Analysis Of Regulatory Network Of Reversible Phosphorylated Proteins In Puccinellia Tenuiflora In Response To Na₂CO₃

Land salinization is a major environmental factor affecting crop yield.Osmotic stress and ionic toxicity caused by salt stress are important factors affecting plant growth.Puccinellia tenuiflora,widely distributed in northern China,has well adapted to stress environments and is a suitable model plant for the study of plant salinity responsive mechanism.Meanwhile,the high-quality gene mapping and the genetic transformation system of Puccinellia tenuiflora have laid a solid foundation for further research on plant salinity response mechanism.In this study,50-day-old Puccinellia tenuiflora seedlings were treated with 100m M Na₂CO₃ for 5 min,15 min,30 min,and 1 h,respectively.The changes of protein phosphorylation levels in Puccinellia tenuiflora leaves under different salt treatment times were analyzed using a quantitative proteomics strategy.The holoprotein was extracted by phenol extraction at each salt treatment time,and 11357 phosphorylated peptides of 4044 phosphorylated modified proteins were identified by Ni-NTA column combined with high resolution mass spectrometry.A total of 2124 peptides from 1393phosphorylated proteins showed significant changes in phosphorylation levels at different salt treatments.These proteins weree mainly involved in gene expression and transcription,signal transduction,photosynthesis,stomatal movement,membrane and transport,protein phosphorylation and dephosphorylation,stress and defense,cell cycle,and ROS scavenging.The pattern of changes in the phosphorylation levels of these proteins at different salt treatment times preliminarily elucidated the molecular regulatory mechanims in Puccinellia tenuiflora at the early stage in response to Na₂CO₃stress:（1）reduce chlorophyll content and regulate chloroplast relocalization for reducing excess light energy uptake;（2）regulate salt response gene expression by altering transcription factor phosphorylation levels;（3）promote Na⁺compartmentalization to maintain intracytoplasmic K⁺homeostasis;（4）increase antioxidant enzyme activity to mitigate oxidative stress injury;（5）alter Ca²⁺channel protein phosphorylation levels to transmit salt signals;and（6）regulate chloroplast vesicle membrane structure to avoid damage to photosynthetic machinery.In this study,a phosphorylated differential peptide screening program was written in R language and a user-interface was built using Shiny to improve the efficiency and accuracy of analyzing phosphorylated proteomics data.Our results provide useful information for understanding plant salt responsive mechanism using proteomics approaches.