free Proline in Cannabis sativa (L) Plant Under

Various Treatments of Fertilizers, Plant Growth Regulators and Sodium Salt. International

Journal of Phytoremediation 17, 56-65.

Ahmad P, Jhon R (2005) Effect of salt stress on growth and biochemical parameters of Pisum

sativum L. Archives of Agronamy and Soil Science 51, 665-672.

Ahmad P, Jhon R, Sarwat M, Umar S (2008) Responses of proline, lipid peroxidation and

antioxidative enzymes in two varieties of Pisum sativum L. under salt stress.

International Journal of Plant Production 2, 353-366.

Akesson A, Julin B, Wolk A (2008) Long-term dietary cadmium intake and postmenopausal

endometrial cancer incidence: a population-based prospective cohort study. Cancer

Research 68, 6435-6441.

REFERENCES

152

Akhtar M1, Jaiswal A, Taj G, Jaiswal JP, Qureshi MI, Singh NK (2012) DREB1/CBF

transcription factors: their structure, function and role in abiotic stress tolerance in plants.

Journal of Genetics 91(3), 385-95.

Ali G, Srivastava PS, Iqbal M (2001) Responses of Bacopa monniera cultures to cadmium

toxicity. Bulletin of Environmental Contamination and Toxicology 66: 342–349.

Ali N, Hadi F (2015) Phytoremediation of cadmium improved with the high production of

endogenous phenolics and free proline contents in Parthenium hysterophorus plant

treated exogenously with plant growth regulator and chelating agent. Environmental

Science and Pollution Research 22, 13305-13318.

Allen SE (1974) Chemical Analysis of Ecological Materials. Blackwell Scientific Publication,

Oxford, London.

Al-Wakeel SAM, Hamed AA, Dadoura SS (1995) Interactive effects of water stress and

gibberellic acid on mineral composition of fenugreek plant. Egyptian Journal of

Physiological Sciences 18, 269-282.

Ambedkar G, Muniyan M (2013) Bioaccumulation of heavy and essential metals in the selected

freshwater fish species from Preumallake Cuddalore District, Tamilnadu, India. Indian

streams research journal 2(12), 1-7.

Arnon DI (1949) Copper enzymes in isolated chloroplasts, Polyphenoloxidae in Beta vulgaris

Plant Physiology 24, 1- 15.

Arnon DI, Stout PR (1939) Molybdenum as an essential element for higher plants. Plant

Physiology 14, 599–602.

Arora A, Byrem TM, Nari MG, Strasburg GM (2000) Modulation of liposomal membranes

fluidity by flavonoids and isoflavonoids. Archives of Biochemistry and Biophysics 373(1),

102-109.

Arteca RN (1996) Plant Growth Substances: Principles and Applications, Chapman and Hall Inc,

New York.

Arun KS, Carlos C, Herminia LZ, Avudainayagam S (2005) Chromium toxicity in plants.

Environment International 31, 739-753.

http://www.ncbi.nlm.nih.gov/pubmed/23271026

REFERENCES

153

Astolfi S, Zuchi S, Passera C (2005) Effect of cadmium on H+ATPase activity of plasma

membrane vesicles isolated from roots of different S-supplied maize (Zea mays L.) plants.

Plant Sciences 169, 361–368.

Baker AJM Brooks RR (1989) Terrestrial higher plants which hyperaccumulate metallic

elements – a review of their distribution ecology and phytochemistry. Biorecovery 1,

811–826.

Balaknina T, Kosobryukhov A, Ivanov A, Kreslauskii V (2005) The effect of cadmium on CO2

exchange, variable fluorescence of chlorophyll and the level of antioxidant enzymes in pea

leaves. Russian Journal of Plant Physiology 52, 15-20.

Bandurska H (2001) Does proline accumulated in leaves of water deficit stressed barley plants

confine cell membrane injuries? II. Proline accumulation during hardening and its

involvement in reducing membrane injuries in leaves subjected to severe osmotic stress.

Acta Physiol. Plant 23, 483-490.

Barcelo J, Poschenriede C (1990) Plant water relations as affected by heavy metal stress: A

review. Journal of Plant Nutrition 13, 1-37.

Bates LS, Waldren RP, Teare ID (1973) Rapid determination of free proline for water stress

studies. Journal of Plant Soil 39, 205–207.

Bavi K, Kholdebarin B, Moradshah A (2011) Effect of cadmium on growth, protein content and

peroxidase activity in pea plants. Pakistan Journal of Botany 43, 1467-1470.

Beak KH, Chang JY, Chang YY, Bae BH, Kim J, Lee IS (2006) Phytoremediation of soil

contaminated with cadmium and/or 2,4,6- Trinitrotoluene. Journal of Environmental

Biology 27, 311-316.

Benjerano NR, Lips SH (1970) Hormonal regulation of nitrate reductase activity in leaves. New

Phytologist 69(1), 165-169.

Berglund M, Akesson A, Nermell B, Vahter M (1994) Intestinal absorption of dietary cadmium

in women depends on body iron stores and fiber intake. Environmental Health

Perspectives 102, 1058-1066.

Bhattacharjee S, Mukherjee AK (1994) Influence of cadmium and lead on physiological and

biochemical responses of Vigna unguiculata (L). WalP. Seedling germination

REFERENCES

154

behaviour, total protein, proline content and protease activity. Pollution Research 13,

269-277.

Bhawmilk PC, Sarkar D (2005) Parthenium hysterophorus: Its world status and potential

management. pP. 1-6. In: Proceeding of the Second International Conference on

Parthenium Management 5-7 December 2005. University of Agricultural Science

Bangolore, India.

Blokhina O, Virolainen E, Fagerstedt KV (2003) Antioxidants, oxidative damage and oxygen

deprivation stress: a review. Annals of Botany 91, 179-194.

Bors W, Heller W, Michel C, Saran M (1990) Flavonoids as antioxidants: determination of

radical-scavenging efficiency. Methods in Enzymology 186, 343-355.

Broughton WJ, McComb AJ (1971) Changes in the pattern of enzyme development in

gibberellin-treated pea internodes. Annual Botany journal 35, 213-228.

Buchanan BB, Gruissem W, Jones RL (2000) Biochemistry and Molecular Biology of Plants,

American Society of Plant Physiologists, Rockville, Maryland, USA.

Century K, Reuber TL, Ratcliffe OJ (2008) Regulating the regulators: the future prospects for

transcription-factor-based agricultural biotechnology products. Plant Physiology 147, 20–

29.

Chai MW, Li RL, Shi FC, Liu FC, Pan X, Cao D, Wen X (2012) Effects of cadmium stress on

growth, metal accumulation and organic acids of Spartina alterniflora Loisel. African

Journal of Biotechnology 11(22), 6091-6099.

Chaudiere J, Ferrari-Iliou R (1999) Intracellular antioxidants: from chemical to biochemical

mechanisms. Food and Chemical Toxcology 37, 949-962.

Chen H, Cutright T (2001) EDTA and HEDTA effects on Cd, Cr and Ni uptake by Helianthus

annus. Chemosphere 45, 21–28

Chen Y, Shen Z, Li X (2004) The use of vetiver grass (Vetiveria zizanioides) in the

phytoremediation of soils contaminated with heavy metals. Applied Geochemistry 19,

1553-1565

REFERENCES

155

Cho U Seo N (2004) Oxidative stress in Arabidopsis thaliana exposed to cadmium is due to

hydrogen peroxide accumulation. Plant Sciences 168, 113-120.

Choi YE Harada E (2005) Roles of calcium and cadmium on Cd containing intra and

extracellular formation of Ca crystals in tobacco. Journal of Plant Biology 48, 113–119.

Choudhary NL, Sairam RK, Tyagi A (2005) Expression of delta1-pyrroline-5-carboxylate

synthetase gene during drought in rice (Oryza sativa L.). Indian Journal of Biochemistry

and Biophysics 42, 366–370.

Chugh LK Sawhney SK (1999) Photosynthetic activities of Pisum sativum seedlings grown in

presence of cadmium. Plant Physiology and Biochemistry 37, 297-303.

