Effects of Toxic Concentrations of Copper on Growth and Metabolism of Rice Seedlings

Originalarbeiten . Original Papers

Department of Botany, University of Calcutta, Calcutta, India

Effects of Toxic Concentrations of Copper on Growth andMetabolism of Rice Seedlings

BISHNUPRIYA DAS GUPTA and SUBHENDU MUKHERJI

With 9 figures

Received May 31,1976' Accepted June 28,1976

Summary

Metabolic alterations in germinating rice seeds as influenced by toxic concentrations ofcupric sulphate were studied with respect to nucleic acids, proteins and hydrolytic enzymes,i.e, a-amylase, ribonuclease (RNase), adenosine triphosphatase (ATPase), phytase andprotease. Root growth inhibition was more pronounced than was shoot growth inhibition.At 10-2 M cupric sulphate the magnitude of the reduction in length in shoot and rootwas 37 % and 85 % respectively, and germination stopped altogether at 10-1 M. RNAcontent decreased in the embryo but increased in the endosperm, whereas DNA contentdecreased in both these plant parts. The amount of alkali-soluble protein increased in bothembryo and endosperm under all the growth-inhibitory concentrations of copper, resultingin reduced RNA/protein and DNA/protein ratios.

A stepwise decrease in the a-amylase activity of the endosperm and in the RNaseactivity of the embryo and endosperm took place which was proportional to increasingCUS04 concentrations. The inhibition could be reversed by joint applications of copperwith gibberellic acid (GA3) and with adenosine 3,5-cyclic monophosphate (cAMP). ATPaseand phytase activities in the embryo did not vary substantially from those of the control,but gradually decreased in the endosperm. Protease activity in the embryo increased withincreasing copper concentrations, but progressively decreased in the endosperm.

As compared with the UV absorption spectra of authentic GAs and cAMP, some changesin the absorption patterns of these hormones was detected when they were in equimolarmixture with CUS04'

Key words: Copper, metabolism, growth, germination, Oryza sativa.

Introduction

In a previous paper, MUKHERJI and DAS GUPTA (1972) described the effects oftoxic concentrations of copper on the growth of lettuce seedlings and on the activities

Z. PJlanzenphysiol. Bd. 82. S. 95-106. 1977.

96 BISHNUPRIYA DAS GUPTA and SUBHENDU MUKHERJI

of certain of their component enzymes. It was shown that excess copper resulted inan increase in the activities of catalase, peroxidase and IAA oxidase, the effectincreasing with increasing copper concentrations. It was obvious that growth wasinversely proportional to the enzyme activity. The increased level of these enzymeshas been attributed to an accelerated synthesis of protein.

In this paper, some further questions pertaining to the characterization of coppertoxicity are broached with respect to rice (Oryza sativa L.) seeds. An understandingof the significance of the deleterious effect of copper on seedling growth requires thatinformation be obtained about the effect of this metal on the alterations in the levelsof nucleic acids and proteins and in the activities of hydrolases, i.e. a-amylase, RNase,ATPase, phytase and protease.

Material and Methods

The experiments were carried out using rice (Oryza sativa L. cv. Rupsail) seeds suppliedby the State Agricultural Research Institute, Chinsurah, West Bengal. The seeds weregerminated in Petri dishes lined with filter papers soaked with different concentrationsof cupric sulphate (CUS04, 5 H 20 ) and other test solutions. They were kept under dark,humid conditions at a constant temperature of 30°C, and after 4 days germination seedlinggrowth and other measurements were made. Water controls were maintained for eachexperiment.

Nucleic acid and protein (alkali-soluble) contents were estimated in both embryo andendosperm, the methods for which have been previously described (PAUL et al., 1971;MUKHERJI et al., 1975). The a-amylase assay was performed only on endosperm material,whereas both embryo and endosperm were assayed for RNase, ATPase, phytase andprotease. The methods followed here have been previously described (PAUL et al., 1970;PAUL and MUKHERJI, 1973).

