Draft - University of Toronto T-Space › bitstream › 1807 › ... · Draft 3 51 52 Introduction 53 Salinity is a global environmental problem particularly in arid climatic regions

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Physiological and biochemical characterization of Acacia

stenophylla and Acacia albida exposed to salinity under hydroponic conditions

Journal: Canadian Journal of Forest Research

Manuscript ID cjfr-2016-0499.R2

Manuscript Type: Article

Date Submitted by the Author: 21-Jun-2017

Complete List of Authors: Abbas, Ghulam; COMSATS, Environmental Sciences; UAF, ISES

saqib, muhammad; UAF, ISES Akhtar, Javaid; uaf, ises Murtaza, Ghulam; UAF, ISES

Keyword: Acacia, CAT, MSI, K+: Na+ ratio, Salinity

Is the invited manuscript for consideration in a Special

Issue? : N/A

https://mc06.manuscriptcentral.com/cjfr-pubs

Canadian Journal of Forest Research

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Physiological and biochemical characterization of Acacia stenophylla and Acacia albida 1

exposed to salinity under hydroponic conditions 2

Ghulam Abbas1, 2*

· Muhammad Saqib 2, 3

· Javaid Akhtar 2 · Ghulam Murtaza

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4 1 Department of Environmental Sciences, COMSATS Institute of Information Technology, Vehari, Pakistan;

2 Institute 5

of Soil and Environmental Sciences, University of Agriculture, Faisalabad, Pakistan; 3 Institute of Plant Nutrition, 6

Justus-Liebig University, Giessen, Germany. 7 8 *Corresponding author: Dr. Ghulam Abbas, Assistant professor 9

Mailing address: Department of Environmental Sciences, COMSATS Institute of Information Technology, Vehari 10

(61100), Pakistan 11

E-mail: [email protected]; [email protected] 12

Contact# +920673018082 13

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Abstract 25

Soil salinity is considered a serious environmental issue in many countries of the world including Pakistan. A 26

hydroponic experiment was carried out to study different mechanisms of salinity tolerance in two acacia species 27

namely Acacia stenophylla and Acacia albida. Uniform seedlings of both the species were grown for 28 days in half 28

strength Hoagland’s nutrient solution with 0, 100 or 200 mM NaCl concentrations. The results revealed that shoot 29

biomass was decreased by 21% and 29% at the lower salinity level (100 mM NaCl), and by 44% and 55% at the higher 30

salinity level (200 mM NaCl) in A. stenophylla and A. albida, respectively. The respective reductions in root biomass 31

of both the species were 20% and 29% at the lower, and 36% and 54% at the higher salinity level. The physiological 32

attributes such as chlorophyll and relative water contents were decreased to a greater extent in A. albida as compared to 33

A. stenophylla. As a result of oxidative stress, membrane stability index (MSI) was decreased in both the species with a 34

greater reduction in the case of A. albida. Among different antioxidant enzymes such as superoxide dismutase (SOD), 35

peroxidase (POD) and catalase (CAT), the highest increase (6 folds) was observed in SOD activity in the case of A. 36

stenophylla. This study concludes that A. stenophylla is more salinity tolerant than A. albida as it maintained better 37

ionic balance and higher activities of antioxidant enzymes, and resultantly higher biomass production. 38

Keywords Acacia, CAT, MSI, K+: Na

+ ratio, Salinity, SOD 39

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Introduction 52

Salinity is a global environmental problem particularly in arid climatic regions of the world (Murtaza et al. 2011). 53

Approximately 20% of the world’s arable lands have high concentrations of soluble salts (FAO 2005). Pakistan is also 54

facing this problem with about one third of its total cultivated area (6.67 mha) affected by salinity (Khan 1998). 55

Plant species face a plethora of problems when exposed to salinity stress. These include reduction in shoot and 56

root growth (Zhang et al. 2013; Abbas et al. 2015; Abbas et al. 2016a), photosynthesis (Saqib et al. 2013; Theerawitaya 57

et al. 2015), leaf chlorophyll and relative water contents (Hassan and Ali 2014; Abbas et al. 2016 a), and disturbed 58

ionic homeostasis (Saqib et al. 2005; Wang et al. 2015). The degree of damage is related to growth stages of plants, 59

type and concentrations of salts, duration of stress and the plant species (Lauchli and Grattan 2007; Saqib et al. 2013; 60

Abbas et al. 2015). 61

Osmotic stress inducing water deficiency, and toxicity of ions (Na+ and Cl

–) are the main causes of growth 62

reduction of plants under salinity stress (Munns and Tester 2008; Flowers et al. 2015). Plants have a variety of 63

mechanisms to overcome the osmotic and ionic effects of salinity (Meloni et al. 2004; Sekmen et al. 2007). The 64

osmotic stress is mitigated by osmotic adjustment, which is attained either by the uptake of inorganic ions or by the 65

accumulation of compatible solutes (Flowers et al. 2015). When exposed to salinity, plants typically accumulate higher 66

levels of toxic ions in their cells (Munns and Tester 2008). Higher accumulation of Na+

and Cl– ions in the cytoplasm is 67

not tolerable by most of the plants and for normal metabolic activities, salt tolerant plant species undergo cellular 68

compartmentation of these toxic ions into vacuoles or restrict them in older tissues (Flowers and Colmer 2008). These 69

toxic ions also restrict the uptake of some essential plant nutrients such as Ca2+

and K

+ (Vicente et al. 2004). Potassium 70

is an important macronutrient which plants need in large quantities (Mahajan and Tuteja 2005). It plays important role 71

in many crucial cellular processes such as photosynthesis, enzyme activation, protein synthesis (Marschner 1995), 72

osmotic balance and stomatal movements (Mahajan and Tuteja 2005). Therefore, a higher K+: Na

+ ratio is 73

indispensable for normal functioning of the plants under salinity stress (Abbas et al. 2013). Plants maintain a higher 74

