Halopriming Imparts Salt Tolerance by Reducing Oxidative, Osmotic Stress and DNA Damage in Five Different Legume Varieties

Salinity, a brutal abiotic stress, limits crop productivity worldwide (Gupta and Huang 2014). India, the largest producer and consumer of pulses, contributes to 54% of total production. Legumes are rich in proteins and can fix atmospheric nitrogen to improve soil fertility. Plant growth is hampered under NaCl stress due to osmotic imbalance which eventually hinders water uptake from soil.  Increased Na⁺ uptake inhibit protein synthesis and disrupt various enzymatic functions (AbdElgawad et al., 2016). These cumulatively attribute to generation of reactive oxygen species (ROS) within plants causing oxidative damages to macromolecules thereby interrupting plant cellular functions (El-baky et al., 2003). To withstand such consequences, plants recruit ROS scavenging enzymes for detoxification and elevate levels of compatible solutes to cope with salinity stress.
       
Seed invigoration is a controlled hydration process where seeds are exposed to low water potential that restricts germination, but permits pre-germinative biochemical and physiological changes that favours the germination process and post-germination events (Jisha and Puthur 2014; Chakrabarty et al., 2022). Upon rehydration primed seeds may exhibit faster rates of germination and greater tolerance to environmental stresses Seed halopriming imparts stress tolerance to plants by enhancing biochemical events under salt-stressed conditions (Biswas et al., 2022). Apart from its cytotoxic effects, compartmentalization of sodium ions serves as energetically cheap osmoticum, during conditions when osmolyte production is energetically impossible for plants. (Chakrabarty et al., 2020).
          
Present research work was focussed on assessing influence of NaCl on growth, physio-biochemical parameters and oxidative stress-induced DNA damage in nonprimed and haloprimed seedlings of five legumes having differential sensitivity towards NaCl. The results of the study may help to improve our knowledge on the mechanism(s) taking place within haloprimed seedlings imparting improved salt tolerance.

Seeds of Cajanus cajan L. var. Rabi, Cicer arietinum L. var. Anuradha, Lathyrus sativus L. var. Nirmal, Lens culinaris Medik. var. Ranjan and Vigna mungo L. var. Sulata were collected from Pulse and Oilseed Research Institute, Behrampore, West Bengal, India. Seeds were surface sterilized with 5% NaOCl solution for 10 minutes and rinsed with distilled water. For priming, seeds were soaked in 50mM NaCl for 2 hours and dried back to its original moisture content. The time duration for seed priming was standardized under laboratory conditions based on trial and error method. Primed and nonprimed seeds were allowed to grow in hydroponic solution (Biswas et al., 2022) with corresponding concentration of NaCl (50 mM, 100 mM and 150 mM). Seedlings grown only in hydroponic solution (0 mM NaCl) served as experimental control. Plants were grown under controlled experimental conditions (200 μmol m^-2s^-1 light intensity, 16/8 hours light/dark photoperiod, relative humidity (RH) of about 80-85%) to understand the influence of NaCl on different legume varieties.
       
Plant growth parameters viz., root and shoot lengths, biomass and water contents were recorded from shoot and root of tested legume varieties (Majumder et al., 2018). H₂O₂ content was estimated according to Velikova et al., (2000), MDA content was estimated according to Hodges et al., (1999) and proline estimation was done according to Bates et al., (1973). Comet Assay was performed according to Arya and Mukherjee (2014) to assess DNA damage.
               
Statistically significant values were determined using one-way analysis of variance (ANOVA) and Dunnett’s analysis. p-values were considered significant at p≤0.05, p≤0.01, p≤0.001 and p<0.0001. Pearson’s bivariate correlation analysis was done using PASW Statistical software SPSS 18.0 (Version 18.0.0.282).

Influence on growth, biomass contents and water contents
 
All legume seedlings exposed to NaCl stress encountered significant reduction in growth, biomass and moisture contents. On an average, root lengths diminished by about 10%, 13%, 32%, 49% and 36% and shoot length decreased  by about 9%, 9%, 24%, 39% and 34%, respectively in Vigna, Lathyrus, Cicer, Cajanus and Lens over corresponding nonprimed controls. Pretreatment of seeds before germination, prevented growth inhibition by about 3%, 9%, 12%, 34% and 12% in root and 5%, 6%, 15%, 27% and 14% in shoot of Vigna, Lathyrus, Cicer, Cajanus and Lens, respectively over corresponding nonprimed controls. Dunnett’s multiple comparison revealed significant and maximum growth inhibition of nonprimed (p<0.0001) root and shoot of Lens under 50mM NaCl unlike primed ones (p>0.05). Primed root and shoot of Cicer, Lathyrus and Vigna exposed to 150mM NaCl, showed non-significant growth reduction (p>0.05) contrasting to nonprimed seedlings (p<0.001) (Fig 1).
 