An experiment to test the possibility that copper builds a complex with hormones wasdone by mixing equal volumes of ethanolic solutions of 20 mM CUS04 and of hormones,i.e. IAA, GAg, kinetin and cAMP, at a concentration of 20 mM. The mixtures were keptat 10°C for 3 days, whereby they were exposed to UV radiation for 15 min each day.Optical absorbance of the mixtures was measured in «Zeiss» Spectrophotometer between210 nm to 380 nm and compared with the UV absorption spectra of the individual hormones.

Results and Discussion

The effects of various concentrations of cupric sulphate on rice seedling elongationare presented in Table 1. Inhibition of elongation began at 10-4 M. At thisconcentration shoot inhibition was rather negligible, whereas the roots were inhibitedto an extent amounting to approximately 12 % of the control length. The inhibitionincreased with higher dosages of copper and root growth inhibition was morepronounced than was shoot growth inhibition. No germination occurred at themaximum concentration tested, i.e. 10- 1 M.

The effects of various concentrations of CUS04 on the nucleic acid andalkali-soluble protein contents are depicted in Figs. 1-3. Nucleic acid synthesis waslargely inhibited in the embryo by the growth-inhibitory doses of CUS04' the amount

Z. Pjlanzenph ysiol. Bd. 82. S. 95-106. 1977.

Toxic concentrations of copper 97

of loss of DNA being relatively more severe than the RNA loss. It appears that thereis a close correlation between the nucleic acid content of the embryo and growthinhibition. The growth rate is also known to be controlled by the RNA content(INGLE and HAGEMAN, 1964) and the influence on RNA metabolism should bereflected in the growth rate (HABER et al., 1964). Conversely, the effects which arecorrelated with growth rate are known to affect RNA metabolism (KESSLER and

Table 1: Effect of varrous levels of CUS04 on the growth of rice seedlings after 4 daysgermination.

Concentration Shoot Inhibition Root InhibitionM cm 0/0 cm 0/0

Water control 2.80 ± 0.23 6.53 ± 0.42

CUS0410-4 M 2.73 ± 0.21 2 5.77 ± 0.33 1210-3 M 2.58 ± 0.17 8 2.34 ± 0.13 64

5 X 10-3 M 1.93 ± 0.17 31 1.07 ± 0.16 8410-2 M 1.76 ± 0.10 37 0.96 ± 0.11 8510-1 M No germination

500---0 EMBRYO

----. ENDOSPERM

45

~3:J:

40CJ)W0::u,

C'<, 3~

~

Za:C' 30E

25

0 0'5 5

CuS04, mM

Fig. 1: Effect of various levels of CUS04 on RNA content of rice seedlings.

Z. Pflanzenphysiol. Bd. 82. S. 95-106. 1977.


0·3

r-:~

:I:(J) 0·2Wa:u,

Ol<,

<tZ0

01 0·1E

~ EMBRYOe----e ENDOSPERM

5

CuS04 ' m M

Fig. 2: Effect of various levels of CUS04 on DNA content of rice seedlings.

MONSELISE, 1959; RAUSER and HANSON, 1965). In the endosperm, however, there wasan alteration in the relative distribution of RNA and DNA, the RNA content of theendosperm exhibiting an increase over that of the control. This is not surprising, sincethe nucleic acid of endosperm can function as a reserve material and serve to provideprecursors for the synthesis of nucleic acid in the developing axis. Growth inhibitionresulting from copper treatment is thought to be associated with a disruption in therate of mobilization and the RNA reserves of the endosperm thus tend to remain at ahigher level. This is in agreement with the lower activity of the endosperm RNase ofsuch treated seedlings, which is indicative of the manner in which the endospermRNA is hydrolyzed (Fig. 5). The DNA content of the endosperm, on the contrary,exhibited a step-wise decrease with increasing CUS04 concentrations. In contrast to

the nucleic acids, the alkali-soluble protein content of the embryo and endospermincreased at all the tested CUS04 concentrations.