K+: Na

+ ratio in their cells by regulating the uptake of K

+, preventing Na

+ influx and increasing Na

+ efflux from the 75

cells (Flowers et al. 2015). 76

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Salinity induced overproduction of reactive oxygen species (ROS) cause oxidative damage to plants. The 77

most important ROS include; superoxide radicals (O2•-), hydroxyl radicals (OH

•) and hydrogen peroxide (H2O2) 78

(Ashraf 2009). These ROS are produced in chloroplast, apoplast, cytosol and mitochondria (Shahid et al. 2014; Abbas 79

et al. 2015), and cause lipid peroxidation, changes to DNA structure, damage to proteins and injury to cell membranes 80

(Shahid et al. 2015). Many antioxidant enzymes such as SOD, CAT and POD detoxify these ROS in plants (Pourrut et 81

al. 2011). Superoxide free radicals are converted to hydrogen peroxide and oxygen in the presence of SOD (Abbas et 82

al. 2015). Catalase and POD further convert this hydrogen peroxide to water and oxygen (Amjad et al. 2014). Plant 83

species differ greatly regarding these antioxidants activities when exposed to salinity stress (Abbas et al. 2015), and 84

plants with higher activities of these antioxidants are considered more tolerant to salinity (Morais et al. 2012). 85

With changing climate, it is expected that there will be a 10% increase in the salt-affected areas globally 86

(Saboora et al. 2006). Resultantly, food and fodder production is expected to be decreased in the years to come 87

particularly in the arid and semiarid regions (El-Kharbotly et al. 2003). Considering this whole scenario, it is 88

indispensable to have plant species which are highly tolerant against salinity and can produce high biomass on such 89

saline lands. In the past, some acacia species had been successfully grown on highly salt-affected soils under arid 90

climatic conditions of Pakistan (Ashraf et al. 2012; Abbas et al. 2016 b). Considering the changing climatic conditions, 91

more species need to be explored for their potential use on salt affected soils. A. stenophylla and A. albida are 92

important tree species which may be grown on salt-affected soils in Pakistan. The relative abundance of both these 93

species on salt affected areas in Pakistan is very less in comparison to other species. In addition to soil 94

phytoremediation (Fathi et al. 2014), A. albida has high medicinal value as well. Its various edible parts have high 95

antioxidant activities of phenolic compounds. Therefore, it is regarded as a rich source of antioxidants (Karoune et al. 96

2015). A. stenophylla is mostly used for site rehabilitation and has good potential for fodder and fuel wood production 97

(Griffin et al. 2011). The comparative salinity tolerance potential (the ability of plants to withstand high salt 98

concentrations), and various salt tolerance mechanisms of A. stenophylla and A. albida are not well known. Therefore, 99

the present study has been conducted to compare the salinity tolerance potential of these tree species by exploring 100

various physiological and biochemical mechanisms of salinity tolerance. 101

Materials and methods 102

Experimental conditions and treatments 103

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This study has been carried out at the Institute of Soil and Environmental Sciences, University of Agriculture, 104

Faisalabad, Pakistan (latitude 31.26o, longitude 71.06

o, altitude of 184.4 m, average annual rainfall 408 mm). The seeds 105

already available at the institute has been used in this study. These seeds were previously collected from five 106

populations which were far away from each other. Such collection of seeds ensured wide representation of the genetic 107

material. Approximately 100 seeds of each species were sown in two-inch layer of sand contained in iron trays. The 108

sand was washed three times with 3% HCl, and two times with distilled water before use. The sand was kept moist 109

with distilled water until seed germination, and thereafter, half-strength Hoagland's nutrient solution (Hoagland and 110

Arnon 1950) was applied to the seedlings for three weeks. Uniform seedlings of both the species with 16 ± 2 cm plant 111

height were transplanted into foam plugged holes in polystyrene sheets floating on half strength Hoagland's nutrient 112

solution contained in 25 L plastic tubs. These tubs were placed on iron stands in the wire house of the Institute of Soil 113

and Environmental Sciences, University of Agriculture, Faisalabad, Pakistan. The wire house was rain protected with a 114

glass roof and sides covered with iron net. The average weather conditions during the experimental period has been: 37 115

± 2°C ambient temperature, 70 ± 4% relatively humidity and 8 ± 1 h d−1

sunlight photoperiod. Seven days after 116

transplantation, the measured quantity of NaCl salt was added to nutrient solution in two increments (one per day) to 117

achieve the desired salinity levels (100 and 200 mM NaCl). The nutrient solution without NaCl salt served as control 118

treatment. The solution was changed after 14 days, and in new solution the required amount of NaCl was added as a 119

single dose. The solution was aerated using aeration pumps and pH of the solution was adjusted daily at 6.0 ± 0.5 with 120

dilute NaOH or HCl. For each species, there were three treatments and four replications with two plants per replication. 121

One plant from each replication was used for growth and ionic measurements, and the other one plant was used for 122

physiological and biochemical measurements. 123

Physiological measurements 124

Chlorophyll content index (CCI) of the second leaf from the top of each plant was measured before harvesting using 125

SPAD-502 Chlorophyll Meter (Amjad et al. 2015). Relative water contents (RWC) and membrane stability index 126

(MSI) of the same leaf were determined as described by Sairam et al. (2002). 127

Plant growth measurements 128

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After 28 days of growth under salinity stress, shoots and roots of each plant were harvested, thoroughly washed with 129

distilled water and their growth attributes including shoot and root lengths and fresh masses were measured. Shoot and 130

root dry masses were recorded after oven drying the samples at 65°C for 72 hours. 131

Plants ionic measurements 132

Oven dried shoot and root samples were ground to powder, passed through 1mm sieve and digested in H2SO4 and 133

H2O2 as described by Wolf (1982). The concentrations of Na+ and K

+ were measured using Sherwood- 410 Flame 134

Photometer whereas, Sherwood- 926 Chloride Analyzer was used for measuring Cl– concentration. 135

Enzyme activities 136

Fully expanded young leaf samples were collected from each plant before harvesting. Immediately after collection, 137

these samples were wrapped in aluminium foils and put into liquid nitrogen. Until further analysis, these samples were 138

stored at -20 °C to stop enzyme activities. For measuring enzyme activities, 0.5 g leaf sample was ground in 5 mL of 139

cold phosphate buffer (50 mM having pH 7.8). The homogenate obtained after grinding was centrifuged at 15000 g for 140