       
Biomass degradation was highest in nonprimed seedlings of Lens (54%), while it was lowered in primed seedlings (44%).  Similar trend was observed for nonprimed Cajanus (50%) and Cicer (44%), compared to primed seedlings (40%, 35% respectively) over control. Primed seedlings of Vigna and Lathyrus showed minimum biomass degradation compared to their nonprimed seedlings. Degradation was least in Vigna (19%) (Fig 2). Similar observation has been reported in ryegrass species having differences in innate tolerance level under salinity (Nizam 2011).
 

       
Plant water status is an important attribute identifying plant tolerance level. Among the test varieties, Vigna showed least water loss under salinity followed by Lathyrus, Cicer, Cajanus and Lens. Seedlings raised from nonprimed seeds of Vigna, Lathyrus, Cicer, Cajanus and Lens exhibited, on an average, 4%, 7%, 7%, 9% and 9% depletion in water contents, respectively, in comparison with their respective nonpretreated control. Seedlings raised from haloprimed seeds were less vulnerable and water contents was replenished, on an average, to about 2% in Vigna and Cajanus; about 5% in Cicer, Lens and Lathyrus .Significant reduction in water contents occurred in nonprimed seedlings of Vigna (p=0.0061), Lathyrus (p<0.0001), Cajanus (p<0.0001) and Cicer (p=0.0319) under 50 mM NaCl. This was reversed in primed seedlings (p>0.05). Significant changes occurred under 100 mM (p<0.001) and 150 mM (p<0.0001) salt exposure in all nonprimed varieties. Primed seedlings of Cajanus (p=0.0001) and Lens (p=0.0029) significantly restored a part of water loss under 100 mM NaCl (Fig 2).
       
Amongst the five varieties, Vigna showed the maximum tolerance when grown under salinity followed by Lathyrus, Cicer, Cajanus and Lens. Previous report related plant water status with inherent salt tolerant capacity of plants, which could be correlated to above findings (Vaktabhai and Kumar 2017). Biswas et al., (2022) reported halopriming relieved salt stress, similar to our results where seedlings raised from primed seeds exhibited improved plant growth and biomass. Additionally, in our study, halopriming proved more beneficial for the more sensitive varieties, Lens and Cajanus compared to the less sensitive ones, Vigna, Lathyrus and Cicer. A positive correlation among growth, biomass and plant water status of primed seedlings (r=0.98 in Lens, r=0.962 in Vigna, r=0.94 in Cicer, Lathyrus, r=0.823 in Cajanus) was obtained.
 
Influence on oxidative stress markers and antioxidant enzyme activity
 
H₂O₂ contents increased in nonprimed seedlings to 18%, 25%, 30%, 66% and 64% in root and to 16%, 24%, 27%, 42% and 55% in shoot of Vigna, Lathyrus, Cicer, Cajanus and Lens respectively over nonprimed control. Haloprimed root and shoot, exhibited lower H₂O₂ accumulation accounting to about 8%, 14%, 18%, 35%, 33% in root,  while in shoot, it was reduced to 7%, 12%, 14%, 21%, 21% in Vigna, Lathyrus, Cicer, Cajanus and Lens respectively, on an average, over nonprimed control (Fig 3a). Statistical analysis revealed significant rise in H₂O₂ level at 50 mM NaCl in nonprimed root of Cicer, Lens and Cajanus (p<0.001) but the significance level reduced in primed (p<0.01) root. In 150 mM NaCl-treated nonprimed root of Lathyrus (p<0.0001) and Vigna (p<0.001), increase in H₂O₂ level was significantly altered. But, under 150 mM NaCl, elevation was much lesser (p<0.01) in primed root of Lathyrus and Vigna.
 

       
Negative correlation amongst H2O2 contents and growth (r=-0.7 in Vigna and Cicer, r=-0.9 in Lens, Lathyrus and Cajanus) and water content (r = 0.9 in all varieties) (Fig 4) indicated that H₂O₂ imposed toxic influence under salinity. However, halopriming prevented H₂O₂ accumulation exhibiting improved growth in all cultivars. Less amount of H₂O₂ accumulation in salt-tolerant Lycopersicon pennellii (Mittova et al., 2002), corroborates with our present observation. Vigna accumulated least H₂O₂ and thus was found to get minimally affected by NaCl, followed by Lathyrus, Cicer, Cajanus while Lens showed maximum accumulation (Fig 3a). Surprisingly, by limiting H₂O₂ accumulation to greater extent, halopriming was more effective for the probable sensitive cultivars viz. Lens, Cajanus, Cicer and Lathyrus as compared to Vigna.
 