Chugh LK, Sawhney SK, Ghorbal MN, Ferjani EE (1997) Cadmium and zinc induction of lipid

peroxidation and effects on antioxidant enzymes activities in bean (Phaseolus vulgaris

L.). Plant Sciences 127, 139-147.

Citterio S, Santagostino A, Fumagalli P, Prato N, Ranalli P, Sgorbati S (2003) Heavy metal

tolerance and accumulation of Cd, Cr and Ni by Cannabis sativa L. Plant and Soil

256:243-252.

Cluis C (2004) Junk-greedy greens: phytoremediation as a new option for soil decontamination.

Biotechnology Journal 2:60–67.

Costa G, Morel J (1994) Water relations, gas exchange and amino acid content in Cd-treated

lettuce. Plant Physiology and Biochemistry 32, 561–570.

Crozier A, Turnbull CGN, (1984) Gibberellins: Biochemistry and action in extension growth.

What‘s New in Plant Physiology 15, 9-12.

Cunningham SD, Shann JR, Crowley DE, Anderson TA (1997) Phytoremediation of

contaminated water and soil. In E. L. Kruger, T. A. Anderson, & J. R. Coats (Eds.),

Phytoremediation of soil and water contaminants. ACS Symposium series 664 (pP. 2–19).

Washington, DC: American Chemical Society.

Curtis D, Klaassen JL, Choudhuri S (1999) Metallothionein: An Intracellular Protein to Protect

Against Cadmium Toxicity. Annual Review of Pharmacology and Toxicology 39, 267–294.

REFERENCES

156

De B, Mukherjee AK (1998) Mercury induced metabolic changes in seedlings and cultured cells

of tomato. Geobios 23, 83-88.

Debruyne D, Albessard F, Bigot MC, Moulin M (1994) Comparison of three advanced

chromatographic techniques for Cannabis identification. Bulletin on Narcotics 46, 109-

121.

Delauney AJ, Verma DPS (1993) Proline biosynthesis and osmoregulation in plants. Plant

Journal 41, 215–223.

Demiral T, Turkan I (2005) Comparative lipid peroxidation, antioxidant defense systems and

proline content in roots of two rice cultivars differing in salt tolerance. Environmental and

Experimental Botany 53, 247-57.

Demirevska-Kepava K, Simova-Stoilova L, Stoyanova Z, Feller U (2006) Cadmium stress in

barley: Growth, leaf pigment and protein composition and detoxification of reactive

oxygen species. Journal of Plant Nutrition 29, 451-468.

Devi SR, Prasad MNV (1998) Copper toxicity in Ceratophyllum demeresum L. (Coontail), a free

floating macrophyte: Response of antioxidant enzymes and antioxidants. Plant Sciences,

138:157.

Dhawan SR, Dhawan P (1996) Regeneration in Parthenium hysterophorus L. World Weeds 3,

181–182.

Dheri GS, Singh Brar M, Malhi SS (2007) Influence of phosphorus application on growth and

cadmium uptake of spinach in two cadmium-contaminated soils. Journal of Plant Nutrition

and Soil Sciences 170, 495–499.

di Toppi LS, Gabbrielli R (1999) Response to cadmium in higher plants. Environmental and

Expimental Botany 41, 105–130.

Diaz J, Bernal A, pomar F, Merino F (2001) Induction of shikimate dehydrogenase and

peroxidase in pepper (Capsicum annum L.) seedlings in response to copper stress and its

relation to lignifcation. Plant Sciences 1, 161-179.

Dubouzet JG, Sakuma Y, Ito Y, Kasuga M, Dubouzet EG, et al (2003) OsDREBgenes in

rice, Oryza sativa L., encode transcription activators that function in drought-, high-salt-

REFERENCES

157

and cold-responsive gene expression. Plant J 33: 751–763. doi: 10.1046/j.1365-

313x.2003.01661.x

Dushenkov V, Kumar PBAN, Motto H, Raskin I (1995) Rhizofiltration: The use of plants to

remove heavy metals from aqueous streams. Environmental Science and Technology 29,

1239–1245.

Ekmekci Y, Tanyolac D, Ayhan B (2008) Effects of cadmium on antioxidant enzyme and

photosynthetic activities in leaves of two maize cultivars. Journal of Plant Physiology

165, 600–611.

Entry JA, Watrud LS, Reeves M (1999) Accumulation of 137Cs and 90Sr from contaminated

soil by three grass species inoculated with mycorrhizal fungi. International Journal of

Environmental Pollution 104, 449-457.

Epelde L, Hernández-Allica J, Becerril JM, Blanco F, Garbisu C (2008) Effects of chelates on

plants and soil microbial community: comparison of EDTA and EDDS for lead

phytoextraction. Science of Total Environment 401, 21–28.

Evangelou MWH, Ebel M, Schaeffer A (2007) Chelate assisted phytoextraction of heavy metals

from soils; effect, mechanism, toxicity, and fate of chelating agents. Chemosphere 68,

989-1003.

Evins WH, Varner JE (1972) Hormonal control of polyribosome formation in barley aleurone

layers. Plant Physiology 49, 348-352.

Falkowska M, Pietryczuk A, Piotrowska A, Bajguz A, Grygoruk A, Czerpak R (2011) The effect

of gibberellic acid (GA3) on growth, metal biosorption and metabolism of the green algae

Chlorella vulgaris (chlorophyceae) Beijerinck exposed to cadmium and lead stress.

Polish Journal of Environmental studies 20, 53–59.

Faller P, Kienzler K, Krieger-Liszkay A (2005) Mechanism of Cd2+

toxicity: Cd2+

inhibits

photoactivation of Photosystem II by competitive binding to the essential Ca2+

site.

Biochimica et Biophysica Acta 1706, 158–164.

Farago ME, Mullen WA (1979) Plants which accumulate metals. Part IV. A possible copper-

proline complex from the roots of Armeria maritima. Inorg. Chim. Acta 32, L93–L94.

REFERENCES

158

Fortescue JAC (1992) Landscape geochemistry: retrospect and prospect. Applied Geochemistry

7, 1–53.

Fritioff A, Kautsky L, Greger M (2005) Influence of temperature and salinity on heavy metal

uptake by submersed plants. Environmental pollution 133, 265–274.

Fukutaku Y, Yamada Y (1984) Source of proline nitrogen in waterstressed soybean (glycine

max) ii. Fate of isn-labelled protein. Physiol. Plant 61, 622-628.

Genrich I, Burd D, George D, Glick BR (2000) Plant growth promoting bacteria that decrease

heavy metal toxicity in plants. Canadian Journal of Microbiology 46, 237–245.

Ghosh M, Singh SP (2005) A review on phytoremediation of heavy metals and utilization of its

byproducts. Applied Ecology and Environmental Research 3(1), 1-18.

Gilmour SJ, Sebolt AM, Salazar MP, Everard JD, Thomashow MF (2000) Overexpression of

Arabidopsis CBF3 transcriptional activator mimics multiple biochemical changes

associated with cold acclimation. Plant Physiology 124, 1854–1865.

Gilmour SJ, Zarka DG, Stockinger EJ, Salazar MP, Houghton JM, Thomashow MF (1998) Low

temperature regulation of the Arabidopsis CBF family of AP2 transcriptional activators as

an early step in cold-induced COR gene expression. The Plant Journal 16, 433–442.

Gonick HC (2008) Nephrotoxicity of cadmium & lead. Indian Journal of Medical Research 128,

335-352.

Gouia H, Ghorbala HH, Meyer C (2000) Effects of cadmium on activity of nitrate reductase and

on other enzymes of the nitrate assimilation pathway in bean. Plant Physiology and

Biochemistry 38, 629-638.

Goyer RA, Liu J, Waalkes MP (2004) Cadmium and cancer of prostate and testis. Biometals 17,

555-558.

