Proteomics Analysis Of Developing Xylem In Response To Artificial Bending In Betula Platyphylla

Protein is the material basis of life,which is related with life and various forms of life activities closely.In recent years,the study in plant proteome has become one of the hot spot of the gene era.To investigate the molecular regulation mechanism of tension wood formation in birch and identify key proteins which regulate the secondary xylem and secondary cell wall formation,comparative proteomics of developing xylem in response to artificial bending and gravitational stimuli in Betula Platyphylla were analysized.The proteomics analysis were carried out using iTRAQ-MS/MS methods.Meanwhile,the protein extraction system for xylem of birch were established.In this study,the total proteins of birch xylem were isolated using the TCA-acetone,mthod,phenol method and extraction kit methods to select the optimum method.The extraction rate and extraction quality of the proteins using different methods was analyzed.The results indicated that the protein concentration and quality using phenol extraction method was largest,and protein bands were clearly and abundantly.The background of the gel was clean and the protein was clear in 2-DE electrophoresis.Therefore the modified phenol protocol is more suitable for the protein extraction of B.platyphylla xylem.The reaction wood system is an effective tool when studying the genes relate to the cell wall formation and the wood forming process.In order to investigate the molecular regulation mechanism of the tension wood formation responding to the artificial bending and gravity stimulation in birch,the trees were bended and tied to their neighbor trees in such a way that the midpoint of the stem was at an angle of approximately 45°.Four weeks later,the xylem samples were collected from tension wood(TW),opposite wood(OW)and normal wood(NW).Soluble protein of TW,OW and NW was extracted by phenol extraction and proteomics analysis was performed using iTRAQ method.The sections of developing xylem from TW,OW and NW were harvested.Staining results showed that TW exhibited higher cellulose contents and lower lignified than OW and NW.TW contained fewer vessels with smaller lumens than OW or NW.The express changes of proteins in B.platyphylla Suks xylem responding to artificial bending and gravity stimulation were analyzed comparatively.A total of 1104 significantly differentially expressed proteins were identified.Among them,there were 639,481 and 460 proteins that were respectively differentially expressed between OW vs TW,NW vs TW,and NW vs OW.281 and 358 proteins were respectively up-regulated and down-regulated in TW when compared with those in OW.291 and 190proteins respectively displayed increased and decreased abundance between NW vs TW,and 237 and 223 proteins were up-regulated and down regulated in OW compared those in NW.There are 173 proteins were differentially expressed among all of the three tissues.To further analyze the function of differentially expressed proteins,the GO classification was carried out.Among them,the biological processes was most,accounting for 44.70%in total.Followed by the cellular components,accounting for 41.48%,and molecular functions,accounting for 13.82%.Further annotation analysis of the differentially expressed proteins showed that they are classed into the lower order categories relate to xylem and cell wall development,such as plant-type cell wall organization,plant-type cell wall modification,lignin biosynthetic process,cellulose biosynthetic process and xylen biosynthetic process,and so on.Meanwhile,biological pathways of the differentially expressed proteins were examined.The results indicated that the proteins related to cellulose and cell wall synthesis and metabolism were up-regulated,and this suggested that the cellulose synthesis was improved in TW;the proteins in lignin synthesis and metabolism pathway were down-regulated,which suggested the lignifications were inhibited in TW.Meanwhile,proteins related to TCA cycle,fatty acid metabolism and amino acid synthesis pathway were altered expressed,indicating that these metabolic pathways are involved in the metabolic regulation during birch tension wood formation.This study identified the metabolic pathways and the related proteins involved in the tension wood formation in birch,elaborated the protein expression mechanism of birch xylem responding to bending and gravity stimulation,and laid the foundation for protein regulation research of wood formation in B.platyphylla Suks.