The effect of CUS04 on the RNA/protein and DNA/protein ratios is presented inTable 2. It is evident that the RNA/protein ratio in the embryo became progressivelysmaller with progressive increases in the CUS04 concentration. The DNA/proteinratio of the embryo, on the other hand, showed no change and remained constant in

Z. Pflanzenphysiol. Ed. 82. S. 95-106. 1977.


both the control and treatment sets. As compared to that of the control, theDNA/protein ratio of the endosperm attained its lowest value at the highest CUS04dosage.

25 0---0 EMBRYO

....-. ENDOSPERM

...,;3:I: 2 0CIlUJa:IJ..

CI<,

zUJ:; 15

a:Q.

0'

E

10

o 0 ·5

CuS04 , m M

Fig. 3: Effect of various levels of CuS04 on alkali-soluble protein content of rice seedlings.

Table 2 : Effect of various levels of CUS04 on RNA/protein and DNA /protein ratios ofrice seedlings after 4 days germination.

Treatment Plant pans

Embryo Endosperm

RNA DNA RNA DNAX 100 X 100

Protein Protein Protein Protein

Water control 4.4 1.6 2.5 1.8

CUS040.5mM 3.1 1.6 0.8 1.11 mM 2.4 1.6 0.5 0.65 mM 2.1 1.6 0.5 0.4


100 BISHNUPRIYA DAS GUPTA and SUBHENDU MUKHER]I

5o

3·7

t-=3:I:(J)UJ 3·5a:u,

Cl

"0WI-exa: 3'3UJCD

..J

W(J)

0I- 3' 1..Jex~

C'E

2·9

0·5 1

CuS04, mM

Fig. 4: Effect of various levels of CUS04 on a-amylase activity of rice endosperm.

CUS04 treatments resulted in an inhibition of the a-amylase acnvity in riceendosperm (Fig. 4). Similarly, the RNase activity in both embryo and endospermdecreased continuously with increasing CUS04 concentrations and the endospermRNase was maintained at higher level than was the embryo RNase (Fig. 5). It is,however, not difficult to associate a CuSOrinduced growth inhibition with a lowcontent of nucleic acids, but it cannot be correlated with the RNase activity in theembryo, which is also depressed by copper. The relationship of the RNase activity ofthe embryo to the depletion of embryo RNA is rather confusing because there issimultaneous decrease in both RNase and RNA contents. A possible explanation isthat during copper toxicity the general decline in metabolism can contribute to lowerlevels of RNA synthesizing enzyme as well as to lower levels of RNase. Enzymaticdegradation by RNase thus may not be rapid, but the coincident rate of RNAsynthesis may be slow. High levels of endosperm RNA under copper treatment,however, parallel the low nuclease activity. Loss of endosperm RNA is thusprevented by a blockage of the degradation of this compound by RNase and this maybe the reason for the RNA accumulation. High levels of endosperm protein can be



3·0~ EMBRYO

~ ____ ENDOSPERM~

J:enWa:u,

2'5C'<,Ec:

0({)(\J

I- 2·0<t

>-I-

>I-<..><t 1·5

0 0·5 5

CuS04,mM

Fig. 5: Effect of various levels of CuSO4 on RNase activity of rice seedlings.

Table 3: Effect of CUS04 applied alone or in combination with GA 3 and cAMP ona-amylase and RNase activity in rice seedlings after 4 days germination. a-amylase expressedas mg maltose released per 5 min and g fresh weight. RNase expressed as absorbency at260 nm per g fresh weight.

Treatment a-amylaseof intact

endosperm

Inhibition(-) or

promotion(+), Ofo

RNaseof intactembryo

Inhibition(-) or

promotion(+), Ofo

RNase of Inhibitionintact (-) or

endosperm promotion(+), Ofo

Water control5 mM CUS040.1 mM GA 30.1 mM cAMPCUS04 + GA 3

CuS04 + cAMP

3.662.904.064.033.663.26

-21+11+10nil-11

1.931.522.202.361.781.91

-22+14+22-8-1

2.741.783.133.412.191.99

-35+14+24-20-28

correlated with growth inhibition, which necessitates poor mobilization of the reservesto the developing axis.

