The Molecular Mechanism Of SiPTI1-5 Regulated Salt Stress Response In Foxtail Millet Revealed Phosphorylation Proteomics

Foxtail millet（Setaria italica）is one of the important food crops in China,which has high stress resistance.Foxtail millet possesses attractive qualities,such as small diploid genome,lower repetitive DNA,short life cycle,and C4 photosynthesis.These characteristics promote it become a model species for exploring C4 crop growth,development and stress response.In-depth analysis of the molecular mechanism of tolerance in foxtail millet is great significance for germplasm improvement of foxtail millet by molecular design breeding.We analyzed the changes of proteins abundance of salt-tolerant cultivar"Yugu 2"（YG2）and salt-sensitive cultivar"AN04"（AN04）under salt treatment by phosphorylation proteomics.The results showed that the phosphorylated proteins of YG2 and AN04 in response to salt stress were mainly involved in signal transduction,transcription,translation,ion transport and metabolism.Compared with AN04,YG2 had unique salt signal sensing and signal transduction characteristics.Among 35 differentially phosphorylated proteins specifically present in YG2,Pto-interacting 1-5（PTI1-5）,mitogen-activated protein kinase（MPK6）,and protein phosphatase 2C（PP2C）were significantly increased.These suggested that PTI1-5 may regulate signaling and metabolic pathways in response to salt stress.PTI1 protein kinase belongs to the receptor-like cytoplasmic kinase（RLCK）,which is involved in oxidative stress and hypersensitivity response during stress process.However,the signaling pathway of salt response regulated by PTI1 is not clear.We used bioinformatics methods to analyze the gene structure,chromosomal localization and cis-acting elements of 12PTI1s in foxtail millet,and preliminarily analyzed the organs expression and stresses response characteristics of PTI1s.These results suggested that Si PTI1-5 may play a key role under salt response.Furthermore,Si PTI1-5 was heterologous overexpressed in yeast and E.coli,and Si PTI1-5 enhanced their tolerance to salt stress.In order to further analyze the biological function of Si PTI1-5 in foxtail millet under salt stress,we used genetic transformation and CRISPR-Cas9 gene editing technology to obtain Si PTI1-5 and sipti1-5 mutant foxtail millet materials,respectively,and analyzed the salt response characteristics of different genetic materials at germination stage.The results showed that the germination rate of Si PTI1-5 overexpressed foxtail millet under 150 m M Na Cl was higher than that of wild-type and sipti1-5 mutant.The root length,leaf length and leaf width of the three materials were inhibited by salt stress,but the overexpression Si PTI1-5 lines were the least inhibited,and the sipti1-5 mutant was the most inhibited.Si PTI1-5 overexpressed lines accumulated more osmotic protective substances such as proline,with less damage to membrane structural integrity and stronger antioxidant enzyme activity.These indicated that Si PTI1-5 overexpression was beneficial to improve salt tolerance of foxtail millet.To further explore the members of Si PTI1-5 regulating salt response signaling pathway in foxtail millet,we analyzed the changes of phosphorylated proteins in wild-type,Si PTI1-5overexpression and sipti1-5 mutant materials during salt response using phosphorylated proteomics.These results showed that the proteins with changed phosphorylation levels were mainly involved in signal transduction,membrane and transport,transcription,and various metabolic processes.Among them,phosphorylation levels of several proteins involved in processes such as signal transduction and carbon metabolism are induced in Si PTI1-5overexpression foxtail.Such as MPK6,salt overly sensitive 1（SOS1）,sucrose non-fermenting-related kinase（Sn RK）,PP2C,potassium transporter（HAK1）,phosphoenolpyruvate（PEP）and UDP-glucose 6-dehydrogenase（UGDH）,etc.indicating that Si PTI1-5 plays an important role in the salt response of foxtail millet.These suggested that Si PTI1-5 may regulate a series of downstream metabolic processes and improve salt tolerance of foxtail millet by regulating MPK kinase cascade signaling pathway.Furthermore,Si MPK6was selected as the target protein of Si PTI1-5 for in-depth analysis.The experiment of luciferase complementation assay and yeast two-hybrid showed that Si PTI1-5 interacted with Si MPK6.In vitro phosphorylation analysis showed that Si PTI1-5could phosphorylate Si MPK6.The subcellular localization analysis showed that Si MPK6 was localized in the nucleus.At the same time,we found that the expression pattern of Si MPK6under salt stress was similar to Si PTI1-5,with the highest expression at 12 h after Na Cl treatment,and the expression level of Si MPK6 in Si PTI1-5 overexpressed foxtail millet was significantly higher than that of wild-type and mutant.In addition,a series of salt response related genes in Si PTI1-5 overexpressing plants were significantly induced by salt stress,for example,the expressions of SOS1,SOS2,SOS3,late embryogenesis abundant proteins 14（LEA14）and delta-1-pyrroline-5-carboxylate synthase（P5CS）were higher than wild-type and mutant,and the differences were more significant under salt stress conditions.These results suggested that Si PTI1-5 may regulate plant salt tolerance through Ca²⁺dependent signaling pathway and MPK signaling pathway.In conclusion,we analyzed the salt response mechanism of different salt-tolerant varieties of foxtail millet by phosphorylation proteomics,bioinformatics,molecular genetics,biochemistry and other research strategies,analyzed the characteristics of PTI1s gene family in foxtail millet,and revealed that Si PTI1-5 may regulate various signals and metabolic processes by regulating MPK signaling pathway to improve salt tolerance of foxtail millet.These results provided important information for further understanding the salt response function of PTI1s gene in foxtail millet,and laid a theoretical foundation for further carrying out molecular design breeding.