Legume Research

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Research Article

Legume Research, volume 45 issue 1 (january 2022) : 73-81

Potential of Seed Halopriming in Mitigating NaCl-induced Adversities on Nitrogen Metabolism in Legume Crops

Sabarni Biswas1, Alivia Paul1, Asok K. Biswas1,*
1Plant Physiology and Biochemistry Laboratory, Centre of Advanced Studies, Department of Botany, University of Calcutta, Kolkata-700 019, West Bengal, India.

  • Submitted05-11-2019|

  • Accepted25-02-2020|

  • First Online 28-07-2020|

  • doi 10.18805/LR-4276

Cite article:- Biswas Sabarni, Paul Alivia, Biswas K. Asok (2022). Potential of Seed Halopriming in Mitigating NaCl-induced Adversities on Nitrogen Metabolism in Legume Crops . Legume Research. 45(1): 73-81. doi: 10.18805/LR-4276.

ABSTRACT

Background: Salinity is a major threat that impairs legume growth and development worldwide. Therefore, present study was aimed to determine the potential of seed halopriming in relieving NaCl-induced disturbances on nitrogen metabolism of seedlings of six legume crops viz., Lens culinaris, Cajanus cajan, Cicer arietinum, Lathyrus sativus, Vigna radiata and Vigna mungo that were detected to have differential sensitivity to NaCl. 

Methods: Nonprimed and haloprimed seeds were grown hydroponically under varying NaCl doses for three weeks. Harvested samples were utilised to characterize the toxic effects of NaCl on nitrogen metabolism of nonprimed and haloprimed seedlings.    

Results: Nonprimed seedlings exhibited reduced nitrate uptake by virtue of which other assimilatory processes of nitrogen fixation were adversely affected. Haloprimed seedlings experienced lesser toxicity under NaCl stress due to elevated activities of nitrate assimilatory enzymes on account of improved nitrate uptake from solution. Lesser ammonium accumulation and lower glutamate dehydrogenase activity implied lesser cytotoxicity in primed seedlings. Based on the trends obtained from tested parameters, nitrogen metabolism was maximally affected in Lens and Cajanus followed by Cicer and Lathyrus. Vigna radiata and Vigna mungo were least affected and therefore may be suggested for cultivation in saline prone agricultural fields after seed halopriming.

INTRODUCTION

Salinity depresses nutrient uptake causing nutritional deficiency and productivity loss of plants (Ashraf et al., 2017). Globally, 45 million hectares of irrigated lands (19.5%) are salt affected (Thu et al., 2016) and are unproductive for agriculture. Therefore, to utilize portions of these lands, agriculturists aim at developing salt tolerant crops to grow them in non-arable regions to escalate crop production.
       
Nitrogen is an integral constituent of proteins, cell construct materials and is crucial for plant growth (Arghavani et al., 2017). Nitrogen metabolism is highly affected under salinity and its regulation is crucial for glycophytes to thrive under salinity (Ashraf et al., 2018; Teh et al., 2016).
       
Legumes are cheapest source of protein and used in crop rotation to restore soil nitrogen naturally. Being glycophytes, legumes are highly affected under salinity as nitrogen uptake competes with Na+ and Cl- entry (Abdelgadir et al., 2005).
       
Present study was aimed primarily to assess the deleterious effects of different NaCl concentrations on the intermediates of nitrogen metabolism in six legume cultivars. Based on tested parameters, partial identification of salt sensitive/tolerant nature of legume cultivars could be deciphered preliminarily. The most tolerant variety could be suggested for cultivation in saline prone agricultural lands. Additionally, all legume cultivars were subjected to halopriming to study whether any improvement occurred in their nitrogen assimilatory ability as compared to those of the nonprimed seedlings under salinity.

MATERIALS AND METHODS

Seeds of Lens culinaris Medik. var. Ranjan, Cajanus cajan L. var. Rabi, Vigna mungo L. var. Sulata, Cicer arietinum L. var. Anuradha, Lathyrus sativus L. var. Nirmal and Vigna radiata L. var. Samrat were collected from Pulse and Oilseed Research Institute, Behrampur, West Bengal. The chosen legume cultivars have not been previously characterized on the basis of physiochemical properties as tolerant/sensitive under salt stress.
       
Surface sterilized seeds were divided into two fractions. First fractions were lined on glass plates containing blotting papers, inserted into packets containing suitable concentrations of hydroponic solution substituted with 50 mM, 100 mM and 150 mM NaCl (nonprimed sets). Other fractions of seeds were immersed in 50 mM NaCl for 2 hours for halopriming. Thereafter, haloprimed seeds were allowed to germinate in same way as that of nonprimed sets (Biswas et al., 2018). Setups were exposed to 16 hours photoperiod, 200 μmol m-2s-1 photon irradiance, 27-30°C. After 21 days, samples were harvested to characterize the toxic effects of NaCl on nitrogen metabolism of nonprimed and haloprimed seedlings.   