Guo CN, Liu F, Xu XM (2006) Chlorophyll-b deficiency and photosynthesis in plants. Plant

Physiology Communications 42, 967-973

Guo TR, Zhang GP, Zhou MX, Wu FB, Chen JX (2007) Influence of aluminum and cadmium

stresses on mineral nutrition and root exudates in two barley cultivars. Pedosphere 17,

505–512.

http://www.sciencedirect.com/science/article/pii/S0981942800007750




http://www.sciencedirect.com/science/journal/09819428

http://www.sciencedirect.com/science/journal/09819428

http://www.sciencedirect.com/science/journal/09819428/38/7

REFERENCES

159

Gwozdz EA, Kopyra M (2003) Plant cell responses to heavy metals – biotechnological aspects.

Biotechnologia 3, 107–123.

Hadi F, Ali N, Ahmad A (2014) Enhanced phytoremediation of cadmium-contaminated soil by

Parthenium hysterophorus plant: effect of gibberellic acid (GA3) and synthetic chelator,

alone and in combinations. Bioremediation Journal 18(1), 46–55.

Hadi F, Bano A (2009) Utilization of Parthenium hysterophorus for the remediation of lead-

contaminated soil. Weed Biology and Management 9 (4), 307–314.

Hadi F, Bano A, Fuller MP (2010) The improved phytoextraction of lead (Pb) and the growth of

maize (Zea mays L.): the role of plant growth regulators (GA3 and IAA) and EDTA alone

and in combinations. Chemosphere 80, 457-462.

Hadi F, Gilpin M, FullerMP (2011) Identification and expression analysis of CBF/DREB1 and

COR15 genes in mutants of Brassica oleracea var. botrytis with enhanced proline

production and frost resistance. Plant Physiology and Biochemistry 49, 1323-1332.

Handique GK, Handique AK (2009) Proline accumulation in lemongrass (Cymbopogon

flexuosus Stapf.) due to heavy metal stress. Journal of Environmental Biology 30, 299-

302.

Haouari CC, Nasraoui AH, Bouthour D, Houda MD, Daieb CB, Mnai J, Gouia H (2012)

Response of tomato (Lycopersicon esculentum) to cadmium toxicity: Growth, element

uptake, chlorophyll content and photosynthesis rate. African Journal of Plant Sciences 6,

1–7.

Hardiman RT, Jacoby B (1984) Absorption and translocation of Cd in bush beans (Phaseolus

vulgaris). Physiologia Plantarum 61, 670–674.

Hartzendorf T, Rolletschek H (2001) Effect of NaCl-salinity on amino acid and carbohydrate

contents of Phragmites australis. Aquat. Bot 69, 195-208.

Hasan, S.A., B. Ali, S. Hayat and A. Ahmad (2007) Cadmium induced changes in the growth

and carbonic anhydrase activity of chickpea. Turkish Journal of Biology 31, 137-140.

Haudecoeur E, Tannieres M, Cirou A, Raffoux A, Dessaux Y, Faure D (2009) Proline

antagonizes GABA-induced quenching of quorum-sensing in Agrobacterium

http://onlinelibrary.wiley.com/doi/10.1111/wbm.2009.9.issue-4/issuetoc

REFERENCES

160

tumefaciens. Proceedings of the National Academy of Sciences of the United States of

America 106, 14587–14592.

Hayat S, Ali B, Hasan SA, Ahmad A (2007) Brassinosteroid enhanced the level of antioxidants

under cadmium stress in Brassica juncea. Environmental and Experimental Botany 60, 33-

41.

Hendy GAF, Baker AJM, Evart CF (1992) Cadmium tolerance and toxicity, oxygen radical

processes and molecular damage in cadmium tolerant and cadmium-sensitive clones of

Holcus lanatus. Acta Botanica Neerlandica 41, 271-281.

Hernandez LE, Carpena-Ruiz R, Garate A (1996) Influence of cadmium on the NO 3 −

assimilation of two cultivars of Zea mays. Developments in Plant and Soil Sciences 68,

207-212.

Hesberg C, Haensch R, Mendel RR, Bittner F (2004) Tandem orientation of duplicated xanthine

dehydrogenase genes from Arabidopsis thaliana. Journal of Biological Chemistry 279,

13547–13554.

Holleman AF, Wiberg E (2001), Inorganic Chemistry, San Diego: Academic Press.

Houshmandfar A, Moraghebi F (2011) Effect of mixed cadmium, copper, nickel and zinc on

seed germination and seedling growth of safflower. African Journal of Agricultural

Research 6(5), 1182-1187

Hristozkova M, Geneva M, Stancheva I (2006) Response of Pea Plants (Pisum sativum L.) to

Reduced Supply with Molybdenum and Copper. International Journal of Agriculture

and Biology 8(2), 218-220.

Huang D, Zhang Y, Qi Y, Chen C, Weihong J (2008) Global DNA hypomethylation, rather than

reactive oxygen species (ROS), a potential facilitator of cadmium-stimulated K562 cell

proliferation. Toxicology Letters 179, 43-47.

Huang GT, Ma SL, Bai LP, Zhang L, Ma H, Jia P, Liu J, Zhong M, Guo ZF (2012) Signal

transduction during cold, salt, and drought stresses in plants. Molecular Biology Reports

39, 969–987.

http://link.springer.com/search?facet-creator=%22L.+E.+Hern%C3%A1ndez%22

http://link.springer.com/search?facet-creator=%22R.+Carpena-Ruiz%22

http://link.springer.com/search?facet-creator=%22A.+G%C3%A1rate%22

REFERENCES

161

IARC (1993) Beryllium, Cadmium, Mercury and Exposures in the Glass Manufacturing

Industry, vol. 58. International Agency for Research on Cancer, Lyon, USA, ISBN

9283212584, pP. 119–238.

Inaba T, Kobayashi E, Suwazono Y, Uetani M, Oishi M, Nakagawa H, Nogawa K. (2005)

Estimation of cumulative cadmium intake causing Itai-itai disease. Toxicology Letters

159(2), 192-201.

Ito Y, Katsura K, Maruyama K, Taji T, Kobayashi M, et al (2006) Functional analysis of rice

DREB1/CBF-type transcription factors involved in cold-responsive gene expression in

transgenic rice. Plant Cell Physiol 47: 141–153. doi: 10.1093/pcp/pci230

Jadia CD, Fulekar MH (2009). Phytoremediation of heavy metals: Recent techniques. African

Journal of Biotechnology 8(6), 921-928.

Jaglo-Ottosen KR, Gilmour SJ, Zarka DG, Schabenberger O, Thomashow MF (1998)

Arabidopsis CBF1 overexpression induces cor genes and enhances freezing tolerance.

Science 280, 104–106.

Jarup L, Akesson A (2009) Current status of cadmium as an environmental health

problem. Toxicology and Applied Pharmacology 238, 201-208.

Jarup L, Berglund M, Elinder CG, Nordberg G, Vahter M (1998) Health effects of cadmium

exposure- a review of the literature and a risk estimate. Scandinavian Journal

of Work, Environment & Health 24 (1), 1-5.

John R, Ahmad P, Gadgil K, Sharma S (2008) Effect of cadmium and lead on growth,

biochemical parameters and uptake in Lemna polyrrhiza L. Plant soil environment 54(6),

262–270

John R, Ahmad P, Gadgil K, Sharma S (2009) Heavy metal toxicity: effect on plant growth,

biochemical parameters and metal accumulation by Brassica juncea L. International

Journal of Plant Production 3, 65–76

Jones RW, Abbott AJ, Hewitt EJ, James DM, Best GR (1976) Nitrate reductase activity and

growth in Paul‘s scarlet rose suspension cultures in relation to nitrogen source and

molybdenum. Planta 133, 27–34.

REFERENCES

162

Joseph P (2009) Mechanisms of cadmium carcinogenesis. Toxicology and Applied

Pharmacology 238, 272- 279.

Jung CH, Maeder V, Funk F, Frey B, Sticher H, Frosserd E (2003) Release of phenols from

Lupinus albus L. roots exposed to Cu and their possible role in Cu detoxification. Plant

Soil 252, 301-312.