20 min at 4 °C. The supernatant thus obtained was used for measuring SOD activity as described by Giannopolitis and 141

Ries (1977) whereas, the method of Chance and Maehly (1955) was used for measuring CAT and POD activities. 142

Data analysis 143

All the measurements were done using four replications (n = 4; one plant per replication) of each species per treatment. 144

The obtained data were analyzed using statistical software package ‘‘Statistix 8.1’’. Two- way analysis of variance 145

(ANOVA) was carried out considering salinity treatments and Acacia species as factors (Steel et al. 1997). The 146

significance of differences among different combinations of treatments and species were determined using Least 147

Significant Difference (LSD) test. The relationships among different variables were examined using simple linear 148

regression and correlation. 149

Results 150

Plant growth 151

With increasing salinity levels (100 and 200 mM NaCl) in the nutrient solution, shoot and root fresh weights of both 152

the acacia species were decreased (Table 1). The analysis of variance showed that the effects of treatments, species, as 153

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well as their interaction were significant. Regarding these parameters, the difference between both species increased as 154

the level of NaCl was increased in the nutrient solution. A. stenophylla produced more fresh weights of shoot and root 155

than A. albida at both salinity levels. Shoot and root dry weights of both the species were also decreased significantly 156

as the salt level was increased in the growth medium (Table 1). At both salinity levels, A. stenophylla produced more 157

dry biomass of shoot and root than A. Albida (Table 1). Shoot dry weight was decreased by 21% and 29% at the lower 158

salinity level (100 mM NaCl), and by 44% and 55% at the higher salinity level (200 mM NaCl) in A. stenophylla and 159

A. Albida, respectively. The respective reductions in root dry weight of both the species were 20% and 29% at the 160

lower salinity level, and 36% and 54% at the higher salinity level. For both the species, a decreasing trend was 161

observed for shoot and root lengths with increasing salinity levels (Table 1). However, treatment x species interaction 162

was not significant for shoot length. Shoot lengths of both the species were statistically similar at the lower salinity 163

level whereas, at the higher salinity level A. stenophylla produced significantly more shoot length than A. albida. On 164

the other hand, A. stenophylla produced significantly more root length than A. albida at both the salinity levels. 165

Chlorophyll content index and relative water contents 166

Salinity caused a significant reduction in chlorophyll content index (CCI) and relative water contents (RWC) in both 167

the species (Fig. 1 a, b). There were significant differences for treatments as well as species in the case of RWC. On the 168

other hand, only the treatment effects were significant for CCI. A. stenophylla showed 35% and 23% reduction in CCI 169

and RWC at the higher level of salinity (200 mM NaCl). The reduction in these attributes in the case of A. albida was 170

43% and 31%, respectively. 171

Ionic composition 172

Shoot and root Na+

and Cl–

concentrations in both the species were significantly increased with increasing salt levels 173

(100 and 200 mM NaCl) in the solution (Fig. 2 a, b, c, d). The treatment x species interaction was found non-174

significant for Na+ and Cl

– concentrations in roots of both the species. At both salinity levels, A. albida accumulated 175

significantly higher amounts of Na+ and Cl

– in

the shoots as compared to A. stenophylla. However, in the case of root 176

Na+ and Cl

– concentrations, both the species showed significant difference only at the higher salinity level (200 mM 177

NaCl). At this salinity level, shoot Na+

concentration was increased by 8.5 and 6.8 folds in A. albida and A. 178

stenophylla, whereas, root Na+ concentration was increased by 6.9 and 5.6 folds in both species, respectively over the 179

respective controls. At the higher salinity level, 8.3 and 6.5 folds increase was observed in shoot Cl– concentration in A. 180

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albida and A. stenophylla, respectively whereas, the respective increase in root Cl– concentration was 6.3 and 5.5 folds 181

for both the species. The concentrations of K+

in shoot and root of both the species decreased significantly by 182

increasing salinity levels in the solution (Fig. 2 e, f). At the higher salinity level (200 mM NaCl), shoot and root K+ 183

concentrations in A. albida were decreased by 76 and 66%, respectively whereas, in A. stenophylla the decrease was 66 184

and 53% for shoot and root, respectively. Shoot and root K+: Na

+ ratios decreased in both the species with increasing 185

salinity levels (100 and 200 mM NaCl) in the growth medium (Fig. 3 a, b). Comparison of both the species indicated 186

that A. stenophylla maintained higher shoot and root K+: Na

+ ratios than A. albida at both the salinity levels. Shoot Na

+ 187

concentration and shoot dry weight of both the species showed strong inverse correlation (Fig. 5 a) whereas, shoot K+: 188

Na+ ratio and shoot dry weight of both species showed strong positive correlation (Fig. 5 b). 189

Membrane stability and enzyme activities 190

The stability of the cell membranes against reactive oxygen species (ROS) was measured in terms of membrane 191

stability index (MSI) (Fig. 4 a), which decreased with increasing salt concentrations (100 and 200 mM NaCl) in growth 192

medium. Comparison of both the species showed that A. stenophylla was more tolerant to this membrane damage by 193

maintaining higher MSI value than A. albida under both the salinity levels. The value of MSI was decreased by 15% 194

and 23% at the lower salinity level (100 mM NaCl), and by 25% and 38% at the higher salinity level (200 mM NaCl) 195

in A. stenophylla and A. albida, respectively. In response to this oxidative stress, the activities of antioxidant enzymes 196

including super oxide dismutase (SOD), catalase (CAT) and peroxidase (POD) were increased significantly in both the 197

species with increasing salt levels in the solution (Fig. 4 b, c, d). For SOD and CAT, the treatment x species interaction 198

was significant, whereas, for POD only the treatment effect was significant. A. stenophylla showed significantly higher 199

activities of SOD and CAT than A. albida at both salinity levels however, POD activities were significantly higher in 200

