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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
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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
2 3
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: ghulamabbas@ciitvehari.edu.pk; g.a92pk@gmail.com 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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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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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
Control 100 mM NaCl 200 mM NaCl
A. stenophylla A. albida
Re
lati
ve
wa
ter
con
ten
ts (
%)
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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
Control 100 mM NaCl 200 mM NaCl
A. stenophylla A. albida
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
Control 100 mM NaCl 200 mM NaCl
A. stenophylla A. albida
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
Control 100 mM NaCl 200 mM NaCl
A. stenophylla A. albida
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
Control 100 mM NaCl 200 mM NaCl
A. stenophylla A. albida
Ro
ot
Cl-
con
cen
tra
tio
n
(mm
ol
g-1
dw
)
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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
Control 100 mM NaCl 200 mM NaCl
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
Control 100 mM NaCl 200 mM NaCl
A. stenophylla A. albida
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
Control 100 mM NaCl 200 mM NaCl
A. stenophylla A. albida
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
Control 100 mM NaCl 200 mM NaCl
A. stenophylla A. albida
Ro
ot
K+:
Na
+
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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
Control 100 mM NaCl 200 mM NaCl
A. stenophylla A. albida
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
Control 100 mM NaCl 200 mM NaCl
A. stenophylla A. albida
SO
D a
ctiv
ity
(U
mg
-1p
rote
in)
e
c
a
e
d
b
0
10
20
30
40
50
60
70
Control 100 mM NaCl 200 mM NaCl
A. stenophylla A. albida
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
Control 100 mM NaCl 200 mM NaCl
A. stenophylla A. albida
PO
D a
ctiv
ity
(U
mg
-1p
rote
in)
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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
Shoot Na+ (mmol g-1 dw)
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
Shoot Na+ (mmol g-1 dw)
SO
D a
ctiv
ity
(U
mg
-1p
rote
in)
A.albida
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