Study On Response Mechanism And Regulation Mechanism Of Rice With Different Salt Tolerance To Salt Stress

With the increasing salinization of soil,how to make effective use of saline-alkali land has become an important problem restricting agricultural production.Rice is a moderately sensitive crop to salt stress,and its yield decreases greatly under salt stress.Therefore,elucidating the response and physiological characteristics of rice with different salt tolerance to salt stress is of great significance to explore the physiological mechanism of rice resistance to salt stress.In this study,two experiments were designed:in experiment 1,salt-tolerant rice variety Lianjian 5(J5)and salt-sensitive rice variety Lianjing 7(L7)were selected as test varieties,and two salt concentration treatments(0 g kg-1 and 2 g kg-1)were set up to explore the effects of salt stress on rice yield components,agronomic and physiological characters.RNA-seq technique was used to study the pathway differences of different rice varieties in response to salt stress.To explore the differences of physiological mechanisms of salt tolerance by mining the genes related to salt tolerance.In experiment 2,the above two rice varieties were selected and four exogenous GAB A concentrations(0,50,100,150 μM)were set under salt stress to explore the effects of exogenous GABA on rice growth and development under salt stress.The main results are as follows:Compared with salt-sensitive varieties,the yield of salt-tolerant varieties decreased less after salt stress.The higher yield of salt-tolerant rice varieties benefited from its higher total spikelet number and seed setting rate.At the same time,the higher crop growth rate of salttolerant rice varieties in the two stages from tillering to jointing and heading to maturity is the basis of higher yield under low salt stress.At panicle differentiation stage,heading stage and maturity stage,salt-tolerant varieties increased aboveground nitrogen accumulation and promoted the synthesis of GA3,GABA,GSH,proline and betaine to maintain higher physiological activity of leaves,and the increase of above-mentioned substances in salttolerant varieties was higher than that in salt-sensitive varieties.Lower Na+content and higher K+ content in leaves of salt-tolerant varieties were helpful to maintain cell ion homeostasis.Compared with salt-sensitive varieties,better agronomic characters and higher leaf physiological activity of salt-tolerant varieties are the basis of strong salt tolerance.In order to further explore the differences of salt tolerance mechanism of different salt tolerant rice varieties,leaf samples were taken at panicle differentiation stage,flowering stage and maturity stage,and RNA-seq technique was used to study the salt tolerance mechanism of different salt tolerant rice varieties.By comparing the differences of different growth stages of the same variety among different treatments,the results are as follows:a total of 33296 genes were compared in 12 samples.First of all,the 12 samples were classified into panicle differentiation stage,flowering stage and maturity stage to study the differentially expressed genes of different rice varieties under salt stress at different stages.No salt treatment was used as control,and a total of 6 comparative groups were analyzed.they are:panicle differentiation stage J5 comparison group,panicle differentiation stage L7 comparison group,flowering stage J5 comparison group,maturity stage J5 comparison group,maturity stage L7 comparison group.The differential genes were significantly up-regulated or down-regulated in all varieties under salt treatment.At panicle differentiation stage,there were 767 significant differential genes in J5 and 235significant differential genes in L7,522 significant differential genes in L7,1697 significant differential genes in J5,122significant differential genes in J5 and 1061 significant differential genes in L7 at the maturity stage.After the preliminary results were obtained,the differential genes in these comparison groups were analyzed by KEGG and GO enrichment analysis to study the salt tolerance-related pathway and ion transport mechanism.Ten pathways related to salt tolerance were selected from the results of KEGG enrichment analysis,among which 6 pathways were related to signal transduction regulating rice growth.They are 1,diterpenoid biosynthesis,2,carotenoid pathway,3,brassinoteroid biosynthesis,4,plant hormone signal transduction,5,MAPK signal transduction pathway-plant.6,alanine,aspartate and glutamate metaboli.There are 2 pathways related to osmotic substances regulating rice growth,7,arginine and proline metabolic,8,β-Alanine metabolism,and 2 pathways related to antioxidant system regulating plant growth.9.glycine,serine and threonine metabolic and 10,glutathione metabolic.During panicle differentiation,there were 20 differential genes in J5 and 16 differential genes in L7,among which 10 differential genes were differentially expressed in two tested rice varieties.At flowering stage,there were 14 differential genes in J5 and 27 differential genes in L7,of which 2 genes were differentially expressed in the two tested rice varieties.At the maturity stage,there were 5 differential genes in J5 and 12 differential genes in L7,of which 3 genes were differentially expressed in the two tested rice varieties.The genes related to Na+ and K+ transport in each comparison group were selected from GO enrichment analysis.The transport mechanisms of the two tested rice were different.At the panicle differentiation stage,four genes were enriched in J5 and L7 respectively,which were related to Na+ and K+transport.At flowering stage,6 genes in J5 were related to Na+and K+transport,and 7 genes in L7.In the maturity stage,J5 did not