GA3 and cAMP, when applied singly, promoted a-amylase production in intact rice

endosperm to the extent of only 11 010 and 10010, respectively (Table 3). Varyingamounts of relief from inhibition could be achieved in the case of joint application of

Z. Pjlanzenphysiol. Bd. 82. S. 95-106. 1977.

102 BISHNUPR.IYA DAS GUPTA and SUBHENDU MUKHERJI

25

....3:J:(/)

UJ 20a:u,

c><,CUJ....<ta: 15UJCXl

...J

(/)

::>a:oJ: 10a.(Jl

oJ:a.c:7'::J..,

5

c:>----0 EMBRY0.--. ENDOSPERM

o 0·5 1 5

CuS04 • m M

Fig. 6: Effect of various levels of CUS04 on ATPase activity of rice seedlings.

these hormones with copper. Reversal of inhibition was almost complete with GA 3

treatment and a-amylase activity was restored to about the same level as in thecontrol. In this respect, cAMP proved to be somewhat less effective than GAs inreversing copper induced enzyme inhibition. When applied jointly with copper,cAMP proved capable of reducing the inhibition from 21 % to 11 Ofn.

In the embryo and endosperm, an almost identical pattern of stimulation of RNaseactivity was demonstrated in response to GAs treatment. Irrespective of the plantpart, the enhancement of the activity in comparison to that of the control appearedto be more marked with cAMP. In the embryo, cAMP appeared to be a better agentthan GAs in reversing RNase inhibition, and the RNase activity was almost equal tothat of the control in case of simultaneous application. In the endosperm, on theother hand, GAs was somewhat more effective than cAMP and the RNase activitywas close to the control value. A similar inhibition of a-amylase and RNase bymercury has been reported, whereby the inhibition could be overcome by GAs andcAMP (MUKHER]I and GANGULY, 1974). Thus it is apparent that GAs or cAMP,when applied alone, have no great effect on the endogenous enzyme activity of the

Z. Pjlanzenpbysiol. Bd. 82. S. 95-106. 1977.

..:3 20J:(/)W0::lJ..

0---0 EMBRYO

..-..... ENDOSPERM


-0~-----o

C'<,

ow....c:r 1 50::WCD..J

(/)

::>0::oJ:

~ 10oJ:a..

0'5 1 5

CUS04, mM

Fig. 7: Effect of various levels of CUS04 on phytase activity of rice seedlings.

intact seedl ings. Although a definite conclusion regarding compentrve inhibitioncannot be drawn, our results a re suggestive of a direct interaction between copper,GAs and cAMP. The to xic action of copper may therefore be attributed to a bind ingto a receptor site required by GAs and cAMP for the induction of a-amylase andRNase synthesis, resulting in a limited supply of food materials for germination andembryo growth.

Little di fference between control and treatment sets was observed regarding theATPase act ivit y of embry o (Fi g. 6). At th e highest concent ration of copper, however,an approximately 20 % decrease was noted. Th e effect of the treatment onendosperm ATPase appeared to be somewhat mor e marked and the decline in theenzy me activity of the treatment sets from the control value was almost continuous.It is to be noted that seedling growth under CUS04 treatment is always stunted, afact which is obviously coupled with a decreased ability to mob ilize energyobtainable from the br eakdown of ATP. This is well substantiated by th e fact thatCUS04 is characterized by its eff ect in ma int ain ing ATPase at a level relati vely lowerth an that of th e control. The data on ph yt ase show th at CUS04 treatments could notelicit any response in embry o. The level of endosperm phytase, on the other hand,was reduced by copper (Fig. 7) .

Z. Pflanzenphysiol. Bd . 82. S. 95-1 06. 1977.


9 0--0 EMBRYO

t-= e--e ENDOSPERM

~

IC/)W 8a:u,

01.....Ec

70<XlC\I

I-et

6

>-I-

>I-U 5et

0 0·5 5

CUS04 7 mM

Fig. 8: Effect of various levels of CUS04 on protease activity of rice seedlings.