A.stenophylla only at the higher salinity level. Among the three enzymes, the highest increase was observed in SOD 201

activity in A.stenophylla (6 folds) as well as A.albida which showed 4 fold increase in SOD activity. There was strong 202

positive correlation between the activities of SOD and shoot Na+ concentration of both the species (Fig. 5 c, d). 203

Discussion 204

The present study indicates that A. albida and A. stenophylla differed significantly regarding all the measured 205

physiological and biochemical characteristics. Shoot and root growth and biomass production by both the species was 206

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decreased with increasing salinity levels in the growth medium however, this reduction was more in the case of A. 207

albida as compared to A. stenophylla. Although acacia is considered a salinity tolerant genus; yet, reduction in growth 208

occurs at higher salt concentrations in the growth medium as previously reported by Marcar and Crawford (2011), 209

Abbas et al. (2015) and Theerawitaya et al. (2015). Osmotic as well as ionic effects of salinity are responsible for such 210

reduction in plant growth under saline conditions (Nawaz et al. 2010). Osmotic effect becomes effective with the onset 211

of salinity stress and results in an early growth reduction mainly due to water deficiency in the plant cells. However, 212

the osmotic stress is a transient effect and it is followed by ionic effect if plants are exposed to higher salinity levels for 213

relatively longer period of times (Munns and Tester 2008). The ionic effect is characterized by excessive Na+

and Cl– 214

accumulation in the cells that results in growth reduction by interfering with crucial biochemical processes (Munns and 215

Tester 2008; Tavakkoli at al. 2010). A significant negative correlation between shoot dry weights and shoot Na+ 216

concentrations of both the species (Fig. 5 a, b) confirms that the toxicity of Na+ greatly contributes to growth reduction 217

of both the species under low (100 mM NaCl) and high (200 mM NaCl) salinity. The greater reduction in shoot and 218

root growth of A. albida than A. stenophylla may partly be attributed to ion toxicity due to relatively higher 219

accumulation Na+ and Cl

– ions in the tissues as well as osmotic stress as indicated by relatively lower water contents 220

(RWC) in the shoots of A. albida. Maintenance of higher RWC in the leaf is considered as an important tolerance 221

mechanism against salinity and plant species with higher RWC can grow better under salinity stress (Huang et al. 222

2015; Abbas et al. 2016 a). 223

Leaf chlorophyll contents were significantly decreased in both the species in response to salinity confirming the 224

findings of Hassan and Ali (2014) for jojoba plants. Similarly, Abbas et al. (2016 a) found that salinity and drought 225

caused marked reduction in chlorophyll contents of A. ampliceps and A. nilotica. The reduction in chlorophyll contents 226

might be the result of chloroplast damage due to high production of reactive oxygen species (Smirnoff 1995) as well as 227

ion toxicity (Tavakkoli et al. 2010). The later author found that leaf chlorophyll contents of faba bean were decreased 228

under NaCl salinity and this decrease was mainly due to Cl– ions rather than Na

+ ions. The higher accumulation of Cl

– 229

in plant tissues is one of the reasons of reduced chlorophyll contents as a result of Cl– induced permeability of 230

chloroplast (Heber and Heldt 1981). We also found that A. albida owing to higher accumulation of Cl– ions showed 231

greeter reduction in chlorophyll contents as compared to A. stenophylla (Fig.2). 232

The concentrations of Na+ and Cl

– ions were increased in both the species with increasing salinity levels in the growth 233

medium. However, A. albida accumulated significantly higher concentrations of these ions than A. stenophylla. The 234

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greater accumulation of these toxic ions by the former species resulted in its lower growth and biomass production. 235

The higher accumulation of Na+ and Cl

– ions by various woody species under salinity stress has also been found by 236

other researchers (Isla et al. 2014; Theerawitaya et al. 2015; Abbas et al. 2016 a) in past. 237

When plants are grown under higher salt concentrations, the toxic ions (Na+ and Cl

–) passively enter into cytoplasm 238

through various cations and anion channels in the cell membrane (Demidchik and Maathuis 2007). This passive entry 239

and buildup of Na+ and Cl

– ions disturbs the ionic homeostasis (Zhu 2003; Sun et al. 2009). Hence, the maintenance of 240

relatively lower salt concentrations in the cytosol are responsible for better adaptation of plants to salinity stress (Tester 241

and Davenport 2003; Flower et al. 2015). Active extrusion of Na+ and Cl

– ions to the external environment is very 242

crucial for ionic homeostasis in the cell (Apse and Blumwald 2007; Munns and Tester 2008) and it is regarded as an 243

important mechanism of salinity tolerance in plants (Chen et al. 2003; Munns and Tester 2008). Our results indicate 244

that A. stenophylla maintained relatively lower concentrations of Na+ and Cl

– in its tissues which is one of the reason of 245

its better tolerance against the applied salinity stress (Fig. 2). 246

Potassium is a crucial element for proper growth of plants under salinity stress (Amjad et al. 2015). Salinity is 247

also responsible for potassium deficiency in plants (Morais et al. 2012). We observed that as a result of higher 248

accumulation of Na+, the accumulation of K

+ was significantly decreased in both the species. However, the reduction 249

was greater in the case of A. albida than A. stenophylla. This type of ion uptake may be due to the competition between 250

Na+ and K

+ ions for cation channels, and the restricted uptake of K

+ in the presence of higher levels of Na

+ (Hardikar 251

and Pandey 2008; Sekmen et al. 2012; Adams and Shin 2014). As a result, the capacity for turgor maintenance and 252

osmotic adjustment is reduced which adversely affects various physiological functions (Greenway and Munns 1980). 253

