Biosynthesis And Light-regulation Mechnisms For Precursor Of Brown Pigment For Brown Cotton

Brown cotton is a kind of naturally colored cotton, which accumulates brown pigment in fiber. Because of less processing and dying procedures, it could induce less harmful chemical effects and is friendlier to environment than white cotton. However, in contrast with white cotton, brown cotton has many defects, such as mixed color, inhomogeneous distribution and unstable inheritance of pigments, which could limit its use and popularization.In previous studies, several flavonoid structural genes including encoding dihydroflavonol reductase (DFR), anthocyanidin synthase (ANS), flavanone3-hydroxylase (F3H), and anthocyanidin reductase (ANR) were cloned from brown fiber. The analysis of chemical properties of extracted compounds primarily demonstrated that the pigment in brown fiber might be proanthocyanidins (PAs, also called condensed tannins). However, the reports on synthesis and accumulation rules of PAs are less and the relationship between PAs and brown pigment in fiber are not yet clear by now.For brown cotton, during the middle and late development stages of fiber, the brown pigment was accumulated in fiber. In the accumulated procedure, light might be related to the sun light irradiation. However, it needs to further research whether the light has effects on pigment synthesis or not.In the experiment, on the one hand, the relationship between brown pigment and PAs was studied. On the another hand, the variation of DNA methylation and gene expression were analyzed by using MSAP and cDNA-AFLP methods during the development stages from5to25DPA. On the third hand, the seedlings of brown cotton were treated with different color films, and the effects of light quality on PAs synthesis were discussed. The results of the experiment were as follows:1. A brown fiber line and a white fiber cultivar were selected to determine the factors that affect the pigmentation in brown fiber. Accordingly, the fibers in different developmental stages were collected to verify the presence of PAs by p-dimethylami-nocinnamaldehyde (DMACA) and toluidine blue O (TBO) staining. The PAs content and related gene expressions were determined. As a result, PAs synthesis started at the stage of papillary epidermic cells of seeds and there were obvious differences on the aspect of PAs synthesis in fiber between white cotton and brown cotton. For white fiber, the PAs content reached maximum at5DPA stage, and then gradually decreased to zero. But for brown fiber, the PAs content was increased from5to15DPA stage, and reached the maximum at the15DPA stage, then gradually decreased from15to40DPA stage. In brown cotton, PAs synthesis continued for the whole developmental stage from5to40DPA. PAs content in brown fiber was far more than that in white fiber, which may be the reason why the brown pigment accumulated in brown fiber.2. Brown fibers were collected to analyze the variation of DNA methylation and gene expression by using MSAP and cDNA-AFLP methods during the development stages from5to25DPA. The results showed that by MSAP analysis, a total of1010.5bands per samples and11-27bands per primer combination were amplified by using66pairs of primer combinations. During the development process of fiber, the sum of methylated sites, the ratio of methylated sites and percentage of fully methylated sites were gradually increased with the advancement of fiber development stages. However, by cDNA-AFLP analysis, a total of32-51bands with average of39.8bands per primer combination were amplified by using64pairs of primer combinations. There are75polymorphic transcription derived fragments (TDFs) were detected, and among that,30TDFs were sequenced and analyzed. By blasting at NCBI website,19TDFs were detected to be homologous with genes from cotton. According to blasting results, among30TDFs,13TDFs were homologous with known functional genes; however,17TDFs were homologous with unknown function genes.3. The seedlings of brown cotton were treated with different color films, and the effects of light quality on PAs synthesis, phenotype traits and photosynthetic system were studied. As a result, red film treatment increased the PAs content, however, yellow and blue films treatment inhibited the PAs synthesis. After treatment with different color films, plant growth were accelerated by red and yellow films, root growth was promoted by blue film. The Physiological Parameters such as light use efficiency, net photosynthetic rate and accumulation of photosynthetic products were decreased after the treatment with red, blue and yellow films. The plants treated with white films maintained better light use efficiency and higher net photosynthetic rate.4. The effects of light quality on variation of DNA methylation were analyzed using the seedlings of brown cotton treated with different color films. The results showed that a total of1300.5bands per samples and15-32bands with an average of19.7bands per primer combination were amplified by using66pairs of primer combinations. The ratios of total methylated sites and fully-methylated sites were increased after treatment with red, yellow and blue films, moreover, blue film treatment decreased the percentage of hemi-methylated sites. In addition,10polymorphic fragments were sequenced and cloned. By blasting at NCBI, the sequences alignment revealed that both coding and non-coding regions could be methylated or demethylated by different light quality. The expression of seq1(homologous with pyruvate kinase) and seq2(homologous with MIP family) were regulated by light quality, which were closely related to the procedure of the demethylation of methylated sites for the genes during the activated expression.