Study On Physiological Mechanisms For Exogenous Silicon-Mediated Salt Tolerance In Cucumber

Natural saline soils often contain high concentrations of Na⁺.Irrigation with sewage and seawater also causes the accumulation of Na⁺and other salt ions in the arable soil.In recent years,protected cultivation is developing rapidly.However,secondary soil salinization is increasingly serious in protected facilities,due to excessive application of chemical fertilizers,especially N fertilizer,unreasonable crop rotation,and rising groundwater.In salinized protected areas,the major salt ions are NO₃^-and Ca²⁺.Secondary soil salinization directly affects crop growth and development,and decreases vegetable yield and quality.In addition to breeding salt-tolerant crop varieties,the use of exogenous substances may be an economic and easy means to improve crop yield and quality.Silicon?Si?is beneficial for plant growth and development.Many studies have shown that Si can alleviate biotic and abiotic stress-induced plant damage.Although the alleviative effect of Si on plant damage induced by Na Cl salt has been widely reported,the underlying mechanism is still not very clear.Moreover,information on Si's effect on plant growth under excess nitrate stress in protected soils is very limited.In this study,using cucumber as the main experimental plant materials,in combination with the use of tomato and rice,the effects of Si on seed germination,Na⁺distribution,hormone levels and leaf senescence under salt stress were investigated.In addition,Si's effect on nitrogen assimilation and chlorophyll synthesis was also investigated in cucumber under excess NO₃^-stress.The main results are as follows:?1?Addition of 0.3 mM Si could increase the seed germination percentage,germination index and seedling vigor index in cucumber?‘Jinyou 1'?under 200 mM Na Cl.Twelve hours after germination initiation,the expression of CYP707A1,which encodes abscisic acid?ABA?8?-hydroxylase,was increased by added Si under salt stress;while the expressions of GA20ox,GA3ox and GA2ox,which encode genes involved in gibberellin?GA?metabolism,were not changed in seeds.Thirty-six hours after germination initiation,added Si markedly inhibited the expressions of ABA biosynthesis genes?NCED1 and NCED2?and gibberellin catabolism gene GA2ox.The?-amylase activity was higher in silicon-applied seeds than the control under salt stress.Compared with salt stress alone,added Si improved the growth and plasma membrane integrity of bud seedlings,while decreasing reactive oxygen species accumulation and lipid peroxidation.Added Si decreased the activities of superoxide dismutase,catalase,peroxidase and ascorbate peroxidase,and the concentrations of protein and proline in radicles of bud seedlings under salt stress,implying a stress alleviation.These results suggest that the improvement of cucumber seed germination under salt stress might be associated with the decrease of ABA level,maintenance of high gibberellin level and the increase of?-amylase activity.The alleviation of oxidative damage by added Si contributed to the improvement of bud seedling growth under salt stress.?2?Addition of 0.3 mM Si could increase the biomass of cucumber seedlings and reduce oxidative damage under 65 mM Na Cl stress.Si addition had no significant effect on Na⁺levels in roots and leaves under salt stress.Si addition did not change the expressions of plasma membrane Na⁺/H⁺antiporter gene SOS1,high-affinity K⁺channel gene HKT1and H⁺-ATPase gene HA3 under salt stress,while promoted the expression of vacuolar Na⁺/H⁺exchanger gene NHX1.Analysis on the Na⁺concentrations in different root parts of stressed plants showed that Si significantly decreased the Na⁺concentration in the root tip?0-0.8 cm?,which was consistent with the subcellular distribution of Na⁺using Na fluorescent dye;Si did not affect the Na⁺concentration in 0.5-5 cm sections from the root tip,but increased the concentration in sections of over 5 cm from the root tip.In addition,subcellular localization of Na within the leaf mesophyll cells showed that Si enhanced Na⁺loading into the vacuole under salt stress.Added silicon increased the levels of GA,auxin IAA and cytokinin?CTK?in roots and leaves under salt stress.These results suggest that Si could improve the growth and alleviate oxidative damage of cucumber seedlings under salt stress.Meanwhile,Si could reduce the toxic effect of Na⁺on other organelles by enhancing sequestration of Na⁺into vacuoles in the leaves and mature area of roots.In addition,Si-induced increase in GA,IAA and CTK levels may be involved in the regulation of salt tolerance in cucumber.?3?Tomato,cucumber and rice are Si excluder,intermediate and high Si-accumulating plants,respectively.Added Si could significantly slow down the degradation rate of chlorophyll induced by salt treatment in these plants.Exogenous Si decreased the expression of senescence-related genes and the content of MDA in leaf segments of tomato and rice under salt treatment.Si-mediated responses of hormone levels are different in leaf segments of tomato and rice under salt stress:Si addition decreased the IAA level in rice whereas the change in tomato was associated with added Si concentrations;the levels of GA was further decreased by Si in rice,while the change in tomato was irregular;Si addition did not change the ABA and JA levels in rice leaf segments,whereas in tomato,it increased the ABA levels significantly and the change of JA levels was irregular.Si addition increased the CTK content?i PA and ZR?of leaf segments in tomato and rice under salt stress.CTK synthesis inhibitor inhibited the delay effect of Si on salt-induced leaf senescence.Si could alleviate the salt-induced senescence in leaf segments of wild type Arabidopsis thaliana but not in the ipt1,3,5,7 quadrupole Arabidopsis mutant.These results suggest that Si could significantly delay plant senescence induced by salt,possibly via increased cytokinin levels.?4?Root application,but not foliar application of Si,could improve cucumber growth under nitrate stress[200 mM,provided by equimolar Ca?NO₃?₂ and KNO₃)],and the optimal concentration of Si is 1.0 mM.Root addition of Si increased photosynthetic rate and decreased oxidative damage of stressed plants.Under nitrate stress,Si did not reduce K and Ca absorption in cucumber leaves.Expression analysis of the nitrate transporter NRTs gene family showed that Si had no effect on the expression of NRT1.5A,a highly expressed gene,under excess NO₃^-stress.Si addition promoted the activities of nitrate reductase,nitrite reductase,glutamine synthase,glutamine-2-oxoglutarate aminotransferase and glutamate dehydrogenase,and decreased the accumulation of nitrate,nitrite and ammonium in leaves under NO₃^-stress.The concentrations of glutamic acid,5-aminolevulinic acid,porphobilinogen and uroporphyrinogen?were increased under nitrate stress,while these were decreased by added Si.Added Si increased the levels of chlorophyll and its precursors?protoporphyrin?,Mg-protoporphyrin?and protochlorophyllide?,and expressions of genes encoding enzymes involved in chlorophyll synthesis?CHLH,POR and CAO?under nitrate stress.These results suggest that Si could improve the photosynthesis and alleviate stress damage of cucumber under nitrate stress by enhancing nitrogen assimilation and chlorophyll synthesis.In conclusion,Si could increase salt tolerance of cucumber seedlings by a variety of physiological processes such as alleviating oxidative damage,enhancing Na⁺partitioning into vacuoles,and regulating the levels of various hormones.CTKs play an important role in the delay of leaf senescence induced by salt stress,and it may be involved in Si-mediated salt tolerance response.Si could improve the growth of cucumber under excess NO₃^-stress by enhancing N assimilation and chlorophyll synthesis.