Embryo protease was enhanced by copper, the effect increasing with increasingconcentrations (Fig. 8). Protease and protein in the embryo are thus apparentlyrelated in their simultaneous increase in reaction to copper. It may be suggested thatembryo protease is in some way related to the dynamic state or «turnover» of proteinin the cell rather than to the absolute protein level at anyone time. On this basis,protein levels may rise even though protease activity also increases. Put in anotherway, enzymic degradation by protease may be rapid, but the coincident rate ofprotein synthesis may also be rapid. The protease level in the control endosperm didnot differ greatly from that in control embryo. In the treatment sets, the pattern ofalteration assumed an inverse relationship in these two plant parts. In contrast to theprotease increase in embryo, the decrease in the activity in the endosperm was theresult of copper treatments which contribute to higher levels of proteins and thus actin the regulation of the endogenous protein content.

Since the ultraviolet absorption spectra of authentic IAA and kinetin virtuallycoincide with those of their mixtures with CUS04, the results from treatment withthese mixtures are not presented here. Interesting differences were noted in the caseof GA 3 and cAMP and the curves presented in Fig. 9 indicate that the optical densityof the mixture of GA 3 and CUS04 was higher than that of authentic GA 3 up to 270nm and then appeared to coincide with the latter afterwards. The curves further

Z. Pflanzenphysiol. Ed. 82. S. 95-106. 1977.


-- GA 3--- +CuS04

cAMP

_.- +CuS04

./'\/., .. / \ \! / \ \

'7\\ I: : \P' \\ i i \ \V" I! \ \I \\ I "\Iii \ \

\i I I : \\ij; \ \\ I : \\; \ \

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f-

enZUJC

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0·2

0'- ....... '--- '-----'

200 2~0 300 3~0

WAVELENGTH, nm

Fig. 9: UV absorption spectra of authentic GA 3 and cyclic AMP and their mixtures withCUS04'

indicate that the furrow near 240 nm of GA3 was not visible in the mixture, instead aslope was observed in this region. The second peak at 260 nm characteristic of GA3

was absent in mixture, the spectrum of which had a shoulder in this region.The UV curve of authentic cAMP shows the clear resolution of 2 peaks, one at 215

nm and the second at 265 nm. The mixture of cAMP and cusa4 yielded an almostidentical curve, the only difference being that the mixture showed higher a.D. valuesthan cAMP alone throughout the entire spectral range, the increase in a.D. beingmore prominent at 215 nm than at 265 nm. It is pertinent to suggest that copper mayform complex with GA3 and cAMP before the hormones enter or mobilize within theplant, thus resulting in a partial abolition of the physiological effects of thesehormones. Their translocation within the plant in the form of the complex may alsobe affected. More experimental evidence is necessary before any comment on thenature of the complex or the reaction leading to its formation can be made.

The authors are indebted to Professor A. K. SHARMA, Head of the Department of Botany,University of Calcutta, for providing facilities for this work. This investigation was



supported by grants from the Council of Scientific & Industrial Research and UniversityGrants Commission, New Delhi.

References

HABER, A. H., T. J. LONG, and D. E. FOARD: Nature 201, 470 (1964).INGLE, J., and R. H. HAGEMAN: Plant Physiol, 39, 730 (1964).KESSLER, B., and S. P. MONSELISE: Physiol. Plant. 12, 1 (1959).MUKHERJI, S., and B. DAS GUPTA: Physiol. Plant. 27, 126 (1972).MUKHERJI, S., and G. GANGULY: Indian J. Exp. Biol, 12,432 (1974).MUKHERJI, S., A. BAG, and A. K. PAUL: BioI. Plant. 17, 60 (1975).PAUL, A. K., and S. MUKHERJI: BioI. Plant. 15, 398 (1973).PAUL, A. K., S. MUKHERJI, and S. M. SIRCAR: Osterr. Bot. Z. 118, 311 (1970).- - - Physiol. Plant. 24, 342 (1971).RAUSER, W. F., and J. B. HANSON: Plant Physiol, Suppl, 40, 51 (1965).

Dr. S. MUKHERJI, Plant Physiology Laboratory, Department of Botany, University ofCalcutta, 35 Ballygunge Circular Road, Calcutta 700019, India.


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Effects of Toxic Concentrations of Copper on Growth and Metabolism of Rice Seedlings