Kadpal R P, Rao N A (1985) Alterations in the biosynthesis of proteins and nucleic acids in

finger millet (eleucine coracana) seedlings during water stress and the effect of proline on

protein biosynthesis. Plant science 40, 73 -79.

Kadukova J, Manousaki E, and Kalogerakis N (2006) Lead and cadmium accumulation from

contaminated soils by Nerium oleander. Acta Metallurgica Slovaca 12, 181–187.

Kafel A, Rozpędek K, Szulińska E, Zawisza-Raszka A, Migula P (2014) The effects of

cadmium or zinc multigenerational exposure on metal tolerance of Spodoptera exigua

(Lepidoptera: Noctuidae). Environmental Science and Pollution Research 21, 4705–

4715.

Kaiser BN, Gridley KL, Brady JN, Phillips T, Tyerman SD (2005) The Role of Molybdenum in

Agricultural Plant Production. Annals of Botany 96, 745–754.

Kasuga M, Liu Q, Miura S, Yamaguchi-Shinozaki K, Shinozaki K (1999) Improving plant

drought, salt, and freezing tolerance by gene transfer of a single stress-inducible

transcription factor. Nature Biotechnology 17, 287–291.

Kellen E, Zeegers MP, Hond ED, Buntinx F (2007) Blood cadmium may be associated with

bladder carcinogenesis: the Belgian case-control study on bladder cancer. Cancer

Detection and Prevention 31(1), 77-82.

Kensa VM, Yasmi SS (2011) Phytochemical screening and antibacterial activity on Ricinus

communis L. Plant Sciences Feed 1 (9), 167-173.

Kevresan S, Krstic B, Popovic M, Kovacev L, Peyevic S, Kandrac J, Malencici K (1998)

Biochemical changes in sugar beet lines in dependence of soil moisture. Biologia

Plantarum 40, 245-250.

REFERENCES

163

Khan AG, Kuek TM, Chaudhury TM, Khoo CS, Hayes WJ (2000) Role of plants, mycorrhizae

and phytochelators in heavy metal contaminated land remediation. Chemosphere 41, 197-

207.

Khan MH, Patra HK (2007) Sodium chloride and cadmium induced oxidative stress and

antioxidant response in Calamus tenuis leaves. Indian Journal of Plant Physiology 12, 34-

40.

Khan MH, Singha LB, Panda SK (2002) Changes in antioxidant levels in Oryza sativa L. roots

subjected to NaCl-salinity stress. Acta Physiologiae Plantarum 24, 145-148.

Khan NA, Ansari HR, Samiullah (1998) Effect of gibberellic acid spray during ontogeny of

mustard on growth, nutrient uptake and yield characteristics. Journal

of Agronomy and Crop Science 181, 61-73

Khatamipour M, Piri E, Esmaeilian Y, Tavassoli A (2011) Toxic effect of cadmium on

germination, seedling growth and proline content of Milk thistle (Silybum marianum).

Annals of Biological Research 2 (5), 527-532.

Kidd PS, Domínguez-Rodríguez MJ, Díez J, Monterroso C (2007) Bioavailability and plant

accumulation of heavy metals and phosphorus in agricultural soils amended by long-term

application of sewage sludge. Chemosphere 66: 1458–1467

Kim CG, Bell JNB, and Power SA. 2003. Effects of soil cadmium on Pinus sylvestris L.

seedlings. Plant and Soil 257, 443–449.

Kirkham MB (2006) Cadmium in plants on polluted soils: Effects of soil factors,

hyperaccumulation, and amendments. Geoderma 137, 19–32.

Knight H, Zarka DG, Okamoto H, Thomashow MF, Knight MR (2004) Abscisic Acid

Induces CBF Gene Transcription and Subsequent Induction of Cold-Regulated Genes via

the CRT Promoter Element. Plant Physiology 135, 1710–1717.

Kohan E, Bagherieh-Najjar MB (2011) DRE-binding Transcription factor (DREB1A) as a

master regulator induced a broad range of abiotic stress tolerance in plant. African Journal

of Biotechnology 10(67), 15100-15108.

Kovaeevic G, Kastori R, and Merkulov LJ (1999) Dry matter and leaf structure in young wheat

plants as affected by cadmium, lead, and nickel. Biologia Plantarum 42, 119–123.

REFERENCES

164

Kozuchowski J, Johnson DL (1978) Gaseous emissions of mercury from an aquatic vascular

plant. In: Ebinghaus R, Baeyens W, Vasiliev O, editors. Novosibersk, Siberia, NATO ASI

Series. Nature 274, 468–469.

Kriegel AM, Soliman AS, Zhang Q, El-Ghawalby N, Ezzat F, Soultan A, Abdel-Wahab

M, Fathy O, Ebidi G, Bassiouni N, Hamilton SR, Abbruzzese JL, Lacey MR, Blake DA

(2006) Serum cadmium levels in pancreatic cancer patients from the East Nile Delta region

of Egypt. Environmental Health Perspectives 114, 113-119.

Krocova Z, Macela A, Kroca M, Hernychova L (2000) The immunomodulatory effect(s) of lead

and cadmium on the cells of immune system in vitro. Toxicology in vitro 14, 33-40.

Kudo H, Kudo K, Ambo H, Uemura M, Kawai S (2011) Cadmium sorption to plasma membrane

isolated from barley roots is impeded by copper association onto membranes. Plant

Sciences 180, 300–305.

Kume S, Kobayashi F, Ishibashi M, Ohno R, Nakamura C, Takumi S (2005) Differential and

coordinated expression of Cbf and Cor/Lea genes during long-term cold acclimation in two

wheat cultivars showing distinct levels of freezing tolerance. Genes and Genetic

System 80, 185–197.

Lakshaman KC, Surinder KS (1999) Photosynthetic activities of Pisum sativum seedlings grown

in presence of cadmium. Plant Physiology and Biochemistry 37, 297-303.

Lalk I, Dorfling K (1985) Hardening, ABA, proline and freezing resistance in the winter wheat

varieties. Physiologia Plantarum 63, 287–292.

Larcher W (1995) Physiological Plant Ecology: Ecophysiology and Stress Physiology of

Functional Groups (3rd edition). New York: Springer.

Lata C, Prasad M, (2011) Role of DREBs in regulation of abiotic stress responses in plants.

Journal of Experimental Botany 62, 4731–4748.

Lavola A, Julkunen-Titto R, Delarosa TM, Lehto T, Aphalo PJ (2000) Allocation of carbon to

growth and secondary metabolites in birch seedlings under UV-B radiation and CO2

exposure. Physiologia Plantarum 109, 260-267.

http://www.ncbi.nlm.nih.gov/pubmed/?term=Abdel-Wahab%20M%5BAuthor%5D&cauthor=true&cauthor_uid=16393667

http://www.ncbi.nlm.nih.gov/pubmed/?term=Abdel-Wahab%20M%5BAuthor%5D&cauthor=true&cauthor_uid=16393667

http://www.ncbi.nlm.nih.gov/pubmed/?term=Fathy%20O%5BAuthor%5D&cauthor=true&cauthor_uid=16393667

http://www.ncbi.nlm.nih.gov/pubmed/?term=Ebidi%20G%5BAuthor%5D&cauthor=true&cauthor_uid=16393667

http://www.ncbi.nlm.nih.gov/pubmed/?term=Bassiouni%20N%5BAuthor%5D&cauthor=true&cauthor_uid=16393667

http://www.ncbi.nlm.nih.gov/pubmed/?term=Hamilton%20SR%5BAuthor%5D&cauthor=true&cauthor_uid=16393667

http://www.ncbi.nlm.nih.gov/pubmed/?term=Abbruzzese%20JL%5BAuthor%5D&cauthor=true&cauthor_uid=16393667

http://www.ncbi.nlm.nih.gov/pubmed/?term=Lacey%20MR%5BAuthor%5D&cauthor=true&cauthor_uid=16393667

http://www.ncbi.nlm.nih.gov/pubmed/?term=Blake%20DA%5BAuthor%5D&cauthor=true&cauthor_uid=16393667

REFERENCES

165

Li YM, Chaney R, Brewer E, Roseberg R, Angle JS, Baker AJM, Reeves R, Nelkin J (2003)

Development of a technology for commercial phytoextraction of nickel: economic and

technical considerations. Plant and Soil 249, 107–115.