Total and soluble nitrogen contents were estimated according to Vogel (1961). Nitrate and nitrite contents were determined according to Zhong et al., (2017) and Werber and Mevarech (1978) respectively. Nitrate reductase activity (NR) and nitrite reductase activity (NiR) were done according to Zaghdoud et al., (2013) and Ghosh et al., (2013) respectively. Dissolved ammonia contents were estimated according to Hoshida et al., (2000). Glutamate synthase (GOGAT) activity and Glutamine synthetase (GS) activity was measured according to Chen and Cullimore (1988) and Zhagdoud et al., (2013) respectively. Glutamate dehydrogenase (GDH) activity was assayed according to Magalhaes and Huber (1991).
       
Experiments were performed in triplicates; significant differences among mean values were compared by one-way ANOVA in Sigma Plot 12.0 software. The p-values were considered to be statistically significant at P<0.05.

RESULTS AND DISCUSSION

NaCl exposure decreased total nitrogen contents by 37%, 22%, 20%, 19%, 13% and 4% in root and 14%, 14%, 12%, 8%, 12% and 4% in shoot of Lens culinaris, Cajanus cajan, Cicer arietinum, Lathyrus sativus, Vigna radiata and Vigna mungo respectively. However, in haloprimed root, the decline narrowed down to 12%, 2%, 10%, 7% and 3% in primed root and by 7%, 7%, 5%, 6% and 3% in primed shoot of Cajanus, Cicer, Lathyrus, Vigna radiata and Vigna mungo respectively. In haloprimed Lens, the said content increased by 1% in root and by 5% in shoot (Table 1). Soluble nitrogen contents decreased by 39%, 22%, 18%, 10%, 5% and 5% in nonprimedroot and by 30%, 18%, 11%, 8%, 4% and 4% in nonprimed shoot of Lens, Cajanus, Cicer, Lathyrus, Vigna radiata and Vigna mungo respectively. Haloprimed seedlings recorded lower reduction in soluble nitrogen contents by 12%, 8%, 2%, 2% and 1% in root of Cajanus, Cicer, Lathyrus, Vigna radiata and Vigna mungo respectively. In primed root of Lens, the content increased to 6%. In primed shoots, the said inhibition reduced to 3%, 11%, 5%, 5%, 2% and 1% in shoot of Lens, Cajanus, Cicer, Lathyrus, Vigna radiata and Vigna mungo respectively (Table 2). Nitrate contents decreased by 48%, 43%, 34%,33%, 26% and 26% in nonprimed roots and by 35%, 34%, 30%, 29%, 24% and 25% respectively in nonprimed shoot of Lens, Cajanus, Cicer, Lathyrus, Vigna radiata and Vigna mungo respectively. Depreciation in said contents declined to 30%, 15%, 10%, 11%, 16% and 9% in haloprimed roots and to 14%, 14%, 10%, 10%, 9% and 8% in haloprimed shoots of Lens, Cajanus, Cicer, Lathyrus, Vigna radiata and Vigna mungo respectively (Table 3). NR activity decreased by 53%, 52%, 31%, 30%, 16% and 3% in root and by 30%, 24%, 25%, 23%, 12% and 2% in shoot of Lens, Cajanus, Cicer, Lathyrus, Vigna radiata and Vigna mungo respectively under NaCl treatment. Decrement in NR activity was reduced to 22%, 29%, 16%, 15%, 11% and 2% respectively in primed roots of Lens, Cajanus, Cicer, Lathyrus, Vigna radiata and Vigna mungo respectively. In primed shoot of Lens, Cicer, Lathyrus and Vigna radiata, the reduction in NR activity narrowed to 6%, 10%, 6% and 7% respectively where as in Cajanus and Vigna mungo the activity was promoted by 16% and 1% respectively (Fig 1). Nitrite contents decreased by 55%, 49%, 41%, 40%, 34% and 30% in nonprimed root and by 44%, 47%, 27%, 38%, 31% and 29% in nonprimed shoot respectively of Lens, Cajanus, Cicer, Lathyrus, Vigna radiata and Vigna mungo. However, reduction in nitrite contents was narrowed to 16%, 16%, 17%, 14% and 11% in haloprimed root of Lens, Cajanus, Cicer, Lathyrus and Vigna mungo respectively, whereas in Vigna radiata, it increased by 5%. In haloprimed shoot, the decrement reduced to 18%, 12%, 8%, 14%, 9% and 3% in Lens, Cajanus, Cicer, Lathyrus, Vigna radiata and Vigna mungo respectively (Table 4). NiR activity catalyzes conversion of nitrite into ammonia. The decline in NiR activity was most prominent in Lens (37% in root, 24% in shoot) and Cajanus (36% in root, 26% in shoot), moderate in Cicer (27% in root, 25% in shoot) and Lathyrus (28% in root, 22% in shoot) and least in Vigna radiata (14% in both root and shoot) and Vigna mungo (6% in root, 4% in shoot). Similar effects have been observed in salt stressed Populus simonii (Meng et al., 2016). In haloprimed seedlings, the inhibition was reduced to 21%, 22%, 19%, 20%, 10% and 3% in roots of Lens, Cajanus, Cicer, Lathyrus, Vigna radiata and Vigna mungo respectively. In primed shoot, the inhibition was reduced to 8%, 16%, 17%, 11% and 2% respectively in Lens, Cajanus, Cicer, Lathyrus and Vigna mungo respectively whereas in Vigna radiata, the activity was promoted by 3% (Fig 2). Dissolved ammonia contents increased in nonprimed root by 81%, 75%, 51%, 46%, 19% and 10% and by 57%, 23%, 46%, 32%, 17% and 9% in nonprimed shoot of Lens, Cajanus, Cicer, Lathyrus, Vigna radiata and Vigna mungo respectively. In haloprimed seedlings, it decreased by 42%, 24%, 16%, 18%, 4% and 3% in root and in shoot the increment narrowed down to 17%, 13%, 16%, 13%, 5% and 3% in Lens, Cajanus, Cicer, Lathyrus, Vigna radiata and Vigna mungo respectively (Table 5).
 