       
Catalase activity declined significantly (p<0.0001) with increasing concentrations of NaCl in nonprimed seedlings. Catalase activity declined by about 24%, 25%, 41%, 41% and 48% in nonprimed root and by about  24%, 25%, 41%, 41% and 48% in nonprimed shoot of Vigna, Lathyrus, Cicer, Cajanus and Lens, respectively. In primed root this inhibition could be restricted to 1%, 11%, 11%, 19% and 27% and in primed shoot to about 2%, 3%, 9%, 24% and 27% over their respective nonprimed control seedlings (Fig 3b). Halopriming could revive the hampered catalase activity and thus, scavenged H₂O₂ better, clearly evident in the bivariate plot (Fig 6).
       
Malondialdehyde (MDA) levels were elevated in nonprimed root, by 13%, 18%, 43%, 48% and 61% while in nonprimed shoot the rise was about 9%, 33%, 33%, 39% and 54% in Vigna, Lathyrus, Cicer, Cajanus and Lens over their respective controls. However, in haloprimed root, the elevation decreased to 1%, 13%, 26%, 28% and 35% while in haloprimed shoot, it reduced to about 2%, 13%, 24%, 25% and 30% over their respective controls. In nonprimed seedlings, significant increase was noted at 50mM (p<0.01), 100mM (p<0.0001) and 150mM (p<0.0001) but in Vigna, significant change was only noted in shoot under 150mM NaCl exposure (p<0.0097). Levels of significance were lowered in haloprimed root and shoot of all varieties (p<0.01) and in Vigna changes were non-significant at 100mM and 150 mM NaCl treatments (Fig 5a).
 

       
MDA contents were positively correlated (Pearson’s) to H₂O₂ contents (r=0.538 in Vigna, r~0.8 Lathyrus, Cicer, Cajanus and Lens) and negative correlated to water contents(r=0.9) and growth(r=0.9) in all the varieties. Halopriming efficiently checked H₂O₂ production, limiting lipid peroxidation(r<0.5). A slope value (m) of 42.86 for nonprimed seedlings in the bivariate plot of H₂O₂ content versus MDA content compared to a lower slope value (m=35.71) for primed seedlings indicated stress releasing effect of halopriming (Fig 6).
 

       
Proline accumulation increased under NaCl stress, by 14%, 23%, 37%, 58% and 52% in root and to 13%, 22%, 25%, 40% and 45% in shoot of Vigna, Lathyrus, Cicer, Cajanus and Lens respectively, on an average, over controls. Haloprimed root and shoot exhibited lower proline accumulation amounting to 2%, 5%, 10%, 17% and 16% in haloprimed root,  while in haloprimed shoot, it was reduced to 1%, 15%, 11%, 16% and 19% in Vigna, Lathyrus, Cicer, Cajanus and Lens respectively, over nonprimed controls.  Amongst the test legumes seedlings, root and shoot of Vigna responded least to salinity. Significant rise in proline contents occurred in nonprimed root(p<0.0001) and shoot (p<0.01) of Cajanus, Lens, Cicer and Lathyrus under 50 mM, 100 mM and 150 mM NaCl treatments. Under 100 mM NaCl treatment, rise in proline content was found to reduce in haloprimed root and shoot of Lens and Cicer (p<0.01). In primed root and shoot of Vigna (p<0.01) and Lathyrus (p<0.0001) the increment was much less compared to their respective nonprimed root and shoot (Fig 5b).
 
       
In our study, a positive correlation was obtained amongst proline, MDA and H₂O₂ contents (r»0.9 in all varieties) indicating rise in proline level occurs with increasing oxidative stress in nonprimed seedlings. Proline accumulation was least in nonprimed NaCl-stressed Vigna indicating its salt tolerance ability, followed by Lathyrus, Cicer, Cajanus and Lens (Fig 5b). Similar trend was observed in cotton cultivars exposed to 240 mM NaCl wherein, tolerant variety accumulated less proline (Zhang et al., 2014; Mondal and Chakrabarty, 2020). Mechanistically, under salinity, halopriming possibly triggered proline breakdown rate in the mitochondria to minimize magnitude of osmotic stress conferring tolerance; as observed in KNO₃ primed rice exposed to salinity (Theerakulpisut et al., 2017). Elevation in proline and H₂O₂ contents has been graphically indicated by a higher gradient value (m=171.43) in nonprimed seedlings while enhanced rate of proline breakdown in primed seedlings was marked by lower gradient value (m=142.86) (Fig 6).
 