enrich the genes related to Na+ and K+transport,but L7 enriched 9 genes.40 genes were selected from the above genes and the results of RNA-seq were verified by real-time fluorescence quantitative PCR.There were differences in the expression of 9 genes between RNA-seq and real-time fluorescence quantitative PCR.The results of analysis on the mechanism of plant growth regulation by signal transduction in two tested rice varieties showed that:1.There were differences in GA synthesis related enzymes GA2ox and GA20ox.During panicle differentiation and flowering,the expression of GA2ox in J5 was up-regulated,while the expression of GA2ox in L7 was not expressed at panicle differentiation,but down-regulated at anthesis.There was no difference in GA20ox in J5,but the GA20ox of L7 was up-regulated in both periods.Combined with physiological data,the results showed that the up-regulation of GA2ox and GA20ox could increase the content of GA3 and improve the salt tolerance.2.ABA synthesis related genes and signal transduction genes were up-regulated in different degrees in two rice varieties during panicle differentiation and flowering stage.The ABF in J5 was up-regulated in these two periods,but there was no significant difference in L7.This makes J5 more efficient in regulating leaf stomatal opening and closing,regulating transpiration rate,and improving salt tolerance of rice plants.3.There are differences in the mechanisms of BRs synthesis and signal transduction between the two tested rice varieties.The up-regulation of DWF4 and DET2 related to BRs synthesis in J5 at panicle differentiation and flowering stage promoted BRs synthesis,while the down-regulation of decomposition-related protein BAS1 decreased the degradation of BRs.Higher BRs synthesis activity was beneficial to improve salt tolerance.4.There were differences in GABA synthesis pathway between the two rice varieties at flowering stage.The up-regulation of GAD in J5 promoted the synthesis of GABA,and the increase of GABA content promoted the stomatal closure of J5 leaves,regulated transpiration rate and improved salt tolerance.The results showed that the upregulation of Pip,PAO and ALDH in J5 at panicle differentiation and flowering stage promoted the synthesis of proline and betaine,increased the content of proline and betaine,and improved salt tolerance.The results of the study on the mechanism of antioxidant system of two tested rice varieties showed that the down-regulation of DHAR1 in L7 inhibited the synthesis of GSH at flowering stage,but there was no significant difference in the expression of related genes in J5,which indicated that the decrease of glutathione content in salt-sensitive rice varieties made their cell detoxification ability significantly lower than that of salt-tolerant varieties and were more vulnerable to saline-alkali stress.According to the results of GO enrichment analysis,OsHKT and OsHAK genes in different salt tolerant rice varieties were differentially expressed under salt stress at different stages and treatments.The reason for the difference in salt tolerance between J5 and L7 is the differential expression of genes involved in ion transport across the plasma membrane and tonoplast.At panicle differentiation stage and flowering stage,the up-regulation of KOB1 in J5 promoted the transmembrane transport of Na+ and K+.There was no differential expression of this gene in L7.The down-regulation of KCO1,OsNHX1 and Osrok in L7 inhibited the transmembrane transport of Na+ and K+.These four genes may be one of the reasons for the salt sensitivity of L7.According to the above results,GABA plays an important role in improving salt tolerance of rice.Although exogenous GABA has been widely studied in cucumber,melon,maize and other plants,the research in rice is rarely reported.Therefore,the experiment of spraying exogenous GABA on rice leaves was designed in this experiment.the results showed that there were differences in the effects of exogenous GABA on different salt-tolerant rice varieties.Exogenous GABA reduced the tillering of salt-tolerant varieties,resulting in the decrease of panicles per pot,and finally led to the decrease of yield,while significantly increased the number of panicles per pot,total spikelets and seed setting rate of salt-sensitive varieties,resulting in a significant increase in yield.GABA at 100 and 150 μM decreased endogenous GABA content,SOD and POD enzyme activity,SPAD value and chlorophyll content at panicle differentiation stage.The decrease of Na+ ratio at panicle differentiation stage affected cell ion homeostasis,resulting in a decrease in physiological activity.However,100 and 150μM GABA increased the content of endogenous GABA,the activity of SOD and POD,the content of SPAD and chlorophyll,and the ratio of SPAD to chlorophyll,which was beneficial to maintain cell ion homeostasis,made it show higher physiological activity,and improved the growth and development of salt-sensitive varieties.To sum up,the upregulation of the above genes at panicle differentiation stage and flowering stage of salt-tolerant varieties promoted the synthesis of related substances,and could maintain better ion homeostasis,so that salt-tolerant varieties could maintain their higher physiological activity.it is the basis for salt-tolerant varieties to have better agronomic characteristics.Higher physiological activity and better agronomic characters are helpful for salt-tolerant varieties to obtain higher yield.Exogenous GABA spraying can increase the yield of salt-sensitive varieties,which is of practical significance.The results can be used as a reference for the study of physiological mechanism of salt tolerance in rice,the mining of salt tolerance genes and the regulation of exogenous chemical control substances in rice.