Li Z, Li L, Chen GPJ (2005) Bioavailability of Cd in a soil–rice system in China: soil type versus

genotype effects. Plant and Soil 271, 165–173.

Linger P, Ostwald A, Haensler J (2005) Cannabis sativa L. growing on heavy metal

contaminated soil: growth, cadmium uptake and photosynthesis. Physiologia Plantarum

49(4), 567–576.

Liu J, Zhu Q, Zhang Z, Xu J, Yang J, Wong MH (2005) Variations in cadmium accumulation

among rice cultivars and types and the selection of cultivars for reducing cadmium in the

diet. Journal of the Science of Food and Agriculture 85, 147–153.

Liu Q, Kasuga M, Sakuma Y, Abe H, Miura S, Goda H, Shimada Y, Yoshida S, Shinozaki K,

Yamaguchi-Shinozaki K (1998) Two transcription factors, DREB1 and DREB2, with an

EREBP/AP2 DNA binding domain separate two cellular signal transduction pathways in

drought- and low-temperature-responsive gene expression, respectively, in Arabidopsis.

The Plant Cell 10, 391–406.

Liu Z, He X, Chen W, Yuan F, Yan K, Tao D (2009) Accumulation and tolerance

characteristics of cadmium in a potential hyperaccumulator— Lonicera japonica Thunb.

Water Air Soil Pollution 196, 29–40.

Lone MI, He Z, Stoffella PJ, Yang X (2008) Phytoremediation of heavy metal polluted soils and

water: progresses and perspectives. Journal of Zhejiang University-SCIENCE B 9, 210-

220.

Lou LQ, Ye ZH, Wong MH (2007) Solubility and accumulation of metals in chinese brake fern,

vetiver and rostrate sesbania using chelating agents. International Journal of

Phytoremediation 9, 325–343.

Ma LQ, Komar KM, Tu C, Zhang W, Cai Y, Kennelley ED (2001) A fern that hyperaccumulates

arsenic. Nature 409, 579-579.

Magnani E, Sjölander K, Hake S (2004) From endonucleases to transcription factors: evolution

of the AP2 DNA binding domain in plants. The Plant Cell 16, 2265–2277.

REFERENCES

166

Mamindy-Pajany Y, Sayen S, Guillon E (2014) Impact of lime-stabilized biosolid application on

Cu, Ni, Pb and Zn mobility in an acidic soil. Environmental Science and Pollution

Research 21, 4473–4481.

Mandhania S, Madan S, Sawhney V (2006) Antioxidant defense mechanism under salt stress in

wheat seedlings. Boilogia Plantrium 50(2), 227-231.

Marin A, Marion-Poll A (1997) Tomato flacca mutant is impaired in ABA aldehyde oxidase and

xanthine dehydrogenase activities. Plant Physiology and Biochemistry 35, 369–372.

Marschner (1995) Mineral Nutrition of Higher Plants. New York: Academic Press.

Martelli A, Rousselet E, Dycke C, Bouron A, Moulis JM (2006) Cadmium toxicity in animal

cells by interference with essential metals. Biochimie 88, 1807-1814.

Marth E, Barth S, Jelovcan S (2000) Influence of cadmium on the immune system. Description

of stimulating reactions. Central European Journal of Public Health 8, 40-44.

Masroor M, Khan A, Gautam C, Mohammad F, Siddiqui MH, Naeem M, Khan MN (2006)

Effect of Gibberellic Acid Spray on Performance of Tomato. Turkish Journal of Biology

30, 11-16.

McElroy JA, Shafer MM, Trentham-Dietz A, Hampton JM, Newcomb PA (2006) Cadmium

exposure and breast cancer risk. Journal of the National Cancer Institute's 98(12), 869-

873.

McLaughlin MJ, Whatmuff M, Warne M, Heemsbergen D, Barry G, Bell M, Nash D, Pritchard

D (2006) A field investigation of solubility and food chain accumulation of biosolid-

cadmium across diverse soil types. Environmental Chemistry 3(6), 428-432.

Meers E, Lesage E, Lamsal S, Hopgood M, Vervaekle P, Tack FMG, Verloo MG (2005)

Enhanced phytoextraction: I. Effect of EDTA and citric acid on heavy metal uptake by

Heliantus annus from a calcareous soil. International Journal of Phytoremediation 7,

129–142.

Mehta S K, Gaur J P (1999) Heavy-metal-induced proline accumulation and its role in

ameliorating metal toxicity in Chlorella vulgaris. New Phytol 143, 253–259

REFERENCES

167

Mendel RR, Haensch R (2002) Molybdoenzymes and molybdenum cofactor in plants. Journal of

Experimental Botany 53, 1689–1698.

Metwally A, Safronova VI, Belimov AA, Dietz KJ (2005) Genotypic variation of the response to

cadmium toxicity in Pisum sativum L. Jorunal of Experimental Botany 56, 167–178.

Michalak A (2006) Phenolic Compounds and Their Antioxidant Activity in Plants Growing

under Heavy Metal Stress. Polish Journal of Environmental Studies 15(4), 523-530.

Miller R (1996) Phytoremediation, Technology Overview Report, Ground-Water. Remediatoin

Technologies Analysis Center, Series O, Vol. 3.

Millic BL, Djilas SM, Canadanovic-Brunet JM (1998) Antioxidative activity of phenolic

compounds on the metal-ion breakdown of lipid peroxidation system. Food Chemistry 61,

443-447.

Mishra S, Srivastava S, Tripathi RD, Govindarajan R, Kuriakase SV, Prasad MNV (2006)

Phytochelatin synthesis and response of antioxidants during cadmium stress in Bacopa

monnieri L. Plant Physiology and Biochemistry 44, 25-37.

Mobin M, Nafees AK (2007) Photosynthetic activity, pigment composition and antioxidative

response of two mustard (Brassica juncea) cultivars differing in photosynthetic capacity

subjected to cadmium stress. Journal of Plant Physiology 164, 601-610.

Mohan BS, Hosetti BB (2006) Phytotoxicity of cadmium on the physiological dynamics of

Salvinia natans L. grown in macrophyte ponds. Journal of Environmental Biology 27:701-

704.

Moore TC (1989) Biochemistry and Physiology of Plant Hormones, Springer-Verlag Inc, New

York.

Mubeen H, Naeem I, Taskeen A, (2010) Phytoremediation of Cu (II) by Calotropis Procera

Roots. New York Science Journal 3(3), 1-5

Nakano T, Suzuki K, Fujimura T, Shinshi H (2006) Genome wide analysis of the ERF gene

family in Arabidopsis and rice. Plant Physiology 140, 411–432.

Nakashima K, Shinwari ZK, Sakuma Y, Seki M, Miura S, Shinozaki K, Yamaguchi-Shinozaki K

(2000) Organization and expression of two Arabidopsis DREB2 genes encoding DRE-

REFERENCES

168

binding proteins involved in dehydration- and high-salinity-responsive gene expression.

Plant Molecular Biololgy 42, 657–665.

Navas-Acien A, Silbergeld EK, Sharrett R, Calderon-Aranda E, Selvin E, Guallar E (2005)

Metals in urine and peripheral arterial disease. Environmental Health Perspectives 113,

164-169.

Navie SC, McFadyen RC, Panetta FD, Adkins SW (1996) The Biology of Australian weed

Parthenium hysterophorus L. Plant Protection Quarterly 11, 76-88.

Nawrot T, Plusquin M, Hogervorst J, Roels HA, Celis H, Thijs L, Vangronsveld J, Van Hecke

E, Staessen JA (2006) Environmental exposure to cadmium and risk of cancer: a

prospective population-based study. Lancet Oncology 7(2), 119-126.

