Salt induced inhibition in photosynthetic parameters and polyamine accumulation in two legume cultivars and its amelioration by pretreatment of seeds with NaCl

Salinity affects plant productivity in arid and semi-arid regions. Glycophytes under salinity stress generate reactive oxygen species (Verma and Mishra, 2005) which cause membrane damage and denature macromolecules (Duan et al., 2008). So, to overcome such salt-induced adversities on plant growth and to increase crop productivity, development of salt tolerant crops undertaking simple eco-friendly technique of pretreatment has been adopted to grow more crops in salinity prone agricultural lands (Saha et al., 2012).
 
Salinity affects photosynthesis rate and various bioenergetic processes (Sudhir and Murthy, 2004). The carbon balance of plants during the period of salt stress and recovery depends on the velocity and degree of photosynthetic recovery (Chaves et al., 2009). Stomatal conductance is also affected by salinity. So, the contributory effect of stomata to limit photosynthesis also increases (Sadeghipour, 2017). Transpiration rate has been reported to decrease under salt stressed conditions (Sharma et al., 2005). The amount of chlorophyll and its accessory pigments also decreased under salt exposure (Amuthavalli and Sivasanakaramoorthy, 2012).
 
Polyamines (PAs) play a key role in regulating adaptive response of plants under abiotic stresses (Zhang et al., 2013). Polyamines exhibit anti-senescence, anti-stress effects by acting as free radical scavengers and activate antioxidant enzymes that enhance detoxification and membrane stabilization due to their acid neutralizing properties (Zhang et al., 2013; Kubiś et al., 2014).
 
Under NaCl stress, Spm and Spd content have been reported to be induced (Li et al., 2016). Cadaverine initiates root development by inducing putrescine and spermine accumulation and also reduces Spd content (Liu et al., 2014).
 
Present study investigates the influence of NaCl on chloroplastic pigment contents, photosynthetic gas exchange parameters and variations in polyamine contents in arhar and maskalai seedlings growing under NaCl stress and how these parameters varied when preconditioned legume seeds (pretreated with 50mM NaCl) were allowed to grow under NaCl stressed conditions. Seed pretreatment has already been reported to confer better growth and metabolism in the said legume cultivars (Chatterjee et al., 2018). However, no study has been conducted till date to compare the alterations in pigment composition, gas exchange parameters and polyamine contents in NaCl stressed and NaCl pretreated arhar and maskalai seedlings. The relative salt tolerance ability between the two legumes could also be understood based on the variations in tested biochemical parameters.

Seeds of arhar (Cajanus cajan L. cv. T120) and maskalai (Vigna mungo L. cv. WBU109) were collected from Pulse and Oil Seed Research Institute Beherampore, West Bengal, India. Experiments were carried out in the laboratory, under controlled physiological conditions (27-30oC with influence of 16h photoperiod at 200µmolm^-2s^-1 photon irradiance for 3 weeks) during 2014-2017. Surface sterilized seeds (25) were placed on glass plate lined by blotting papers with 3 replicates containing suitable concentration of test solutions. Different concentrations of NaCl were added to Hoagland solution (Widodo et al., 2009) to prepare 100mM and 150mM salt solutions. The reversal of salt toxicity was determined by pretreating seeds with 50mM of NaCl for 2 hours (pretreated) before salt treatment (Chatterjee et al., 2018). Seedlings were harvested after 3 weeks for further experiments.
 
Chlorophyll contents were estimated according to Arnon et al., (1949). Chlorophyll fluorescence was estimated at excitation wavelength 640nm and emission wavelength 680nm using Hitachi U-2000 spectroflurimeter. Hill activity was measured at 420nm spectrophotometrically according to Vishniac (1957). Carotene and xanthophyll contents were estimated according to Mukherjee and Biswas (1979).
 
Physiological gas exchange parameters viz., photosynthetic rate, stomatal conductance and transpiration rate were measured from well developed expanded leaves using a portable gas exchange system LI-6400 (LI-COR Inc., USA) between 10 am to 11 am in the morning under ambient conditions. CO₂ concentration was measured at 300µmol-mol^-1 and leaf temperature was at 28oC.
 
Polyamine estimation was done using high performance liquid chromatography following the protocol of Flores and Galston, (1982).
 
The biochemical experiments were performed three times (n=3) and the data obtained from the experiments were analysed by one-way analysis of variance (ANOVA) using Sigma Plot software.