Plants try to keep K+: Na

+ ratios to the desired levels in the cytosol by (a) regulating the uptake of K

+ (b) preventing the 254

entry of Na+ and (c) exclusion of Na

+ from the cells (Khan et al. 2009). The K

+: Na

+ ratio is an important criterion for 255

the selection of plant species against salinity stress (Amor et al. 2005; Saqib et al. 2005). We found that shoot dry 256

weights of both the species were positively correlated with shoot K+: Na

+ ratios (Fig. 5 c, d) which were significantly 257

decreased in both the tree species due to differential accumulation of Na+ and K

+ under the applied salinity levels. A. 258

stenophylla maintained relatively higher K+: Na

+ ratios both in roots and shoots which reflects its better capacity to 259

control K+ transport under salinity stress. 260

Oxidative stress caused by salinity results in over production of ROS (Shalata et al. 2001) which causes many 261

damaging effects to plants including injuries to cell membranes (Abbas et al. 2015). The membrane stability index 262

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(MSI) is an important indicator of the membranes stability against the applied stress. In the present study, the values of 263

MSI were progressively decreased in both species. A. stenophylla was better adapted to oxidative stress by maintaining 264

relatively higher values of MSI under the applied salinity levels. To mitigate the oxidative damage, the activities of 265

antioxidant enzymes were increased in both the species with increasing salinity levels. Superoxide dismutase is the key 266

enzyme for detoxification of superoxide radicals and it converts them to H2O2 and O2 in the cell (Shahid et al. 2014). 267

The strong positive correlation between shoot Na+ concentrations and SOD activities indicates that with increasing Na

+ 268

concentrations, SOD activities were also increased in both species (Fig. 5 e, f). According to Mittal et al. (2012), the 269

plant species showing higher activities of SOD are more tolerant to salinity. These observations are in accordance with 270

our results as we observed that A. stenophylla showed considerably more increase (6 folds) in SOD activity and hence, 271

it is more salt tolerant than A. albida which showed relatively less increase (4 folds) in SOD activity. Hydrogen 272

peroxide (H2O2) formed by the dismutation of superoxide is further converted into molecular oxygen and water by the 273

action of CAT and POD (Pourrut et al. 2011; Morais et al. 2012). We observed higher activities of POD and CAT in A. 274

stenophylla than A. albida, particularly at the higher salinity level. The higher activities of CAT and POD caused more 275

detoxification of H2O2 in A. stenophylla than A. albida confirming the previous findings of Zhang et al. (2013). 276

Likewise, Morais et al. (2012) observed that A. longifolia showed more tolerance against salinity than Ulex europaeus 277

due to the higher activities of antioxidant enzymes particularly CAT. The higher salinity tolerance of A. stenophylla 278

than A. albida seems to be related to its better detoxification potential against ROS, due to which this species was able 279

to produce more biomass under salinity stress. 280

Conclusions 281

It is concluded that the growth of both acacia species was reduced under salinity stress due to osmotic and ionic effects 282

as well as oxidative damage to cell membranes. A. stenophylla was more salt tolerant than A. albida due to less 283

accumulation of toxic ions (Na+ and Cl

-) and higher K

+: Na

+ ratios in the tissues. Moreover, the higher activities of 284

antioxidant enzymes particularly SOD, provided more tolerance against oxidative stress. Therefore, A. stenophylla is 285

more suitable species than A. albida for use on saline soils. 286

287

References 288

289

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Abbas, G., Saqib, M., and Akhtar, J. 2016 a. Differential response of two acacia species to salinity and water stress. 290

Pak. J. Agr. Sci. 53: 51–57. 291

Abbas, G., Saqib, M., Akhtar, J., Murtaza, G., Shahid, M., and Hussain, A. 2016 b. Relationship between rhizosphere 292

acidificaton and phytoremediation in two acacia species. J. Soils Sediments 16: 1392–1399. 293

Abbas, G., Saqib, M., Akhtar, J., Murtaza, G., and Shahid, M. 2015. Effect of salinity on rhizosphere acidification and 294

antioxidant activity of two acacia species. Can. J. Forest. Res. 45: 124–129. 295

Adams, E., and Shin, R. 2014. Transport, signaling, and homeostasis of potassium and sodium in plants. J. Integr. Plant 296

Biol. 56: 231–249. 297

Amjad, M., Akhtar, J., Haq, M.A., Riaz, M.A., and Jacobsen, S.E. 2014. Understanding salt tolerance mechanisms in 298

wheat genotypes by exploring antioxidant enzymes. Pak. J. Agr. Sci. 51: 969–976. 299

Amjad, M., Akhtar, S.S., Yang, A., Akhtar, J., and Jacobsen, S.E. 2015. Antioxidative response of quinoa exposed to 300

iso-osmotic, ionic and non-ionic salt stress. J. Agron. Crop Sci. doi:10.1111/jac.12140. 301

Amor, N.B., Hamed, K.B., Debez, A., Grignon, G. and Abdelly, C. 2005. Physiological and antioxidant responses of 302

the perennial halophyte Crithmum maritimum to salinity. Plant Sci. 168: 8898–99. 303

Apse, M.P. and Blumwald, E. 2007. Na+ transport in plants. FEBS Lett 581: 2247–2254. 304

Ashraf, M. 2009. Biotechnological approach of improving plant salt tolerance using antioxidants as markers. 305

Biotechnol. Adv. 27: 84–93. 306

Ashraf, M.A., Awan, A.R., and Mahmood, K. 2012. Rehabilitation of saline ecosystems through cultivation of salt 307

tolerant plants. Pak. J. Bot. 44: 69–75. 308

Chance, M., and Maehly, A.C. 1955. Assay of catalases and peroxidases. Methods Enzymol 2: 764–817. 309

doi:10.1016/S0076-6879(55)02300-8. 310

Demidchik, V. and Maathuis, F.J.M. 2007. Physiological roles of nonselective cation channels in plants: from salt 311

stress to signalling and development. New Phytol 175: 387–404 312

El-Kharbotly, A., Maghloub, O., Al-subhi, A., and Alhalhali, A. 2003. Indigenous grass species with potential for 313

maintaining rangeland and livestock feeding in Oman. Agric. Ecosyst Environ. 95: 623–627. 314

FAO. 2005. Global network on integrated soil management for sustainable use of salt-affected soils. FAO Land and 315

Plant Nutrition Management Service, Rome, Italy. http://www.fao.org/ag/agl/agll/spush 316