Ngayila N, Botineau M, Baudu M, Basly JP (2008) Myriophyllum alterniflorum DC. Effect of

low concentrations of copper and cadmium on somatic and photosynthetic endpoints: A

chemometric approach. Ecological Indicators 9, 379- 384.

Nikolic N, Kojic D, Pilipovic A, Pajevic S, Krstic B, Borisev M, Orlovic S (2008) Response of

hybrid popular to cadmium stress: Photosynthetic characteristics, cadmium and proline

accumulation, and antioxidant enzyme activity. Acta Biologica Cracoviensia series

botanica 50(2), 95-103.

Nikolopoulos D, Manetas Y (1991) Compatible solutes and in vitro stability of salsola soda

enzymes: proline incompatibility. Phytochemistry 30,411-413.

Nriagu J (1988) A silent killer of environmental metal poisoning. Environmental Pollution 50(1-

2), 139-161.

Oono Y, Yazawa T, Kawahara Y, Kanamori H, Kobayashi F, Sasaki H, et al (2014) Genome-

Wide Transcriptome Analysis Reveals that Cadmium Stress Signaling Controls the

Expression of Genes in Drought Stress Signal Pathways in Rice. PLoS ONE 9(5): e96946.

doi:10.1371/journal.pone.0096946

Oporto C, Smolders E, Degryse F, Verheyen L, Vandecasteele C (2009) DGT-measured fluxes

explain the chloride-enhanced cadmium uptake by plants at low but not at high Cd supply.

Plant and Soil 318, 127–135.

http://www.ncbi.nlm.nih.gov/pubmed/?term=Vangronsveld%20J%5BAuthor%5D&cauthor=true&cauthor_uid=16455475

http://www.ncbi.nlm.nih.gov/pubmed/?term=Van%20Hecke%20E%5BAuthor%5D&cauthor=true&cauthor_uid=16455475

http://www.ncbi.nlm.nih.gov/pubmed/?term=Van%20Hecke%20E%5BAuthor%5D&cauthor=true&cauthor_uid=16455475

http://www.ncbi.nlm.nih.gov/pubmed/?term=Staessen%20JA%5BAuthor%5D&cauthor=true&cauthor_uid=16455475

REFERENCES

169

Ozden M, Demirel U, Kahraman A (2009) Effects of proline on antioxidant system in leaves of

grapevine (Vitis vinifera L.) exposed to oxidative stress by H2O2. Scientia

Horticulturae 119, 163–168.

Ozturk L, Eker S, and Ozkutlu F (2003) Effect of cadmium on growth and concentrations of

cadmium, ascorbic acid and sulphydryl groups in durum wheat cultivars. Turkish Journal

of Agriculture and Forestry 27, 161–168.

Padmavathiamma PK, Li LY (2007) Phytoremediation Technology: Hyper-accumulation Metals

in Plants. Water Air Soil Pollution 184, 105–126.

Pal M, Horvath E, Janda T, Paldi E, Szalai G (2006) Physiological changes and defence

mechanisms induce by cadmium stress in maize. Journal of Plant Nutrition and Soil

Science 169, 239-246.

Paleg L G, Stewart G R, Bradbeer J W (1984) Proline and glycine betaine influences protein

solvation. Plant physiology 75, 974-978.

Panda SK, Khan MH (2003) Salt stress influences lipid peroxidation and antioxidants in the leaf

of an indica rice (Oryza sativa L.). Physiology and Molecular Biology of Plants 9,273-278.

Parsons WT, Cuthbertson EG (1992) Noxious Weeds of Australia. Inkata Press, Melbourne.

Perfus-Barbeoch L, Leonhardt N, Vavasseur A, Forestier C (2002) Heavy metal toxicity:

cadmium permeates through calcium channels and disturbs the plant water status. Plant

Journal 32, 539–548.

Pesch B, Haerting J, Ranft U, Klimpel A, Oelschlagel B, Schill W (2000) Occupational risk

factors for renal cell carcinoma: agent-specific results from a case-control study in

Germany. MURC Study GrouP. Multicenter urothelial and renal cancer study.

International Journal of Epidemiology 29, 1014-1024.

Poschenrieder C, Gunse G, Barcelo J (1989) Influence of Cadmium on Water Relations,

Stomatal Resistance, and Abscisic Acid Content in Expanding Bean Leaves. Plant

Physiology 90, 1365-1371.

Prasad MNV, Freitas HMDO (2003) Metal hyperaccumulation in plants-biodiversity prospecting

for phytoremediation technology. Electron Journal of Biotechnology 6(3), 110–146.

REFERENCES

170

Rajkumar M, Freitas H (2008) Influence of metal resistant-plant growth promoting bacteria on

the growth of Ricinus communis in soil contaminated with heavy metals. Chemosphere 71

(5), 834–842.

Ramadevi S, Prasad MNV (1998) Copper toxicity in Ceratophyllum demeresum l. (coontail), a

free floating macrophyte: Response of antioxidant enzymes and antioxidants.

Plant Sciences 138-157.

Rana M, Dhamija H, Prashar B, Sharma S (2012) Ricinus communis L. – A Review.

International Journal PharmTech, Research 4(4), 1706-1711.

Raskin I, Smith RD, Salt DE (1997) Phytoremediation of metals: using plants to remove

pollutants from the environment. Current Opinion in Biotechnology 8, 221–226.

Reddy KJ, Munn LC, Wang L (1997) Chemistry and mineralogy of molybdenum in soils. In:

Gupta UC, ed. Molybdenum in agriculture. Cambridge: Cambridge University Press.

Revathi K, Haribabu TE, Sudha PN (2011). Phytoremediation of Chromium contaminated soil

using Sorghum plant. International Journal of Environmental Sciences 2(2), 417-428.

Rice-Evans CA, Miller NJ, Paganga G (1997). Antioxidant properties of phenolic compounds.

Trends Plant Sciences 2, 152-159.

Rogers NJ, Franklin NM, Apte SC, Batley GE (2007) The Importance of Physical and Chemical

Characterization in Nanoparticle Toxicity Studies. Integrated Environmental

Assessment and Management 3, 303-304.

Ryu DY, Lee SJ, Park DW, Choi BS, Klassen CD, Park JD (2004) Dietary iron regulates

intestinal cadmium absorption through iron transporters in rats. Toxicology Letters 152, 19-

25.

Saco D, Alvarez M, Martin S (1995) Activity of nitrate reductase and the content of proteins in

Nicotiana rustica grown with various levels of molybdenum. Journal of Plant Nutrition

18, 1149-1153.

Sagi M, Scazzocchio C, Fluhr R (2002) The absence of molybdenum cofactor sulfuration is the

primary cause of the flacca phenotype in tomato plants. Plant Journal 31, 305–317.

REFERENCES

171

Sahw BP, Sahu SK, Mishra RK (2004) Heavy metal induced oxidative damage in terrestrial

plants. In: Presad MNV (Ed.), Heavy Metal Stress in Plants: From Biomolecules to

Ecosystems, second ed. Springer, Berlin, pp 84–126.

Sakihama Y, Mano J, Sano S, Asada K, Yamasaki H (2000) Reduction of phenoxyl radicals

mediated by monodehydroascorbate reductase. Biochemical and Biophysical Research

Communications 279, 949-954.

Sakuma Y, Liu Q, Dubouzet JG, Abe H, Shinozaki K, Yamaguchi-Shinozaki K (2002) DNA-

binding specificity of the ERF/AP2 domain of Arabidopsis DREBs, transcription factors

involved in dehydration- and cold-inducible gene expression. Biochemical and Biophysical

Research Communications 29, 998–1009.

Salt DE, Blaylock M, Kumar PBAN, Dushenkov V, Ensley BD, Chet L, Chet I, Raskin I (1995)

Phytoremediation: a novel strategy for the removal of toxic metals from the environment

using plants. Biogeochemistry13, 468–474.

