Analysis Of Phosphorylation In Cellulose Synthase And Purification Of Its Potential Protein Kinases

Cellulose is an important component of plant cell walls and the most abundant biological resources on the earth.Previous studies demonstrate that phosphorylation of cellulose synthases(CESAs)plays an important role in regulating the synthesis and deposition of cellulose microfibrils,leading to the different structures of cell walls and diverse pattens in growth and development of plants.Thus,it is important to understand how CESA proteins are phosphorylated and how the phosphorylation events control plant growth and regulate plant response to environmental cues,which would help improve crops for enhanced productivity and resistance to environmental stresses.In this study,phosphoproteomics data were used to dig and analyze phosphorylated proteins and their phosphorylation sites in response to hormone treatment.With the help of functional clustering and protein interaction network analysis,the function of differentially phosphorylated proteins and their interactions were analyzed in depth.CESA sequences from 13 different species were analyzed to understand their genetic relationships and the conservatism and variation of their potential phosphorylation sites in evolution.Selected protein kinases thatare coexpressed with CESA were expressed and purified from E.coli to examine phosphorylation of CESAs in vitro.In addition,the mcherry-labeled tubulin TUA5 transgenic plants were constructed to study the relationship between CESA and its interacting CC protein as well as microtubules and the functional roles of phosphorylation sites of CESAs to further understand the mechasnisms underlying the regulation of cell expansion by CESA phosphorylation in response to growth and environmental conditions.Functional enrichment analysis revealed that the proteins differentially phosphorylated in response to hormone treatment were mainly involved in protein localization and transport,response to hormonal stimulation and temperature stresses.They mainly affect the cell membrane composition,cytoplasmic components and the composition of the nucleus as well.The most relevant GO term is GO: 0009737,which is worth of further study.Protein interaction network analysis identified those differentially phosphorylated proteins that functionally interact with CESAs.In addition,phosphoproteomics analysis identified some new CESA phosphorylation sites,which may specifically respond to hormonal stimulation.CESA phosphorylation is an ancient event.Newly evolved phosphorylation sites of CESA proteins along with their conserved sites may provide diverse regulatory pathways for the regulation of cellulose biosynthesis in higher plant.For the kinased that are potentially involved in phosphorylating CESA proteins,a SUMO tag was successfully applied to express and purify these kinases in order to further examine their ability to phosphorylate CESA proteins and their phosphorylation sites.