Gradual decrease in total chlorophyll, chlorophyll a and b, carotene contents, xanthophylls as well as in chlorophyll fluorescence intensity was observed in shoots of both the legume cultivars under salt stress (Table 1, Table 2). Similar reports have also been reported in salt stressed Catharanthus roseus (Jaleel et al., 2007). The decrease in total chlorophyll content was by 53% and 34% in nonpretreated arhar and maskalai respectively. In pretreated seedlings, level of chlorophyll depletion narrowed down to about 26% in arhar and 14% in maskalai seedlings respectively. The chlorophyll a and chlorophyll b contents decreased by 49% and 73% respectively in arhar and by 43% and 64% respectively in maskalai as compared to nonpretreated control. Pretreated seedlings showed lower depletion of pigment content in arhar by about 30% and 26% whereas in maskalai the decline minimized to about 15% and 11% for chlorophyll a and chlorophyll b respectively. Carotene contents decreased in arhar and maskalai by about 22% and 12% respectively. However, pretreated seedlings showed reduction in the rate of carotene by 5% in arhar and 4% in maskalai seedlings respectively. Xanthophyll contents decreased by 65% in arhar and 3% in maskalai under NaCl stress. Seed pretreatment reduced the level of decrease by about 4% in arhar whereas in maskalai the said content was increased by 13% over nonpretreated control. Intensity of chlorophyll fluorescence decreased in the test seedlings under NaCl stress by 45% and 22% in arhar and maskalai respectively. In salt pretreated arhar and maskalai, the decrement in chlorophyll fluorescence intensity reduced by 14% and 12% respectively over nonpretreated control. The decline in principal as well as accessory pigments content in the test cultivars under salinity was probably due to increased chlorophyllase enzyme activity that induced damage of pigment protein complexes of chloroplasts (Abeer et al., 2015). Higher rate of chlorophyll b degradation can be correlated with its increased conversion to chlorophyll a under stressed condition (Saha et al., 2010). However, seed pretreatment significantly altered the chloroplastic pigment constitution in both arhar and maskalai probably by neutralizing the toxic effects of NaCl on pigment biosynthesis on account of which intensity of chlorophyll fluorescence increased. Present results are in accordance with those obtained by Saha et al., (2010) on mungbean. NaCl application decreased Hill activity in nonpretreated arhar and maskalai by 49% in arhar and by 52% in maskalai (Fig 1, Fig 2). This happened due to hindered PSII reactions and oxygen dissipation from photosynthetic apparatus. Chloroplasts isolated from pretreated seedlings exhibited reduced inhibition in Hill activity to about 25% in arhar and to 11% in maskalai. This probably occurred because seed pretreatment developed resistance against salt stress that improved Hill activity in the chloroplast of the test seedlings by promoting photolysis of water resulting in improved electron flow from chlorophyll molecules to electron acceptor dye DCPIP causing increased rate of DCPIP reduction. 
 

 

 

 

 
NaCl application reduced stomatal conductance (g_s), net photosynthetic rate (P_n), internal CO₂ concentration (Ci), and transpiration rate (E) compared to control (Table 3, Table 4). A reduction of 83% in stomatal conductance, 80% in photosynthesis rate resulted in reduction of internal CO₂ concentration by 77% in arhar. In maskalai, the decrease in stomatal conductance, photosynthetic rate, internal CO₂ concentration was by about 40%, 68% and 32% respectively over nonpretreated control. Reduction in photosynthetic rate was due to stomatal closure and chlorophyll degradation under salt stress. Report by Sadeghipour (2017) on cowpea showed that reduction in photosynthesis under salinity is attributed to stomatal closure which decreased internal CO₂ concentration. Rate of transpiration diminished in nonpretreated arhar and maskalai seedlings by 81% and by 34% respectively. Similar observation was reported earlier in salt stressed tobacco (Hatamnia et al., 2013).  Pretreatment of seeds before its exposure to NaCl narrowed down the rate of decrease in net photosynthesis (P_n), stomatal conductance (g_s) and internal CO₂ concentration (C_i) to 72%, 68% and 65% respectively over non-pretreated control in arhar seedlings whereas in maskalai seedlings, the decrement was lowered in the respective aforementioned parameters by 21%, 68% and 21% respectively, on an average, for net photosynthesis (P_n), stomal conductance (g_s) and internal CO₂ concentration (Ci) respectively indicating lesser endogenous damage in photosynthetic parameters due to seed pretreatment.
 