Sarkari A, Ravindran G, Krishnamurthy V (2013) A brief review on the effect of cadmium

toxicity: from cellular to organ level. International Journal of Biotechnology and Research

3(1), 17-36.

Schutzendubel A, Schwanz P, Teichmann T, Gross K, Langenfeld-Heyser R, Godbold DL, Polle

A (2001) Cadmium-induced changes in antioxidative systems, hydrogen peroxide

content, and differentiation in Scots pine roots. Plant Physiology 127, 887–898.

Schwartz SS, Leon-Kloosterziel KM, Koorneef M, Zeevart JAD (1997) Biochemical

characterisation of the ab2 and ab3 mutants in Arabidopsis thaliana. Plant Physiology 117,

161–166.

Schwitzguebel JP, Kumpiene J, Comino E, Vanek T (2009) From green to clean: a promising

and sustainable approach towards environmental remediation and human health for the

21 st century. Agrochimica 53, 209-237.

Sharma SS, Schat H, Vooijs R (1998) In vitro alleviation of heavy metal-induced enzyme

inhibition by proline. Phytochemistry 49, 1531–1535.

REFERENCES

172

Shen YG, Zhang WK, Yan DQ, Du BX, Zhang JS, Liu Q, Chen SY (2003) Characterization of a

DRE-binding transcription factor from a halophyte Atriplex hortensis.

Theoretical and Applied Genetics 107, 155–161.

Shi GR, Cai QS (2009) Cadmium tolerance and accumulation in eight potential energy crops.

Biotechnology Advances 27(5), 555–561.

Shinozaki K, Yamaguchi-Shinozaki K (2007) Gene networks involved in drought stress response

and tolerance. Journal of Experimental Botany 58, 221–227.

Siddiqui MF (2010). Cadmium induced renal toxicity in male rats, Rattus rattus. Eastern Journal

of Medicine 15, 93-9.

Singh RK, Gupta MK, Singh AK, Kumar S (2010) Pharmacognostical Investigation Of Ricinus

communis Stem. International Journal of Pharmaceutical Sciences and Research 1(6), 89-

94.

Singleton VL, Rossi JA (1965) Colorimetry of total phenolics with phosphomolybdic-

phosphotungstic acid reagents. American Journal of Enology and Viticulture 16, 144-

158.

Smeets K, Cuypers A, Lambrechts A, Semane B, Hoet P, Vanlaere A, Vangronsveld J (2005)

Induction of oxidative stress and antioxidative mechanisms in Phaseolus vulgaris after Cd

application. Plant Physiology and Biochemistry 43, 437-444.

Smirnoff N, Cumbes Q J (1989) Hydroxyl radical scavenging activity of compatible solutes.

Phytochemistry 28, 1057–1060.

Sogut Z, Zaimoglu BZ, Erdogan R, Sucu YM (2005) Phytoremediation of landfill leachate using

Pennisetum clandestinum. Journal of Environmental Biology 26, 13-20.

Somashekaraiah BV, Padmaja K, Prasad APK (1992) Phytotoxicity of cadmium ion in

germinating seedling of mung bean (Phaseolus vulgaris): Involvement of lipid peroxides in

chlorophyll degradation. Physiologia Plantarum 85, 85-89.

Stiborova M, Doubrovava M, Brezninova A, Frederick A (1986) Effect of heavy metal ion on

growth and biochemical characteristics of photosynthesis of barley Hordeum vulgare L.

Phytosynthetica 20, 418-425.

REFERENCES

173

Stockinger EJ, Gilmour SJ, Thomashow MF (1997) Arabidopsis thaliana CBF1 encodes an AP2

domain-containing transcriptional activator that binds to the C-repeat/DRE, a cis-acting

DNA regulatory element that stimulates transcription in response to low temperature and

water deficit. Proceedings of the National Academy of Sciences USA 94, 1035–1040.

Sumanta N, Haque CI, Nishika J, Suprakash R (2014) Spectrophotometric Analysis of

Chlorophylls and Carotenoids from Commonly Grown Fern Species by Using Various

Extracting Solvents. Research Journal of Chemical Sciences 4(9), 63-69.

Sun Q, Ye ZH, Wang XR, Wong MH (2007). Cadmium hyperaccumulation leads to an increase

of glutathione rather than phytochelatins in the cadmium hyperaccumulator Sedum

alfredii. Journal of Plant Physiology 164, 1489–98.

Sun Y, Zhou Q, Diao CY (2008) Effects of cadmium and arsenic on growth and metal

accumulation of Cd-hyperaccumulator Solanum nigrum L. Bioresource Technology

99(5), 1103–1110.

Sun Y, Zhou Q, Xu Y, Wang L, Liang X (2011) The role of EDTA on cadmium phytoextraction

in a cadmium hyperaccumulator Rorippa globosa. Journal of Environmental

Chemistry and Ecotoxicology 3(3), 45-51.

Suzuki N, Koizumi N, Sano H (2001) Screening of cadmium-responsive genes in Arabidopsis

thaliana. Plant Cell and Environment 24, 1177–1188.

Takahama U, Oniki T (2000) Flavonoid and some other phenolics as substrates of peroxidase:

physiological significance of the redox reactions. Journal of Plant Research 113, 301-309.

Takiguchi M, Achanzar W, Qu W, Li G, Waalkes M (2003) Effects of cadmium on DNA-(-

cytosine-5) methyltransferase activity and DNA methylation status during cadmium

induced cellular transformation. Experimental Cell Research 286, 355-365.

Tamas L, Huttova J, Zigova Z (1997) Accumulation of stress protein in intracellular spaces of

barley leaves induced by biotic and abiotic factors. Biol Plant 39:387-394.

Tassi E, Pouget J, Petruzzelli G, Barbafieri M (2008) The effects of exogenous plant growth

regulators in the phytoextraction of heavy metals. Chemosphere 71, 66–73.



REFERENCES

174

Tausz M, Wonisch A, Grill D, Morales D, Jimenez MS (2003) Measuring antioxidants in tree

species in the natural environment: from sampling to data evaluationm. Journal of

Experimental Botany 54, 1505-1510.

Thapar R, Srivastava AK, Bhargava P, Mishra Y, Rai LC (2008) Impact of different abiotic

stress on growth, photosynthetic electron transport chain, nutrient uptake and enzyme

activities of Cu-acclimated Anabaena doliolum. Journal of Plant Physiology 165, 306-

316

Kume Tordoff GM, Baker AJM, Willis AJ (2000) Current approaches to the revegetation and

reclamation of metalliferous mine wastes. Chemosphere 41(1–2), 219–228.

Tran TA, Popova LP (2013) Functions and toxicity of cadmium in plants: recent advances and

future prospects. Turkish Jouranl of Botany 37, 1-13.

Tuteja N, Gill SS, Tiburcio AF, Tuteja R (2012) Improving crop resistance to abiotic stress.

Wiley-Blackwell publishers, Germany, P. 249.

Umezawa T, Fujita M, Fujita Y, Yamaguchi-Shinozaki K, Shinozaki K (2006) Engineering

drought tolerance in plants: discovering and tailoring genes to unlock the future. Current

Opinion in Biotechnology 17, 113–122.

Uraguchi S, Watanabe1 I, Yoshitomi A, Kiyono M, Kuno K (2006) Characteristics of cadmium

accumulation and tolerancein novel Cd-accumulating crops, Avena strigosa and

Crotalaria juncea. Journal of Experimental Botany 57 2955–2965

USEPA (2007) Integrated Risk Information System (IRIS) home page.

http://www.epa.gov/iris/subst/index.html. U.S. Environmental Protection Agency, Office of

Emergency and Remedial Response, Washington, DC.

Vahter M, Akesson A, Liden C, Ceccatelli C, Berglund M (2007) Gender differences in the

disposition and toxicity of metals. Environmental Research 104, 85-9.

