DNA methylation alterations of upland cotton (Gossypium hirsutum) in response to cold stress

DNA methylation plays an important role in regulating gene expression in plants. In the experiment, we studied effects of cold on DNA methylation variation in upland cotton. Using the methylation-sensitive amplified polymorphism procedure, we chose 66 pairs of selective amplification primers to assess the status and levels of cytosine methylation. The hemimethylation of the external cytosine and the full methylation of the internal cytosine were scored. As a result, cold triggered the demethylation of hemimethylated or internally full methylated cytosine. With the prolongation of cold treatment, the demethylation loci increased and the methylation loci decreased. Nevertheless, this change could be reverted when cotton was subsequently recovered under normal temperature. In addition, 29 polymorphic bands that appeared in the electrophoretogram were sequenced. By homologous alignment analysis, most of these 29 fragments were identified as genes or DNA clones involved in abiotic stress response. The variation in methylation loci existed at both coding and non-coding regions. Furthermore, the expression of the abiotic stress-related genes, GhCLSD (Seq21), GhARK (Seq22), GhARM (Seq15, Seq18, Seq19 and Seq21) and GhTPS (Seq8), were tested. The results revealed that cold treatment induced down-regulation of GhCLSD, GhARK and GhARM, but up-regulated the expression of GhTPS. These changes were in accordance with the alteration of DNA methylation. Thus, cold may affect the gene expression via changing the methylation status in the cytosine nucleotide.     

The accumulation of pigment in fiber related to proanthocyanidins synthesis for brown cotton

Brown cotton is a kind of naturally colored cotton. Because of less processing and little dying, it is more friendlier to environment than white cotton. For brown cotton, pigment accumulation in fiber is one of the most important characteristics. In this study, we selected a brown fiber line and a white fiber cultivar to determine the factor that affects the pigmentation in brown fiber. Accordingly, fibers were collected to verify the presence of PAs by p-dimethylaminocinnamaldehyde (DMACA) and toluidine blue O (TBO) staining. The PAs content and related genes expressions were determined. As a result, 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 at 5 DPA, and then gradually decreased to zero. But for brown fiber, the PAs content was increased from 5 to 15 DPA stage, and reached the maximum at the 15 DPA stage, then gradually decreased from 15 to 40 DPA stage. On the contrary, in white cotton, PAs were synthesized in the whole developmental stage from 5 to 40 DPA. And PAs content in brown fiber were far more than that in white fiber, which may be the reason why the brown pigment accumulated in brown fiber.     

Methylation analysis revealed salicylic acid affects pearl millet defense through external cytosine DNA demethylation

The cytosine DNA methylation and demethylation have a role in regulating plant responses to the environment by affecting the promoter regions of most plant defense-related genes through the CpG islands or the CCGG motifs. Salicylic acid, a defense and signaling plant hormone, is seen playing crucial role in the variation of the methylome. In this study, the effects of salicylic acid and feeding of the millet headminer (Heliocheilus albipunctella de Joannis) on pearl millet DNA methylome changes were evaluated through MSAP epigenotyping during panicle development. The results showed that millet headminer feeding increased the level of genomic methylation while application of salicylic acid caused DNA demethylation occurring mostly at external cytosine and accompanied by a decrease of the number of larvae per panicle. This suggests that hemimethylation (external cytosine methylation) has key role in regulating defense responses and conferring tolerance to pearl millet through salicylic acid application.     

Characterization of restriction-modification enzymes Cfr13 I from Citrobacter freundii RFL13

This communicatiopn describes some properties of RCfr13 I and MCfr13 I, isolated from Citrobacter freundii RFL13. RCFfr13 I restriction enzyme recognizes the 5'-G GNCC sequence and cleaves, as indicated by the arrow. MCfr13 I methylase modifies the internal cytosine producing m5C (5'-GGNm5CC). RCfr13 I is sensitive not only to this type of substrate modification but also to hemimethylation in overlapping sites by MCfr10 I (internal cytosine of RCfr13 I recognition is methylated) and MHpa II (external cytosine is methylated). From these results the sensitivity of RCfr13 I to methylation by dcm methylase of E.coli in overlapping sites is deduced.     