 

 
The ratio of (spermine+spermidine)/putrescine content increased in salt stressed arhar and maskalai seedlings (Fig 5, Fig 6). In salt stressed arhar, the ratio was increased by 1039% in root and by 108% in shoot whereas in maskalai, it increased by 63% in root and 362% in shoot respectively over nonpretreated control. Increased content of (Spermine+Spermidine)/Putrescine ratio under NaCl stress in the present study can be corroborated with earlier reports published on different salt stressed plant species (Zapata et al., 2004). Accumulation of spermine and spermidine probably attributed protection under saline conditions as reported in wheat cultivars grown under salt stress (El-Shintinawy, 2000). In the study, accumulation of higher level polyamines on experiencing salt toxicity was probably an attempt by the seedlings to reduce the NaCl induced toxicity. Moreover, since, spermidine is synthesized from putrescine by the activity of spermidine synthase and synthesis of spermine occurs from spermidine, presence of lower levels of putrescine and higher levels of spermine and spermidine were recorded in salt stressed plants. This indicated that under salinity, conversion of putrescine to spermine and spermidine took place for which putrescine contents was gradually lowered. Similar reduction in putrescine contents was reported in abiotically stressed pigeon pea (Radadiya et al., 2016). Cadaverine which initiates cell division and root growth decreased under salt stress by about 40% and 35% in root and by about 46% and 54% in shoot of arhar and maskalai seedlings respectively (Fig 7, Fig 8). Results obtained from pretreated arhar seedlings showed that the (Spermine+Spermidine)/Putrescine ratio decreased under NaCl stress to about 19% in root and 43% in shoot (Fig 3 and 5). In pretreated maskalai seedlings, the ratio was reduced to about 10% in root and 99% in shoot, indicating less accumulation of spermine and spermidine and more accumulation of putrescine (Fig 4, Fig 6) in pretreated seedlings, conferring stress tolerance. This indicated that low activity of spermidine synthase and spermine synthase resulted in low accumulation of spermine and spermidine and therefore, presence of more putrescine (substrate for spermidine synthase) was recorded. NaCl pretreatment generated seedlings that experienced less toxic cellular environment under lethal levels of salinity due to presence of increased level of putrescine which not only acted as free radical scavenger but also triggered photosynthetic activity. This can be correlated with the hypothesis of improved photosynthesis resulting in accumulation of endogenous putrescine proposed by Ioannidis et al., (2012).  Additionally, in roots of pretreated arhar seedlings, the cadaverine contents was found to increase to 65% in root and to about 89% in shoot on an average, compared to nonpretreated control (Fig 7). In pretreated maskalai root, the decrement in cadaverine content was reduced to about 10% and in shoot by it increased by about 305% over nonpretreated control (Fig 8) for which better root growth occurred that probably helped the plant to regulate unhindered water uptake under salt stress (Chatterjee et al., 2017).
 

 

 

 

 

 


The mean values obtained for all the tested parameters were found to be more statistically significant for arhar indicating its salt-sensitivity. In maskalai, the values obtained were not that statistically significant indicating its tolerant nature.

Increasing concentrations of NaCl interfered with stomatal conductance, affected rate of transpiration and decreased photosynthetic rate in nonpretreated arhar and maskalai seedlings. Polyamines like spermine and spermidine accumulated in salt stressed seedlings which was obligatory for them to continue their growth under NaCl stress. Decrease in putrescine contents under salinity affected rate of photosynthesis. Reduction in cadaverine contents also led to stunted root growth under salt stress. However, pretreatment of seeds prior to germination with sublethal dose of NaCl (50mM) produced seedlings that exhibited improved gaseous exchange and polyamine accumulation indicating stress release leading to better growth and development of the test cultivars.

This research was financially supported by a grant offered by the Department of Science and Technology, Government of West Bengal under sanction no. 1012(Sanc.)/ST/P/S&T/2G-2/2013.

SB and PC from Calcutta University performed all experiments. SB of Calcutta University drafted the manuscript. AM and SB from Jadavpur University assisted by providing portable Li-COR instrument and data generation of gas exchange parameters. AKB helped in experimental designing and data interpretation.