Mechanism Research On Drought Tolerance Of White Clover Induced By Spermidine Involved In Nitric Oxide Signal Transduction

Drought stress is one of the most serious abiotic stresses affect crops growth,development and yields.As a bioactive substance polyamine play an important role in plant stress resistance.In this study a physiological experiment was designed to investigate the function of exogenous spermidine mechanism to improve drought tolerance in white clover(Ladino),and the role of nitric oxide signaling in the process of spermidine-induced drought resistance.Combining with laser confocal microscopy and molecular biology method.The results are as follows:(1)The 20?mol/L spermidine seed-soaking of white clover can effectively improve the seed germination ability and promote the growth of the seed radicle elongation,and increase the content of fresh weight and dry weight,under 10%-15%PEG treatment.Effects of 20?mol/L Spd treatment on antioxidant enzymes activities were not consistent of white clover leaves.Exogenous spermidine treatment can effectively improve the relative water content,decrease the electrolyte leakage and MDA content of white clover leaves in different periods under drought stress.The degree of membrane lipid peroxidation decreased,so as to improve the stability of seedlings under drought stress and protect the structure of cell membrane,reduce the inhibition of growth of white clover seedlings under drought stress.(2)The results showed that treatment with 20?mol/LSpd caused a rapid increase in DAF-FM fluorescence relative to control(H2O).NO fluorescence was visible as early as 15 min after treatment,maximized at 45 min then decreased following the passage of time which indicated that Spd induce an increased generation of NO in mesophyll cells of White clover leaves.Pre-treatment with DCHA substantially blocked the NO accum?lation induced by PEG,meanwhile,this inhibiting effect was almost fully restored by the application of Spd in mesophyll cells of detached leaves.Both Spd and PEG treatment can increase the NR and NOS activities.PEG-induced NR and NOS activities were almost nullified by pre-treatment with DCHA,but the application of Spd co?ld visibly restore the diminished activities of NR and NOS blocked by DCHA.(3)Treatment with 20?mol/L Spd significantly enhanced the activities of SOD,POD,CAT and APX and their genes expression levels respectively.These results were partly prevented by application of Hb,NaVO3 or L-NAME at different levels.(4)Treatment with PEG led to a significant effect on induction of antioxidant enzyme activities including SOD,POD,CAT and APX and their genes transcript levels as compared with controls.Whereas Hb.NaVO3,L-NAME and DCHA effectively broke the increases of 4 antioxidant enzymes activities and genes expression levels induced by PEG.Moreover,the block-effects resulted from Hb,NaV03,L-NAME and DCHA were partly recovered by additional Spd.(5)When the concentration of SNP is 10?mol/L-75?mol/L,play the role of protection,and high concentration of SNP(more than 100?mol/L)is toxic for white clover,indicating that protective effect of sodium nitroprusside(SNP)of white clover has obvious dose effect.The drought stress induced relative water content decrease and EL and MDA content increase rapidly,cell membrane damaged intensified.Under droutght stress,exogenous 50?mol/L SNP significantly improved relative water content,and maintained significant lower electrolyte leakage and MDA content.Exogenous SNP have no effect on white clover under normal conditions.The results showed that exogenous SNP can effectively improve osmotic stress tolerance of white clover.In conclusion,the present study further verified that exogenously applied Spd and NO could serviceably improve drought tolerance of white clover and provided evidences that Spd could rapidly induce NO signal molec?lar releasing in mesophyll cells of white clover leaves as well as involved in NO signal transduction induced by drought stress in white clover.Furthermore,it was also observed that Spd-induced drought tolerance of white clover was associated with antioxidant defense through mediating NO signaling originated from NR and NOS.