Functional Characterization Of Cellulose Synthase Complexes And Related Proteins In Cotton Fibers

Cellulose is the most abundant biomass on the earth, and also is the major compound of plant cell wall. It is composed of the beta-1,4-glucans, which are assembled into the microfibrils by hydrogen bonds. Cellulose biosynthesis is catalyzed by membrane-bound cellulose synthases (CESA), which are believed to form cellulose synthase complex (CSC) for microfibril depositon and assembly. However, much remains unknown about the CSC composition and the related biological functions. In this study, hence, the CSC activities were assayed using anti-GhCESAl immunoprecipitated proteins of mature cotton fibers and chromatography-mass spectrometry (LC-MS/MS). In addition, two UDP-glucose sterol glucosyltransferases (SGT) were characterized by detecting their enzyme activities under various conditions in vitro and observing gene expression patterns in vivo. Here are major points proposed in this study:1. Adding commercial cellulase into cotton fiber extraction process, we determined an enhanced cellulose biosynthesis in vitro.2. Western analysis of the anti-GhCESAl immunoprecipitated proteins demonstrated a reliable approach for CSC member identification.3. LC-MS/MS detection of the anti-GhCESAl immunoprecipitated proteins showed two types of GhCESA and a number of non-CESA proteins, suggesting that they should be members of the CSC in cotton fibers.4. Based on bioinformatic analysis, two GhSGTs contained PSPD and PSPG motifs that were characteristic of SGT, but GhSGT2was unique with transmembrane domain.5. Using recombinant GST-fusion GhSGT proteins, two GhSGT enzyme activities were determined in vitro for the SG production generated from UDP-glucose and beta-sitosterol or total crude sterols.6. GhSGTl and GhSGT2both showed much higher activities for SG production from total sterol substrate than from the purified P-sitosterol, but GhSGT2could even have higher activity than that of GhSGT1in vitro. Compared with the GhSGTl, the GhSGT2remained high activity under various temperatures. Meanwhile, Triton (X-100) addition strongly inhibited GhSGT1activity, but enhanced GhSGT2activity up to8-folds. Supplemented with beta-mercaptoethanol, the GhSGT2activity was slightly reduced, whereas GhSGT1was enhanced. In addition, MgCl2or NaCl could distinctively enhance two GhSGT activities.7. Real time analysis showed that two GhSGT genes were remarkably increased during cotton fibers development. Compared with GhSGT2, GhSGTl displayed relatively lower expression levels in fibers and leaf tissues, but was slightly higher in root and stem tissues. Furthermore, GhSGTl and GhSGT2exhibited distinct expression patterns upon heat shock. Cotton fibers are important raw material for textile industry. The yield and quality of cotton fibers will affect industrial development to a large extent. Gossypium hirsutum and Gossypium barbadense are key cultispecies. Upland cotton shows high yield but general quality. Island cotton shows high quanlity but low yield. Improvement on quality of upland cotton fiber is the key direction to get the breed with high quality and yield. The development of cotton fibers is related to cotton fiber quality. Based on this, we conducted a study to improve upland quality.(1) We cloned two xyloglucan endotransglycosylase gene and analyzed their expressions in the developing fibers and reconstructed them to the expression vector;(2) We cloned a KORRIGAN (AF511408) gene from cotton and the coding region was inserted the expression and repression vectors;(3) We attempted to transformed the recontrcted vectors into cotton. The results are as follows.1. A new xyloglucan endotransglycosylase gene was cloned and submitted to NCBI database. Genbank code is JN968478;2. Semiquantitative expression analysis showed that JN968478had higher expression than another XET gene (AY189971);3. We cloned the KORRIGAN gene, which was recombined to the expression and repression vectors;4. We attempted to transformed these recombinant vectors into cotton by pollen-tube pathway method. No transgenic cotton seedling was detected by PCR identification.