Table 1: Effect of NaCl on total nitrogen contents in 21 days old nonprimed and haloprimed legume seedlings.


 

Table 2: Effect of NaCl on soluble nitrogen contents in 21 days old nonprimed and haloprimed legume seedlings.


 

Table 3: Effect of NaCl on nitrate contents in 21 days old nonprimed and haloprimed legume seedlings.


 

Fig 1: Effect of NaCl on nitrate reductase (NR) activity of 21 days old nonprimed and haloprimed seedlings of legume cultivars.


 

Table 4: Effect of NaCl on nitrite contents in 21 days old nonprimed and haloprimed legume seedlings.


 

Fig 2: Effect of NaCl on nitrite reductase (NiR) activity of 21 days old nonprimed and haloprimed seedlings of legume cultivars.


 

Table 5: Effect of NaCl on dissolved ammonia contents in 21 days old nonprimed and haloprimed legume seedlings.


       
Decline in total nitrogen, soluble nitrogen, nitrate and nitrite contents with reduced activities of NR was recorded in the tested legume cultivars under NaCl stress since, salinity diminished nitrate uptake in tested seedlings. Similar report on reduction of nitrate uptake has been published in salt stressed Lycopersicon esculentum (Debouba et al., 2006). By virtue of lesser nitrate uptake under NaCl stress, subsequent decline in NR and NiR activities were recorded. This occurred because nitrate content is known to regulate NR and NiR gene expressions (Wang et al., 2000). Similar report on decline in NR activity in salt stressed wheat (Ahanger and Agarwal, 2017) evinces that nitrate contents regulate NR activity and determines the flow of nitrate to the active sites of the enzyme. Decreased nitrate uptake in the tested legume cultivars developed an uptake competition between NO3- and Cl- during growth by affecting cellular membranes (Wang et al., 2011; Zhang et al., 2013). Reduction in nitrate uptake and lower NR activity under salt stress in the tested cultivars decreased corresponding nitrite contents. Amongst the tested cultivars, decrease in the total and soluble nitrogen contents, nitrate and nitrite contents assisted by the NR activity were maximally inhibited in Lens and Cajanus indicating their salt sensitivity whereas, it was moderately affected in Cicer and Lathyrus. In Vigna radiata and Vigna mungo, the inhibitory effects were minimal, manifesting their partial salt tolerance. Seed priming helped to overcome such adversities conferring increased nitrate uptake during seedling growth. Present study recorded increased ammonia contents under NaCl stress. Elevated ammonia contents aggravate cytotoxicity, impair osmotic regulation and plant development (Zhang et al., 2013). NiR activity in Lens, Cajanus, Cicer and Lathyrus was much higher as compared to their respective NR activity whereas, in Vigna radiata and Vigna mungo, opposite trend was observed, i.e. higher NR activity and less of NiR activity. This corresponded to higher accumulation of NH4+ in Lens, Cajanus, Cicer and Lathyrus indicating more cytotoxic environments. Lower ammonia accumulation due to low NiR activity resulted in lesser cytotoxicity in Vigna radiata and Vigna mungo indicating that these two cultivars were least salt sensitive.
       
