TaSnRk2.10 From Wheat Phosphorylates TaERD15 And TaENO1 And Confers Drought Tolerance In Rice

Wheat(Triticum aestivum L.)is the most cultivated crop worldwide,and its importance is self-evident.Drought severely restricts wheat production,and wheat is particularly sensitive to drought stress at jointing stage.Drought induces the accumulation of abscisic acid(ABA)in plants,which in turn causes plants to respond to drought stress.ABA signaling pathway plays a key role in stress response.Sucrose Non-fermenting 1-Related protein Kinase 2(Sn RK2),as an important regulatory component in the classical ABA response pathway,is the hub of signal transduction in various biological processes and stress response.However,the mechanism of TaSnRK2.10 in response to drought stress has not been fully elucidated.In this study,the TaSnRK2.10 gene was discovered and cloned,and the functions of TaSnRK2.10 in growth,development and drought resistance were further studied through transgenic rice,and the yeast library was screened.The candidate interacting proteins Ta ERD15 and Ta ENO1 were found and verified by yeast two-hybrid,firefly luciferase reporter,bimolecular fluorescence complementation,co-immunoprecipitation and pull-down assays.The phenotypes of Ta ERD15 overexpressed transgenic rice lines were studied under drought and the main conclusions were as follows:First,transcriptomic analysis of the young panicles at the jointing stage of “Jinmai 47”(JM47)showed that the key regulatory gene TaSnRK2.10 in the ABA signaling pathway was substantially up-regulated.The expression pattern of TaSnRK2.10 in different drought-sensitive wheat types after PEG treatment also demonstrated the role of TaSnRK2.10 in drought tolerance response.Secondly,by comparing the amino acid sequence similarity,it was found that TaSnRK2.10 had high homology(94.5% to 100%)with the corresponding members of Sn RK2 family(especially in grasses),but low homology(79.2% to 85.6%)with Sn RK2 s of dicotyledonous plants.Multiple sequence alignment of amino acids revealed that TaSnRK2.10 protein was highly conserved,including the N-terminal serine/threonine kinase domain and the C-terminal Domain I(response to osmotic stress)and Domain II(response to ABA).Among the derived amino acid sequences,there was only a single amino acid variation(S250A),which was not in the conserved functional domains and caused no differences in the predicted secondary structures.Phylogenetic tree analysis showed that three homoeologs of TaSnRK2.10 were closely related to their ancestral species,and clustered with monocot proteins(especially grasses),including rice(Oryza sativa L.),maize(Zea mays L.),barley(Hordeum vulgare L.)and sorghum(Sorghum bicolor L.).Spatiotemporal tissue-specific expression analysis showed that TaSnRK2.10 were expressed constitutively in all wheat tissues at jointing stage,and the expression level was higher in shoots than that in roots.The expression patterns of TaSnRK2.10 under different abiotic stresses and exogenous hormone treatments showed that TaSnRK2.10 was rapidly induced by ABA,and also induced by PEG,high salt,low temperature,high temperature and Me JA.Thirdly,to study the biological function of TaSnRK2.10 overexpression(OE)transgenic rice lines were constructed.The germination results showed that seed germination of TaSnRK2.10 OE lines with higher overexpression level were significantly sensitive to ABA,while the TaSnRK2.10 OE line with lower overexpression level was not significantly different from wild type(WT).Statistical analysis of growth and yield traits of TaSnRK2.10 OE lines and WT at seedling,booting and maturity stages showed that the plant height,tiller number,maximum root length,root dry weight,and the yield of TaSnRK2.10 OE lines with higher expression were significantly decreased,whereas there were no significant differences between the line OE-144 with relatively lower overexpression level and WT.Fourthly,TaSnRK2.10 improved drought tolerance of transgenic rice through multi-level regulation.At the physiological level,TaSnRK2.10 enhanced drought tolerance of transgenic rice by regulating stomatal opening and closing,reducing leaf water loss rate,increasing leaf water content,improving cell membrane stability,reducing malondialdehyde(MDA)content,and increasing survival rate.Transcriptome analysis of WT and TaSnRK2.10 OE lines before and after drought at seedling stage showed that TaSnRK2.10 enhanced drought resistance by regulating the expression of related genes including ABA signaling pathway(PP2C and ABI5 family members)and JA signaling pathway(TIFY11b).Metabolomic analysis showed that TaSnRK2.10 substantially increased the levels of non-enzymatic antioxidants,including ascorbic acid,6-O-galloylglucose(tannins)and flavonoids,by reducing various basal metabolism,thereby coordinating plant metabolic balance and enhancing drought resistance under drought stress.Fifthly,to further study the signaling pathway of TaSnRK2.10 involved in drought response,two interacting proteins Ta ENO1 and Ta ERD15 were identified by screening the yeast library.And yeast two-hybrid,firefly luciferase reporter,bimolecular fluorescence complementation,co-immunoprecipitation and pull-down assays were used to further verify the results.Phos-tag assays demonstrated that Ta ERD15 and Ta ENO1 were phosphorylated by TaSnRK2.10.Transgenic rice lines overexpressing Ta ERD15 showed a drought-sensitive phenotype with significantly higher MDA content and significantly lower survival rate than WT under drought stress.TaSnRK2.10 could phosphorylate Ser 91 of Ta ERD15,and the phosphorylated Ta ERD15 was degraded through the 26 S proteasome pathway,thereby eliminating its adverse effects of Ta ERD15 on drought resistance.Ta ENO1 was induced by ABA and PEG,and participated in plant drought resistance by regulating the metabolite phosphoenolpyruvate metabolism.In conclusion,the TaSnRK2.10 was identified as a drought responsive protein kinase based on the panicle transcriptome data of wheat under drought stress at jointing stage.And TaSnRK2.10 was found to respond to drought stress at physiological,biochemical,transcriptional and metabolic levels.Meanwhile,two TaSnRK2.10 interacting proteins Ta ERD15 and Ta ENO1 were identified,and the Ta ERD15 OE transgenic rice lines were drought sensitive.TaSnRK2.10 eliminates its adverse effects in the process of drought resistance by phosphorylating Ta ERD15.TaSnRK2.10 OE lines with higher expression level improved drought resistance of transgenic rice,while TaSnRK2.10 OE lines with lower expression showed no significant differences in growth index and drought resistance compared with WT.Therefore,precise regulation of TaSnRK2.10 expression level is of great significance in crop breeding,and droughtinducible promoter may be a better choice.