Fathi, R.A., Godbold, D.L., Al-Salih, H.S., and Jones, D. 2014. Potential of phytoremediation to clean up uranium-317

contaminated soil with acacia species. J. Environ. Earth Sci. 4: 81–91. 318

Page 12 of 22



Draft

13

Flowers, T.J., and Colmer, T.D. 2008. Salinity tolerance in halophytes. New Phytol. 179: 945–963. doi: 319

10.1111/j.1469-8137.2008.02531.x. 320

Flowers, T.J., Munns, R. and Colmer, T.D., 2015. Sodium chloride toxicity and the cellular basis of salt tolerance in 321

halophytes. Annals of botany, 115(3), pp.419-431. 322

Giannopolitis, C.N., and Ries, S.K. 1977. Superoxide dismutases. I. Occurrence in higher plants. Plant Physiol. 59: 323

309–314. doi:10.1104/pp.59.2.309. 324

Greenway, H. and Munns, R. 1980. Mechanisms of salt tolerance in non-halophytes. Ann. Rev. Plant Physiol. 31: 325

1491–90. 326

Griffin, A.R., Midgley, S.J., Bush, D., Cunningham, P.J., and Rinaudo, A.T. 2011. Global uses of Australian acacias–327

recent trends and future prospects. Divers. Distrib. 17: 837–847. 328

Hassan, F., and Ali, E. 2014. Effects of salt stress on growth, antioxidant enzyme activity and some other physiological 329

parameters in jojoba [Simmondsia chinensis (Link) Schneider] plant. Aus. J. Crop Sci. 8: 1615p. 330

Heber, U. and Heldt, H.W. 1981. The chloroplast envelope: structure, function and role in leaf metabolism. Ann. Rev. 331

Plant Physiol. 21: 139–168. 332

Hoagland, D.R., and Arnon, D.I. 1950. The water culture method for growing plant without soil. California Agri. Exp. 333

Station Circular. 347:1_32. 334

Huang, C.J., Wei, G., Jie, Y.C., Xu, J.J., Zhao, S.Y., Wang, L.C., and Anjum, S.A. 2015. Responses of gas exchange, 335

chlorophyll synthesis and ROS-scavenging systems to salinity stress in two ramie (Boehmeria nivea L.) 336

cultivars. Photosynthetica. 53: 455–463. 337

Isla, R., Guillen, M., and Aragues, R. 2014. Response of five tree species to salinity and waterlogging: shoot and root 338

biomass and relationships with leaf and root ion concentrations. Agroforestry systems. 88: 461_ 477. 339

Karoune, S., Falleh, H., Kechebar, M.S.A., Halis, Y., Mkadmini, K., Belhamra, M., Rahmoune, C., and Ksouri, R. 340

2015. Evaluation of antioxidant activities of the edible and medicinal Acacia albida organs related to phenolic 341

compounds. Nat. Prod. Res. 29: 452–454. 342

Khan, G.S. 1998. Soil salinity/sodicity status in Pakistan. Soil Survey of Pakistan, Lahore 59p. 343

Khan, M.A., Sherazi, M.U., Khan, M.A., Mujtaba, S.M., Islam, E., Muntaz, S., Shereen, A.R., Ansari, U. and Ashraf, 344

M.Y. 2009. Role of proline, K/Na ratio and chlorophyll content in salt tolerance of wheat (Triticum aestivum 345

L.). Pak. J. Bot. 41: 6336–38. 346

Page 13 of 22



Draft

14

Lauchli, A., and Grattan, S.R. 2007. Plant growth and development under salinity stress. In: Jenks MA, Hasegawa PM, 347

Jain SM (eds) Advances in molecular breeding toward drought and salt tolerant crops. Springer, Dordrecht, 348

Germany pp, 285–315. 349

Mahajan, S. and Tuteja, N. 2005. Cold, salinity and drought stress: an overview, Arch. Biochem. Biophys. 444: 1391–350

58. 351

Marcar, N.E., and Crawford, D.F. 2011. Intraspecific variation for response to salt and waterlogging in Acacia 352

ampliceps Maslin seedlings. New Forest. 41: 207–219. 353

Marschner, H. 1995. Mineral nutrition of higher plants, second ed. Academic Press, London. 354

Meloni, D.A., Gulotta, M.R., Martinez, C.A. and Oliva, M.A 2004. The effects of salt stress on growth, nitrate 355

reduction and proline and glycinebetaine accumulation in Prosopis alba. Braz. J. Plant Physiol. 16: 39–46. 356

Mittal, S., Kumari, N., and Sharma, V. 2012. Differential response of salt stress on Brassica juncea: photosynthetic 357

performance, pigment, proline, D1, and antioxidant enzymes. Plant Physiol. Biochem. 54: 17–26. 358

Morais, M.C., Panuccio, M.R., Muscolo, A., and Freitas, H. 2012. Salt tolerance traits increase the invasive success of 359

Acacia longifolia in Portuguese coastal dunes. Plant Physiol. Biochem. 55: 60–65. 360

Munns, R. and Tester, M. 2008. Mechanisms of salinity tolerance. Annu Rev Plant Biol. 59: 651–681 361

Murtaza, B., Murtaza, G., Rehman, M.Z., and Ghafoor, A. 2011. Use of brackish water for the reclamation of dense 362

saline-sodic soils by auger hole technology and economic growth of wheat crop. Pak. J. Agr. Sci. 48: 269–363

276. 364

Nawaz, K., Hussain, K., Majeed, A., Khan, F., Afghan, S., and Ali, K. 2010. Fatality of salt stress to plants: 365

morphological, physiological, and biochemical aspects. Afr. J. Biotechnol. 9: 5475–5480. 366

Pourrut, B., Shahid, M., Dumat, C., Winterton, P., and Pinelli, E. 2011. Lead uptake, toxicity, and detoxification in 367

plants. Rev. Environ. Contam. Toxicol. 213: 113–136. 368

Saboora, A., Kiarostami, K., Behroozbayati, F., and Hashemi, S.H. 2006. Salinity (NaCl) tolerance of wheat genotypes 369

at germination and early seedling growth. Pak. J. Biol. Sci. 9: 2009–2021. 370

Sairam, R.K., Rao, K.V., and Srivastava, G.C. 2002. Differential response of wheat genotypes to long term salinity 371

stress in relation to oxidative stress, antioxidant activity, and osmolyte concentration. Plant Sci. 163: 1037–372

