The Effect Of Spd On The SOS Pathway Of Cucumber Roots Under Salt Stress Based On The Results Of Phosphorylated ITRAQ

As the third largest saline-alkali land in the world,soil salinization has severely restricted the construction and development of agriculture and rural areas in China.Therefore,overcoming soil salt damage and improving plant salt tolerance have become key issues in solving current agricultural development.Cucumber root tissue is fragile and its growth is often restricted by salt stress in protected culture.The view that polyamines can improve plant salt tolerance has been generally accepted.In particular,studying the regulation mechanism of polyamines on salt-stressed plants from the level of protein phosphorylation can more directly find proteins and genes related to stress,and improve the biological mechanism of polyamines to alleviate salt-stress damage.The SOS signal pathway is a relatively clear signal pathway that plants respond to salt stress.However,it is not clear whether there is a correlation between polyamines and SOS signaling pathways.In this experiment,‘Jinyou No.4’ cucumber was selected as the material.Based on the results of phosphorylated proteome identification,we used transcriptomics technology to study the effects of exogenous Spd on the expression of related proteins and genes in the SOS pathway of cucumber seedlings under Na Cl stress.And combined with signal molecule inhibitor processing to construct the signal transduction pathway of polyamine-regulated SOS pathway,which provides new insights for polyamines to relieve salt stress of cucumber seedlings.The main research results are as follows:1.Compared with the control,a total of 133 differentially phosphorylated proteins were identified under salt stress,among which the up-regulated proteins were mainly kinase proteins,ion transport proteins,glycolysis and amino acid metabolism related proteins.They all play an important role in plant salt tolerance.Compared with salt stress,the increased application of polyamines under salt stress can enhance the salt tolerance of cucumber seedlings by up-regulating STK,ribosomal protein,DNA replication or protein repair,sugar transporter and cellulose synthesis related proteins.2.Compared with the control,STK gene,MAPK gene,receptor kinase gene,PPL gene,calciumdependent protein gene and K⁺ transporter gene were significantly up-regulated on the third day of salt stress treatment.By the fifth day of salt stress treatment,the gene up-regulation was weakened,but increased application of polyamines could significantly promote the gene up-regulation.Compared with the control,salt stress up-regulated the expression of STK,PPL and MAPK,and increased application of polyamines could further promote the up-regulation of STK and MAPK.This shows that polyamines can alleviate the salt stress of cucumber seedlings by promoting the transcription and translation of STK and MAPK.3.Analysis of the transcriptomics sequencing results showed that a total of 50 differentially expressed genes related to the SOS pathway were screened.Compared with the control,salt stress up-regulated 11 SOS pathway-related genes,and increased application of polyamines further up-regulated gene expression.These genes are mainly involved in catalysis,transport,cell membrane composition,growth and development,stimulus response,amino acid metabolism,carbohydrate metabolism and vitamin metabolism.Enrichment analysis showed that the differential genes mainly include calcium ion binding and serine/threonine active metabolic pathways.4.Based on the SOS protein sequences of Arabidopsis thaliana,there are five CsSOS1,fifteen CsSOS2 and seven CsSOS3 gene family members were identified in the whole-genome database of cucumber.They are unevenly distributed on 7 chromosomes.The subcellular location shows that CsSOS1 and CsSOS2 are mainly located in the plasma membrane,and CsSOS3 is located in the cytoplasm.Phylogenetic evolution shows that CsSOS1-2,CsSOS2-2,CsSOS2-6 and CsSOS3-5 are closely related to the Arabidopsis SOS protein.The prediction of cis-elements showed that the promoter regions of CsSOS1,CsSOS2 and CsSOS3 mainly contained defense stress and drought-induced response elements.Structural analysis showed that the protein sequences of CsSOS1,CsSOS2 and CsSOS3 contained Na-H Exchanger,STKc and EFh domains,respectively,and were highly conserved.q RT-PCR showed that compared with the control,the salt stress treatment significantly increased the expression of CsSOS gene family in different tissues on the first day.By the fifth day of treatment,compared with salt stress,increased application of Spd can significantly promote the expression of CsSOS1-1^CsSOS1-5,CsSOS2-4,CsSOS2-5,CsSOS2-8,CsSOS2-9,CsSOS2-11,CsSOS2-12 and CsSOS3-1^CsSOS3-7 in roots,root tips and leaves.5.Adding Spd for 6h-24 h under salt stress significantly increased the H₂O₂,NO and Ca²⁺ content in cucumber root tissue.When the H₂O₂ content of root tissue was inhibited under the condition of salt and polyamine,the degradation of polyamine was inhibited.At this time,the NO content decreased significantly from 12h-24 h,and the Ca²⁺ content of the downstream signal molecule of NO also decreased significantly.Under the condition of salt and polyamine supplemented with exogenous H₂O₂ for 6h,Ca²⁺increased compared with the condition of salt and polyamine plus H₂O₂ inhibitor.It shows that H₂O₂ can promote the increase of Ca²⁺ content in root tissue.When the root Ca²⁺ content was inhibited under the condition of salt and polyamine,the changes of H₂O₂ and NO content were unstable,but the expression of CsSOS gene family decreased significantly.Supplementing exogenous Ca²⁺ under the condition of salt and polyamine can significantly up-regulate CsSOS1-4,CsSOS2-5,CsSOS2-8 at 24 h,and CsSOS3-7 at 6h-12 h.This shows that under salt stress conditions,polyamines promote the Ca²⁺ content of cucumber root tissue through H₂O₂ and NO signaling molecules,thereby up-regulating the expression of SOS signaling pathway genes and maintaining intracellular ion balance.