Vajpayee P, Tripathi RD, Rai UN, Ali, Singh SN (2000) Chromium (VI) accumulation reduces

chlorophyll biosynthesis, nitrate reductase activity and protein content in Nymphaea alba

L. Chemosphere 41, 1075-1082

http://www.epa.gov/iris/subst/index.html

REFERENCES

175

Vargas R, Ramirez C (1989) Response of soyabeans and groundnuts to Rhizobium and to N, P

and Mo fertilizer on a Typic Pellustert from Canas, Guanacaste. Agronomia

Costarricense 13(2), 175-181.

Vassilev A, Perez-Sanz A, Semane B, Carteer R, Vangronsveld J (2005) Cadmium

accumulation and tolerance of two salix genotypes hydrophonically grown in presence of

cadmium. Journal of Plant Nutrition 28, 2159-2177.

Venekamp JH (1989) Regulation of cytosolic acidity in plants under conditions of drought.

Physiol Plant 76, 112-117.

Venekamp JH, Lampe JE, Koot JTM (1989) Organic acids as a sources of drought-induced

proline synthesis in field bean plants, vicia faba. International Journal of Plant physiology

133, 654 - 659.

Verstraeten SV, Keen CL, Schmitz HH, Fraga CG, Oteiza PI (2003) Flavan-3-ols and

procyanidins protect liposomes against lipid oxidation and disruption of the bilayer

structure. Free Radical Biology and Medicine 34, 84-92.

Waalkes M (2003) Cadmium carcinogenesis. Mutation Research 533, 107-120.

Waalkes M, Misra R (1996) Cadmium carcinogenicity and genotoxicity. In: Chang, L., editor.

Toxicology of metals. Boca Raton, FL: CRC Press pp- 231-244.

Waisberg M, Black WD, Chan DY, Hale BA (2005) The effect of pharmacologically altered

gastric pH on cadmium absorption from the diet and its accumulation in murine tissues.

Food and Chemical Toxicology 43, 775-782.

Wang Q, Guan Y, Wu Y, Chen H, Chen F, Chu C (2008) Overexpression of a rice OsDREB1F

gene increases salt, drought, and low temperature tolerance in both Arabidopsis and rice.

Plant Molecular Biology 67, 589–602.

Weast RC (1984) CRC Handbook of Chemistry and Physics. 64th Edn., CRC Press, Baca Raton.

Wei SH, Zhou QX (2004) Discussion on basic principles and strengthening measures for

phytoremediation of soils contaminated by heavy metals. Chinese Journal of Ecology 23,

65–72.

REFERENCES

176

WHO (1989) Evaluation of Certain Food Additives and Contaminants. Thirty-third Report of the

Joint FAO/WHO Expert Committee on Food Additives. World health organization

Technical Report Series 776.

Wild A, Schmitt V (1995) Diagnosis of damage to Norway spruce (Picea abies) through

biochemical criteria. Physiologia Plantarum 93, 357-382.

Williams RJP, Frausto da Silva JJR (2002) The involvement of molybdenum in life.

Biochemical and Biophysical Research Communications 292, 293–299.

Winkel-shirley B (2002) Biosynthesis of favonoids and effects of stress. Current Opinion in

Plant Biology 5, 218-223

Wojtaszek P (1997) Oxidative burst: an early plant response to pathogen infection. Biochemical

Journal 322, 681-692.

Xiong L, Ishitani M, Lee H, Zhu KJ (2001) The Arabidopsis LOS5/ABA3 locus encodes a

molybdenum cofactor sulfurase and modulates cold stress-and osmotic stress-responsive

gene expression. Plant Cell 13, 2063–2083.

Xu J, Yin X, Li X (2009) Protective effects of proline against cadmium toxicity in

icropropagated hyperaccumulator, Solanum nigrum L. Plant Cell Reports 28, 325–333.

Yamaguchi-Shinozaki K, Shinozaki K (1993) The plant hormone abscisic acid mediates the

drought-induced expression but not the seed-specific expression of rd22, a gene responsive

to dehydration-stress in Arabidopsis thaliana. Molecular Genetics and Genomics 238, 17–

25.

Yang SL, Lan SS, Gong M (2009) Hydrogen peroxide-induced proline and metabolic pathway of

its accumulation in maize seedlings. Journal of Plant Physiology 166, 1694–1699.

Yang W, Liu XD, Chi XJ, Wu CA, Li YZ, Song LL, Liu XM, Wang YF, Wang FW, Zhang

C, Liu Y, Zong JM, Li HY (2011) Dwarf apple MbDREB1 enhances plant tolerance to low

temperature, drought, and salt stress via both ABA-dependent and ABA-independent

pathways. Planta 233, 219–229.

Zadeh BM, Savaghebi-Firozabadi GR, Alikhani HA, Hosseini HM (2008) Effect of Sunflower

and Amaranthus Culture and Application of Inoculants on Phytoremediation of the Soils

http://www.ncbi.nlm.nih.gov/pubmed/?term=Liu%20XM%5BAuthor%5D&cauthor=true&cauthor_uid=20967459

http://www.ncbi.nlm.nih.gov/pubmed/?term=Wang%20YF%5BAuthor%5D&cauthor=true&cauthor_uid=20967459

http://www.ncbi.nlm.nih.gov/pubmed/?term=Wang%20FW%5BAuthor%5D&cauthor=true&cauthor_uid=20967459

http://www.ncbi.nlm.nih.gov/pubmed/?term=Zhang%20C%5BAuthor%5D&cauthor=true&cauthor_uid=20967459

http://www.ncbi.nlm.nih.gov/pubmed/?term=Zhang%20C%5BAuthor%5D&cauthor=true&cauthor_uid=20967459

http://www.ncbi.nlm.nih.gov/pubmed/?term=Liu%20Y%5BAuthor%5D&cauthor=true&cauthor_uid=20967459

http://www.ncbi.nlm.nih.gov/pubmed/?term=Zong%20JM%5BAuthor%5D&cauthor=true&cauthor_uid=20967459

http://www.ncbi.nlm.nih.gov/pubmed/?term=Li%20HY%5BAuthor%5D&cauthor=true&cauthor_uid=20967459

REFERENCES

177

Contaminated with Cadmium. American-Eurasian Journal of Agricultural

& Environmental Sciences 4, 93–103.

Zalups RK, Ahmad S (2003) Molecular handling of cadmium in transporting epithelia.

Toxicology and Applied Pharmacology 186 163-188.

Zarka DG, Vogel JT, Cook D, Thomashow MF (2003) Cold induction of Arabidopsis CBF genes

involves multiple ICE (Inducer of CBF Expression) promoter elements and a cold-

regulatory circuit that is desensitized by low temperature. Plant Physiology 133, 910– 918.

Zeng X, Jin T, Jiang X, Kong Q, Ye T, Nordberg GF (2004) Effects on the prostate of

environmental cadmium exposure - a cross-sectional population study in China. Biometals

17(5), 559-565.

Zengin FK, Munzuroglu O (2006) Toxic effects of cadmium (Cd++

) on metabolism of sunflower

(Helianthus annuus L.) seedlings. Acta Agriculturae Scandinavica, Section B —

Soil & Plant Science 56, 224-229.

Zhao FJ, Hamon RE, Lombi E, McLaughlin MJ, McGrath SP (2002) Characteristics of cadmium

uptake in two contrasting ecotypes of the hyperaccumulator Thlaspi caerulescens. Journal

of Experimnetal Botany 53, 535–543.

Zheng HP, Gao LS, Wang SR (2010) Effects of cadmium on growth and antioxidant responses

in Glycyrrhiza uralensis seedlings. Plant Soil and Environment 56(11), 508-515.

Zhou QX, Song YF (2004) Principles and Methods of Contaminated Soil Remediation. Science

Press, Beijing, China.

Zhou WB, Qiu BS (2005) Effects of cadmium hyperaccumulation on physiological

characteristics of Sedum alfredii Hance (Crassulaceae). Plant Science 169, 737-745.

Zhu YL, Zayed AM, Quian JH, De Souza M, Terry N (1999) Phytoaccumulation of trace

elements by wetland plants: II. Water hyacinth. Journal of Environmental Quality 28, 339–

344.

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