1046. 373

Saqib, M., Zorb, C., Rengel, Z., and Schubert, S. 2005. Na+ exclusion and salt resistance of wheat (Triticum aestivum) 374

are improved by the expression of endogenous vacuolar Na+/H

+ antiporters in roots and shoots. Plant Sci. 169: 375

959–965. 376

Page 14 of 22



Draft

15

Saqib, M., Akhtar, J., Abbas, G., and Nasim, M. 2013. Salinity and drought interaction in wheat (Triticum aestivum L.) 377

is affected by the genotype and plant growth stage. Acta Physiol. Plant. 35: 2761–2768. 378

Sekmen, A.H., Türkan, I. and Takio, S. 2007. Differential responses of antioxidative enzymes and lipid peroxidation to 379

salt stress in salt-tolerant Plantago maritima and salt-sensitive Plantago media. Physiol. Plant. 131: 399–411. 380

Sekmen, A.H., Turkan, I., Tanyolac, Z.O., Ozfidan, C. and Dinc, A. 2012. Different antioxidant defense responses to 381

salt stress during germination and vegetative stages of endemic halophyte Gypsophila oblanceolata Bark. 382

Environ. Exp. Bot. 77: 637–6. 383

Shahid, M., Dumat, C., Pourrut, B., Abbas, G., Shahid, N., and Pinelli, E. 2015. Role of metal speciation in lead-384

induced oxidative stress to Vicia faba roots. Russ. J. Plant Physiol. 62: 448–454. 385

Shahid, M., Pinelli, E., Pourrut, B., and Dumat, C. 2014. Effect of organic ligands on lead-induced oxidative damage 386

and enhanced antioxidant defense in the leaves of Vicia faba plants. J. Geochem. Explor. 144: 282–289. 387

Shalata, A., Mittova, V., Volokita, M., Guy, M., and Taj, M. 2001. Response of cultivated tomato and its wild salt-388

tolerant relative Lycopersicon pennellii to salt-dependent oxidative stress: the root antioxidative system. 389

Physiol. Plant. 112: 487–494. 390

Smirnoff, N. 1995. Antioxidant systems and plant response to the environment. In: Smirnoff V (ed) Environment and 391

plant metabolism: Flexibility and acclimation. BIOS Scientific Publishers, Oxford, UK. 392

Steel, R.G.D., Torrie, J.H., and Dickey, D.A. 1997. Principles and procedures of statistics: a biometrical approach, 3rd 393

edn. McGraw Hill Co., New York, USA. 394

Tavakkoli, E., Rengasamy, P. and McDonald, G.K. 2010. High concentrations of Na+ and Cl

–ions in soil solution have 395

simultaneous detrimental effects on growth of faba bean under salinity stress. J. Exp. Bot. 61: 4449-4459. 396

Tester, M. and Davenport, R. 2003. Na+ tolerance and Na

+ transport in higher plants. Ann Bot (Lond) 91: 503–527. 397

Theerawitaya, C., Tisarum, R., Samphumphuang, T., Singh, H.P., Cha-Um, S., Kirdmanee, C., and Takabe, T. 2015. 398

Physio-biochemical and morphological characters of halophyte legume shrub, Acacia ampliceps seedlings in 399

response to salt stress under greenhouse. Front. Plant Sci. 6: 630. doi: 10.3389/fpls.2015.00630. 400

Vicente, O., Boscaiub, M., Naranjoa, M.A., Estrelles, E., Belles, J.M. and Soriano, P. 2004. Responses to salt stress in 401

the halophyte Plantago crassifolia (Plantaginaceae). J. Arid Environ. 58: 4634–81. 402

Wang, J., Li, S., Meng, Y., Ma, X., Lai, Y., and Yang, K. 2015. Transcriptomic profiling of the salt-stress response in 403

the halophyte Halogeton glomeratus. BMC Genomics. 16: 169. doi: 10.1186/s12864-015-1373-z. 404

Wolf, B.A. 1982. Comprehensive system of leaf analysis and its use for diagnosis crop nutrient status. Commun. Soil 405

Sci. Plant Anal. 13: 1035–1059. 406

Page 15 of 22



Draft

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Zhang, M., Fang, Y., Ji, Y., Jiang, Z., and Wang, L. 2013. Effects of salt stress on ion content, antioxidant enzymes, 407

and protein profile in different tissues of Broussonetia papyrifera. S. Afr. J. Bot. 85: 1–9. 408

Table 1

Effects of various levels of NaCl on shoot and root growth of A.stenophylla and A.albida.

Control 100mM NaCl 200 mM NaCl Control 100mM NaCl 200 mM NaCl

Shoot fresh weight (g plant-1) Shoot dry weight (g plant

-1)

A.stenophylla 3.20±0.12 a 2.30±0.10 b 1.57±0.12 d 0.57±0.02 a 0.45±0.02 b 0.32±0.01 d

A.albida 3.10±0.16 a 1.93±0.10 c 1.00±0.14 e 0.55±0.02a 0.39±0.03 c 0.25±0.02e

Root fresh weight (g plant-1) Root dry weight (g plant

-1)

A.stenophylla 1.70±0.08 a 1.30±0.07 b 0.78±0.06 d 0.25±0.007 a 0.20±0.007 b 0.16±0.006 c

A.albida 1.62±0.04 a 1.10±0.05 c 0.50±0.06 e 0.24±0.009 a 0.17±0.005 c 0.11±0.007 d

Shoot length (cm) Root length (cm)

A.stenophylla 26.0±1.08 a 19.1±1.08 b 13.3±1.22 c 21.0±0.85 a 17.1±0.71 b 10.2±1.0 d

