Comparative Proteomics Study Of Natutally Colored Fiber And Characterization Of GhANR Gene

1. Object: Naturally colored cotton is cotton endowed with natural colors. Because the color exist in the fiber,dyeing is not required during the fabric manufacturing process. The use of naturally colored cotton couldeliminate dyeing costs and the disposal of toxic dye waste. As concerns for human health and the environmentincrease, naturally colored cotton has become an environmentally friendly option. However, the colors ofnaturally colored cotton primarily include green and several shades of brown. Cross-breeding can only generatenew shades of brown and green, which are insufficient for clothing versatility. Therefore, the color types havenot been altered by conventional breeding due to the lack of genetic resources for the generation of color.Genetic engineering is recognized as a way to generate new fiber color types and has just begun. For the geneticengineering approach, it is important to characterize the genes related to pigment biosynthesis. In this study, weused a proteomic approach to compare the protein profiles of brown color fiber (BCF) and white color fiber(WCF) to identify functional proteins involved in pigment synthesis. The comparison of the protein profiles ofBCF and WCF is of critical significance for the elucidation of the molecular mechanism of pigment biosynthesisand the development of new fiber color types via genetic manipulation.2. Results and Conclusions2.1A comparative proteomic analysis was performed to identify the differences between brown cotton fiber anda white near-isogenic line, and78differential spots were identified at three time points (12-,18-, and24-daypost-anthesis [DPA]) using MALDI-TOF/TOF. Our data illustrate several aspects of pigment synthesis and fiberdevelopment in brown color fiber (BCF). First,21spots were associated with secondary metabolism;15of thesewith high abundance in BCF were involved in flavonoid biosynthesis. DMACA staining and qPCR of7genesencoding enzymes involved in proanthocyanidins demonstrated that a multitude of proanthocyanidins weredeposited in BCF, which, when taken together with our comparative proteomic analysis and previous studies,suggested proanthocyanidins should be the pigment products in BCF and confirmed the crucial role offlavonoids in pigment synthesis in brown color fibers. This is the first time a proteomic approach has providedevidence to support the hypothesis that flavonoids are responsible for pigment synthesis in BCF. Second, severalspots with lower abundance in BCF were found, including glycolytic pathway, redox homeostasis, cytoskeleton,and protein metabolism related proteins. Our results implied that flavonoid synthesis was prevalent, and manymetabolic pathways that are active during fiber development may have been repressed in BCF, which may partlyaccount for the inferior fiber qualities of BCF. The identification of these protein spots with lower abundance inBCF, including proteins that are critical for fiber development, such as ascorbate peroxidase, superoxidedismutase, actin, annexin and heat shock protein, shows molecular evidence for explaining the inferior fiberqualities of color genotypes and provides insights for the fiber quality improvement of color types by geneticmanipulation.2.2A comparative proteomic analysis was performed to identify the differences between green cotton fiber and awhite near-isogenic line, and25differential spots were identified at three time points (12-,18-, and24DPA)using MALDI-TOF/TOF. These proteins were related to secondary metabolism, energy/carbohydrate metabolism,cell wall-related, protein metabolism, cytoskeleton, amino acid metabolism, cell response, lipid metabolism.Several spots with significantly changed abundance were identified as phospholipase D, phenylcoumaranbenzylic ether reductase and s-adenosylmethionine synthase, suggested that they may play very important role inpigment biosynthesis. In addition, the proteome of21DPA (Days Post Anthesis) green cotton fiber was analyzedusing two-dimensional gel electrophoresis to yield a protein map. A total of220individual spots were excisedand analyzed by MALDI-TOF/TOF MS. A total of156proteins were identified and cataloged according to theirfunctions. Twelve different classes of proteins were identified in21DPA green cotton fiber. Many of theseproteins were related to carbohydrate metabolism and energy production. Other notable functional classesincluded oxidoreductases, cell wall-related and cytoskeleton. This study provides insight into the majorphysiological events in21DPA green cotton fiber, and advances our understanding of molecular mechanismsrelated to pigment biosynthesis in green cotton fiber. Although pigment metabolism is an additional process innaturally colored cotton fiber that differs from white cotton fiber, we did not find specific proteins related topigment biosynthesis. In addition, flavonoid biosynthesis related protein spots showed no any changes in abundance, suggesting that the pigment in green cotton fiber was different from that in brown cotton fiber.2.3Anthocyanidin reductase (ANR) is a key enzyme which catalyzes the anthocyanidins into the correspondingepicatechin, an initiating monomer of proanthocyanidin synthesis. Using2D-PAGE comparisons, a protein spotwere found to be significantly up-regulated in brown color cotton fiber but hardly detectable in the white colorcotton fiber. The spots were excised from the gel, partially sequenced and identified to be ANR. GhANR cDNAcontained an open reading frame of1008bp encoding a protein of336amino acid residues. Real-time PCRanalysis showed that the expression level of GhANR was higher in BCF compared with WCF. When GhANR wasexpressed in Escherichia coli, the GhANR protein was showed possessing ANR activity and can catalyze theconversion of cyanidin to epicatechin.