GOGAT activity decreased by 36%, 32%, 22%, 17%, 12% and 2% in root and by 22%, 20%, 17%, 11%, 5% and 1% in shoot of Lens, Cajanus, Cicer, Lathyrus, Vigna radiata and Vigna mungo respectively. However, in haloprimed roots, the inhibition narrowed down to 34%, 12%, 20%, 9% and 1% in Lens, Cajanus, Cicer, Vigna radiata and Vigna mungo respectively. In haloprimed Lathyrus root, the activity was promoted to 14%. In shoot of primed seedlings, the inhibition narrowed down to 6%, 9%, 16%, 8% and 2% in Lens, Cajanus, Cicer, Lathyrus and Vigna radiata respectively. In haloprimed shoot of Vigna mungo, the activity was promoted to 2% (Fig 3). GS activity declined in root by 42%, 36%, 29%, 27%, 18% and 16% and in shoot by 37%, 26%, 24%, 13%, 8% and 2% respectively in Lens, Cajanus, Cicer, Lathyrus, Vigna radiata and Vigna mungo respectively. The inhibition in GS activity was decreased to 22%, 18%, 10%, 15%, 12% and 10% in root and by 16%, 14%, 9%, 4%, 1% and 1% in shoot of Lens, Cajanus, Cicer, Lathyrus, Vigna radiata and Vigna mungo (Fig 4).
 

Fig 3: Effect of NaCl on glutamate synthase (GOGAT) activity of 21 days old nonprimed and haloprimed seedlings of legume cultivars.


 

Fig 4: Effect of NaCl on glutamine synthetase (GS) activity of 21 days old nonprimed and haloprimed seedlings of legume cultivars.


       
Decline in GS and GOGAT activities caused NH4+ accumulation under salinity in the tested cultivars. Inhibition was notable in Lens and Cajanus, followed by Cicer and Lathyrus. This perhaps generated cytotoxicity because cellular pH was disrupted and photophosphorylation got uncoupled (Ashraf et al., 2018). The inhibitory effect of GS and GOGAT was least in Vigna radiata and Vigna mungo, probably hinting their tolerance towards NaCl. Similar report has been published in arsenic stressed wheat seedlings (Ghosh et al., 2013). Seed halopriming helped to alleviate such NaCl induced toxicity, facilitating better nitrate assimilation. Enhanced GDH activity catalyzes conversion of glutamate into ammonia (Skopelitis et al., 2006). Salinity provoked significant elevation in GDH activity by 70%, 47%, 35%, 31%, 18% and 4% in nonprimed root and by 58%, 44%, 23%, 28%, 11% and 6% in nonprimed shoot of Lens, Cajanus, Cicer, Lathyrus, Vigna radiata and Vigna mungo respectively. The activity was maximum in Lens and Cajanus, moderate in Cicer and Lathyrus and least in Vigna radiata and Vigna mungo, which could be justified with their respective average percentages of ammonia accumulation recorded in nonprimed root and shoot. High NH4+is harmful for cells and needs quick assimilation. Similar changes have been noticed in saline-alkaline stressed tomato (Zhang et al., 2013). Seed priming decreased GDH activity to 31%, 18%, 17%, 11%, 12% and 7% in roots and to  23%, 19%, 3%, 10%, 5% and 2% in shoots of Lens, Cajanus, Cicer, Lathyrus, Vigna radiata and Vigna mungo respectively. Present results corroborate with the reduced NH4+ accumulation in respective haloprimed seedlings. Similar ameliorative change has been observed in selenate administered arsenic stressed wheat (Ghosh et al., 2013) (Fig 5).
 

Fig 5: Effect of NaCl on glutamate dehydrogenase (GDH) activity of 21 days old nonprimed and haloprimed seedlings of legume cultivars.


 
Fig 6 schematically illustrates NaCl-induced alterations on nitrogen metabolism in nonprimed seedlings and efficacy of seed halopriming in overcoming such adversities facilitating better nitrate assimilation.
 

Fig 6: Illustration depicting alterations in intermediates of nitrogen metabolism and its assimilatory enzymes in nonprimed and haloprimed seedlings of legumes cultivars subjected to NaCl stress.

CONCLUSION

The potency of seed halopriming to overcome NaCl-induced disturbances on nitrogen metabolism depended on differential resistance mechanisms of tested cultivars. Pre-germination halopriming probably aroused a ‘memory response’ in seeds for which better nitrogen assimilation was noted in tested primed seedlings under salinity. The efficacy of halopriming was more effective in salt sensitive cultivars and lesser in partially salt-tolerant cultivars like Vigna radiata and Vigna mungo. This hinted species-specific efficiency of seed halopriming. Although, the halopriming efficacy was less in partially tolerant cultivars (Vigna radiata and Vigna mungo) but, it helped them to grow better under NaCl stress, no matter how much less the efficiency was. Based on present results, these two cultivars may thus be haloprimed and suggested for cultivation to increase pulse production in saline prone agricultural fields.

ACKNOWLEDGEMENT

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

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