Regulation of cotton fiber elongation by xyloglucan endotransglycosylase/hydrolase genes

Ligon lintless mutant (li1li1) with super-short fibers (5-8 mm in length) and its wild type (Li1Li1) with normal fibers (30 mm in length) were used to study the function of xyloglucan endotransglycosylase/hydrolase (XTH) genes during fiber elongation in cotton. Wild-type cotton attained the fiber elongation stage earlier (5 days post-anthesis, DPA), than the Ligon lintless mutant (12 DPA) with a higher fiber elongation velocity of about 1.76 mm/day. Xyloglucan contents in Ligon lintless mutant fibers were 5-fold higher than the wild type during 9-15 DPA. It was also observed that the activity of XTH in wild-type cotton fibers was about 2-fold higher than that of the Ligon lintless mutant with a peak at 12 DPA. DNA blot analysis indicated that the XTH gene in the Ligon lintless mutant and its wild type belonged to a multiple allelic series. However, RNA blot analysis and quantitative real-time PCR exhibited an earlier expression (10 DPA) of XTH in wild type as compared to delayed (15 DPA) expression in the Ligon lintless mutant. The study also revealed that 9-15 DPA might be a key phase for upregulation of fiber elongation via increasing XTH activity. Higher XTH activity can cleave down the xyloglucan-cellulose chains thus loosening fiber cell wall and promoting fiber cell elongation in wild type as compared to its mutant.     

Exogenous Jasmonic Acid Inhibits Cotton Fiber Elongation

Jasmonic acid (JA) is a well-characterized phytohormone that acts in various ways to influence plant development. Its role in cotton fiber development, however, has not yet been thoroughly explored. In this study, JA was proven to be an inhibitor of ovule and fiber development in vitro. Continuous exogenous JA application inhibited fiber elongation. This effect was dependent on development stage and dosage. Fibers and ovules at three different stages of development and different JA dosages were compared. The most serious suppression was detected when ovules 1?day before anthesis (–1?DPA) were cultured in medium with 2.5?μM JA. Genes related to trichome and fiber development responded differently to JA treatment between –1?DPA and 1?day post anthesis (1 DPA). JAs (JA and JA-Ile) quantification showed that JAs content was sharply decreased from –1?DPA to 5?DPA ovules, which indicated that JA was negatively associated with fiber elongation in vivo. In addition, gene expression analysis showed the same trend. Our results demonstrate that there was a negative relationship of JA with fiber elongation in vitro and in vivo. These results are meaningful for uncovering the mechanism of fiber elongation in cotton.     

Infraspecific DNA methylation polymorphism in cotton (Gossypium hirsutum L.)

Cytosine methylation is important in the epigenetic regulation of gene expression and development in plants and has been implicated in silencing duplicate genes after polyploid formation in several plant groups. Relatively little information exists, however, on levels and patterns of methylation polymorphism (MP) at homologous loci within species. Here we explored the levels and patterns of methylation-polymorphism diversity at CCGG sites within allotetraploid cotton, Gossypium hirsutum, using a methylation-sensitive amplified fragment length polymorphism screen and a selected set of 20 G. hirsutum accessions for which we have information on genetic polymorphism levels and relationships. Methylation and MP exist at high levels within G. hirsutum: of 150 HpaII/MspI sites surveyed, 48 were methylated at the inner cytosine (32%) and 32 of these were polymorphic (67%). Both these values are higher than comparable measures of genetic diversity using restriction fragment length polymorphisms. The high percentage of methylation-polymorphic sites and potential relationship to gene expression underscore the potential significance of MP within and among populations. We speculate that biased correlation of methylation-polymorphic sites and genes in cotton may be a consequence of polyploidy and the attendant doubling of all genes.