A.albida 25.2±1.15 a 18.0±0.91 b 9.10±0.90 d 20.2±0.70 a 14.8±0.80 c 7.30±0.90 e

Each value is the mean ± standard error of four replications. Mean values for each parameter sharing different letters are statistically different at p ≤ 0.05

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17

409

410

Fig. 1.

a b 411

412

413

414

415

416

417

418

419

420

421

422

423

424

425

a

b

c

a

b

d

0

5

10

15

20

25

30

35

40

45

50

Control 100 mM NaCl 200 mM NaCl

A. stenophylla A. albida

Ch

loro

ph

yll

con

ten

t (S

PA

D u

nit

)

a

b

c

a

c

d

0

10

20

30

40

50

60

70

80

90



Re

lati

ve

wa

ter

con

ten

ts (

%)

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18

Fig. 2. 426

427

a b 428

c d 429

e

d

b

e

c

a

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8

2



Sh

oo

t N

a+

co

nce

ntr

ati

on

(mm

ol

g-1

dw

)

d

c

b

d

c

a

0

0.2

0.4

0.6

0.8

1

1.2



Ro

ot

Na

+ c

on

cen

tra

tio

n

(mm

ol

g-1

dw

)

e

d

b

e

c

a

0

0.5

1

1.5

2

2.5



Sh

oo

t C

l-co

nce

ntr

ati

on

(mm

ol

g-1

dw

)

d

c

b

d

c

a

0

0.2

0.4

0.6

0.8

1

1.2

1.4



Ro

ot

Cl-

con

cen

tra

tio

n

(mm

ol

g-1

dw

)

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19

e f 430

431

Fig. 3. 432

a b 433

434

435

436

437

a

b

d

a

c

e

0

0.2

0.4

0.6

0.8

1

1.2

1.4


A. stenophylla A. albidaS

ho

ot

K+

co

nce

ntr

ati

on

(mm

ol

g-1

dw

)

a

b

c

a

b

d

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7



Ro

ot

K+

co

nce

ntr

ati

on

(mm

ol

g-1

dw

)

a

b

d

a

c

e

0

1

2

3

4

5

6



Sh

oo

t K

+:

Na

+

a

b

c

a

b

d

0

0.5

1

1.5

2

2.5

3

3.5

4

4.5



Ro

ot

K+:

Na

+

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20

Fig. 4.

438

a b 439

440

c d 441

442

443

444 445 446 447 448 449 450 451

a

b

c

a

c

d

0

10

20

30

40

50

60

70

80

90

100



Me

mb

ran

e s

tab

ilit

y i

nd

ex

(%)

e

c

a

e

d

b

0

2

4

6

8

10

12

14

16

18



SO

D a

ctiv

ity

(U

mg

-1p

rote

in)

e

c

a

e

d

b

0

10

20

30

40

50

60

70



CA

Ta

ctiv

ity

(U

mg

-1p

rote

in)

d

c

a

d

c

b

0

0.5

1

1.5

2

2.5

3

3.5



PO

D a

ctiv

ity

(U

mg

-1p

rote

in)

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21

452 Fig. 5. 453 454 455 456 457

a b 458

c d 459

y = -0.188x + 0.5983

R² = 0.9089

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0 0.5 1 1.5 2

Sh

oo

t d

ry w

eig

ht(

g p

lan

t-1)

Shoot Na+ (mmol g-1 dw)

A.stenophylla y = -0.1824x + 0.5727

R² = 0.8765

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0 1 2 3S

ho

ot

dry

we

igh

t (g

pla

nt-1

) Shoot Na+ (mmol g-1 dw)

A.albida

y = 0.0416x + 0.3519

R² = 0.8187

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0 2 4 6

Shoot K+: Na+

Sh

oo

t d

ry w

eig

ht

(g p

lan

t-1)

A.stenophylla y = 0.0478x + 0.2948

R² = 0.7994

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0 2 4 6

Shoot K+: Na+

Sh

oo

t d

ry w

eig

ht

(g p

lan

t-1)

A.albida

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e f 460

461

Figure captions

Fig. 1. Chlorophyll contents (a) and relative water contents (b) of A.stenophylla and A.albida subjected to NaCl stress.

Each vertical bar represents the mean value ± standard error of four replications. Values sharing a common letter are

statistically similar at 5% probability level

462

Fig. 2. Shoot Na+ (a), root Na

+ (b), shoot Cl

- (c), root Cl

- (d) shoot K

+ (e) and root K

+ (f) concentrations of

A.stenophylla and A.albida subjected to NaCl stress. Each vertical bar represents the mean value ± standard error of

four replications. Values sharing a common letter are statistically similar at 5% probability level

463

Fig. 3. Shoot (a) and root (b) K+: Na

+ ratios of of A.stenophylla and A.albida subjected to NaCl stress. Each vertical bar 464

represents the mean value ± standard error of four replications. Values sharing a common letter are statistically similar 465

at 5% probability level 466

Fig. 4. Membrane stability index (a), superoxide dismutase (b), catalase (c) and peroxidase (d) activities of 467

A.stenophylla and A.albida subjected to NaCl stress. Each vertical bar represents the mean value ± standard error of 468

four replications. Values sharing a common letter are statistically similar at 5% probability level 469

Fig. 5. Correlations between shoot dry weights and shoot Na+

concentrations (a, b) and shoot K

+: Na

+ ratios (c, d), and 470

between shoot Na+ concentrations and superoxide dismutase activities (e, f) of A.stenophylla and A.albida subjected to 471

salinity stress 472

y = 9.6559x + 0.7109

R² = 0.992

0

2

4

6

8

10

12

14

16

18

0 0.5 1 1.5 2


SO

D a

ctiv

ity

(U

mg

-1p

rote

in)

A.stenophylla

y = 5.7662x + 1.4068

R² = 0.9593

0

2

4

6

8

10

12

14

16

0 1 2 3


SO

D a

ctiv

ity

(U

mg

-1p

rote

in)

A.albida

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Draft - University of Toronto T-Space › bitstream › 1807 › ... · Draft 3 51 52 Introduction 53 Salinity is a global environmental problem particularly in arid climatic regions