Influence on DNA damage
 
In our study, all varieties responded significantly at 150 mM NaCl; therefore, comet assay was performed with root and shoot grown under 150 mM NaCl. Our results depicted that DNA damage was less than 10% in nonprimed Vigna but, priming limited it to less than 5%. Nonprimed Lathyrus, Cajanus and Cicer showed nearly 25% DNA damage. Primed seedlings of same varieties showed significant decline in DNA damage to about less than 10% for Lathyrus and Cicer and 15% for Cajanus. DNA degradation was highest in nonprimed root and shoot of Lens, however, primed seedlings recorded lower tail % DNA under stress (10%) jointly. DNA damage was statistically significant in Lens, Cajanus, Cicer and Lathyrus at p<0.0001, while in Vigna it was much lower (p=0.0049). Lower level of DNA damage (p<0.01) after pretreatment in Lens, Cajanus, Cicer and Lathyrus indicated halopriming induced stress release (Fig 7a).
 

       
Previous reports showed that, DNA recombination/DNA repair mechanism gets induced on applying different salt concentrations which could be a possible way to recover or survive under stress (Collins 2004). This was probably achieved by halopriming technique. In our study, primed seedlings showed reduced DNA damage as their nuclei formed comets that visually resembled class 0 comets, similar to nuclei of untreated seedlings (Kumaravel et al., 2007). This was probably achieved either by blocking excess H₂O₂ accumulation or by effectively inducing catalase activity. Positive impact of halopriming on salt-induced DNA damage could be correlated with the amount of H₂O₂ and MDA along with CAT activity and proline contents. Statistically, a lower slope value of H₂O₂ and MDA contents versus DNA damage plot (m=1.0) for pretreated samples, indicated reduction in DNA damage after halopriming over nonprimed seedlings (m=1.2) (Fig 7b). On the other hand (Fig 8) reveals the positive effect of proline and catalase in form of a matrix scatterplot.
 

               
Amongst the test varieties, Vigna and Lathyrus exhibited lower percentages of NaCl-induced inhibition on growth, biomass and moisture contents that resulted in lower accumulation of H₂O₂ in roots and shoots as compared to other three legumes. Also, lower levels of H₂O₂ accumulation in Vigna and Lathyrus limited the degree of cell membrane injury that corresponded with the lower extent of DNA damage as compared to Lens, Cajanus and Cicer. Thus, present findings suggest that based on the test parameters, Vigna and Lathyrus responded least to NaCl stress as compared to other varieties, probably indicating their partial salt-tolerant nature. Similar physiological responses has been observed in salt-tolerant varieties of common bean genotypes viz., KEB-049, KEB-CB053 and Mac-33 over salt-sensitive genotypes viz., KEB-055 and KEB-050 under salinity (Kouam et al., 2017). Reports by Chen et al., (2007) in 23 asparagus bean cultivars under salinity also corresponded to our observations. However, in the present work, seed halopriming helped to overcome the NaCl^- induced adversities by reducing oxidative stress and DNA damage in the test legume varieties to variable extents.

Salinity retarded growth by triggering oxidative stress in all legumes, impact being more on nonprimed seedlings. Present study revealed that amongst the five tested legume varieties, growth of Lens, Cajanus and Cicer were more adversely affected under NaCl stress as compared to Lathyrus and Vigna. This not only indicates salt-sensitive nature of Lens, Cajanus and Cicer but also partial salt tolerant nature of Vigna and Lathyrus seedlings grown under salt stress. Also, seed halopriming was able to alleviate the adversities experienced by all the test seedlings grown under NaCl stress. Salinity tolerance in nonprimed seedlings was achieved by osmolyte accumulation whereas in haloprimed seedlings, it was achieved by increasing catalase activity that promoted cellular detoxification. This lowered membrane degradation causing limited interaction of DNA with Na⁺ and Cl^- ions reducing the extent of DNA damage. Based on our study, enhanced rate of ROS detoxification by catalase was the major mechanism adopted by haloprimed seedlings to survive under salinity. Thus, pre-germination treatment acclimatised seeds favouring germination and post-germinative events conferring improved growth under salinity.

The work was financially supported by The Department of Science and Technology, Government of West Bengal under Sanction no. 1012 (Sanc.)/ST/P/SandT/2G-2/2013.