BIOCHEMICAL AND MOLECULAR CHANGES UNDER SALINITY STRESS IN ARACHIS HYPOGAEA

Abstract

In the present work attempts have been made to understand the biochemical and molecular basis of salt stress response in groundnut (Arachis hypogaea) at seedling stage. The groundnut seedlings used in all experiments were grown hydroponically under salinity stress of 50-200 mM NaCl for seven days. The effect of NaCl stress on the growth of groundnut seedlings was studied. The remarkable reduction in the growth of root as well as shoot was seen in the seedlings under salinity stress from salt level 50 to 200 mM. The inhibition of growth was also supported by reduction in fresh weights of shoot and root of seedlings. Further, marked differences were also noted in anatomical features under salt stress. The decreased in cell size due to thickening of cell wall and disappearance of intercellular space in both roots and shoots of NaCl treated seedlings were seen. Reduction in cell size and thickening of cell wall resulted in stunted growth due to reduction in overall extensibility of cell. These are common salt stress responses shown by most of the plants. There was increase in proline accumulation in roots and shoots of salt stressed seedlings. The accumulation of proline under salinity stress by plant is one of the major responses and it plays important role in salt tolerance by acting as osmoprotectant. Besides, proline was also reported to play protective role by stabilizing various cellular proteins and enzymes. Itmaybe likely thatthe proline may play a similar role in groundnut as well. Peroxidases and various hydrolases are found to play important role in cell growth. The effect of salt stress on peroxidases and hydrolases was studied. There was significant increase in peroxidase activity and reduction in hydrolase activity ii under salt stress. The most prominent increase was found in ionicallybound cell wall peroxidase fraction. The purification of ionically bound fraction resulted in identification of salt stress induced ionically bound anionic peroxidase of molecular weight 39 kDa and pi 5.5. An inverse relationship was found in peroxidase activity and growth. Peroxidases reduce cell growthby altering cell extensibility by increasing cell wall rigidity due to cross linking of cell wall polysaccharides and structural proteins like extensin. A number of cationic and anionic peroxidase isoforms cause cell wall rigidity by ferulic acid mediated lignification and suberinization and cross linking of extensin. It is likely that the salt stress induced increase in activity of ionically bound anionic peroxidase in present study may be involved in reduced growth by catalyzing ferulic acid mediated cross linking of cell wall polymers. This was confirmed by the purified peroxidase specificity for ferulic acid and ferulic acid polymer formation. A number of ionically bound anionic and cationic peroxidases induced under stress condition from various plants have been reported in earlier studies. Effect of NaCl on the overall protein profile using SDS-PAGE and 2-D PAGE was studied. There found to be change in protein expression profile under salt stress, the expression of a number of proteins induced while a few were repressed. Several new proteins were synthesized under salt stress those were not present in normal seedling. A 31.6 kDa protein band identified to be the most prominently induced while a 48 kDa protein most prominently repressed as revealed from SDS-PAGE. Polyclonal antibodies were generated against protein band of 31.6 and 48 kDa in rabbit and the antisera were used for expression studies. Expression profile of these two proteins under salt stress and various abiotic stresses, and ABA were studied. The 31.6 kDa salt induced protein expression increased with increasing salt concentration iii while the expression of 48 kDa protein repressed with salt increase as expected. Under different abiotic stresses like NaCl, KCl and PEG expression of 31.6 kDa protein was found to be most prominent and is supposed to be major osmotic stress induced protein. However, it was induced by ABA though the level of expression was relatively low. Therefore, it seems to be ABA responsive as well. Expression of 31.6 kDa protein was also studied in 10 cultivars of groundnut having different salt sensitivity. By comparing the expression pattern of 31.6 kDa protein in control and salt treated seedlings, it was found that the expression of this protein was more in salt treated seedlings of T-64, JAWAN, KAUSHAL, TG-1, TIRUPATI-1, KADRI-4, TIRUPATI-4 and KRG-1 cultivars of groundnut. Differences were also observed in the expression of 31.6 kDa protein among the control seedlings of different cultivars. The expression of this protein was high in control as well as in salt treated seedling of ICGS-37 cultivar which indicates its possible role in salt tolerance. Onthe other hand the expression of 48 kDa protein was seen to be repressed under various abiotic stresses like NaCl, KCl, mannitol and PEG but no remarkable change was observed in presence ofABA. The expression of48 kDa protein was also decreased significantly in salt treated seedling ofTMV-7, ICGS-37, JAWAN, KRG-1, KAUSHAL, TG-1, TIRUPATI-1, KADRI-4 and TIRUPATI-4. There found to be little variations in the various cultivars. The maximum reduction was found in ICGS-37. Tissue specific expression of 31.6 kDa and 48 kDa proteins showed that 31.6 kDa band is more intense in case of shoot treated with salt in comparison with the root, while the expression of48 kDa protein was equally repressed in salt treated roots and shoots. IV MALDI-TOF peptide mass fingerprinting studies of 31.6 kDa polypeptide showed its similarity with apple (Mains domestica) NADP-dependent sorbitol -6-phosphate dehydrogenase (S6PDH). Since this enzyme has a key role in the accumulation of sorbitol in the tissue which has a role of osmoprotection during salt stress. NADP-dependent sorbitol-6-phosphate dehydrogenase nature of 31.6 kDa protein was further confirmed by the sorbitol accumulation and NADP-dependent sorbitol-6-phosphate dehydrogenase activity. There was found to be a strong correlation between sorbitol accumulation and NADP-dependent sorbitol-6-phosphate dehydrogenase enzyme activity with increase in salt concentration. The NADPdependent sorbitol-6- phosphate dehydrogenase was partially purified and analyzed on SDS-PAGE and Native PAGE. The immuno-reactivity of antibody raised against 31.6 kDa protein band recognized approximately a 31.6 kDa band in SDS-PAGE and 63 kDa in Native-PAGE, indicating the dimeric nature of the enzyme. The immunoflourescence study using FITC labeled conjugates showed the difference in expression of 31.6 kDa protein in root and shoot of salt treated and control seedling. The protein is more abundant in shoot tissue of salt treated compared to control and it more abundant in shoot tissue than root. The protein was found to be localized in cell organelle particularly in plastidsy Therefore, the accumulation of sorbitol under salt stress may be one of the mechanisms of salt tolerance in groundnut seedling and NADP-dependent sorbitol-6- phosphate dehydrogenase plays important role in it. Role of sorbitol accumulation in salt tolerance has been reported from other plants like Japaneese persimmon (Diospyros kaki),Plantago maritime and tobacco.