Identification And Functional Analysis Of MiRNAs Involved In Cotton Fiber Initiation

Cotton is one of the most important cash crops, since it provides lots of fibers, andeven proteins and oils. Cotton fiber development undergoes four distinctive andoverlapping stages: initiation, elongation, secondary thickening, and maturation. Amongthem, initiation is regarded as the determining stage for lint yield because it has aprofound impact on the number of fibers. MicroRNA (miRNA), an important regulatoryfactor, plays crucial roles in plant organ development and stress response. However, nosystematic study on miRNA’s role during fiber initiation was reported yet. Here, weperformed high throughput sequencing, employed a comparative small RNAomeanalysis between Xuzhou142upland cotton and its fiberless (fl) mutant, screened forpotential functional miRNAs in cotton fiber initiation, and attempted to uncover theirfunctions during this process.In total,5896335and3747020sequences which could be used for analysis wereobtained from the wild-type and fl mutant, respectively. From the sequencing data,41conserved miRNA families and36novel miRNAs were identified. Among them, theaccumulation of31miRNAs was statistically different between the wild-type and the flmutant. Comparative miRNAome analysis combined with Northern blotting andRACE-PCR revealed seven fiber initiation-related miRNAs expressed in cotton ovulesand experimentally validated targets of these miRNAs are involved in different cellularresponses and metabolic processes, including transcriptional regulation, auxin andgibberellin signal transduction, actin bundles, and lignin biosynthesis. This studydescribes a complex regulatory network consisting of these miRNAs expressed incotton ovules to coordinate fiber initiation responses.To further explore the role of GhmiR156during early fiber development, weanalyzed its accumulation in5dpa,10dpa,15dpa,20dpa, and25dpa fibers by deepsequencing. GhmiR156expression gradually increased from5dpa to20dpa and slightlydecreased from20dpa to25dpa, whereas the expression level of the target gene,GhSPL9, was negatively correlated to GhmiR156’s accumulation. Further, GhDFR,GhF3H, and GhANS, which are required in anthocyanin synthesis,showed very similarexpression patterns with GhSPL9. The gel mobility shift assays indicated that GhSPL9could bind to the GhDFR and GhF3H promoters in vitro. In the transient expression assay on tobacco leaves, GhSPL9could increase the expression of the reporter genedriven by GhDFR or GhF3H promoter by over three-fold. Together, these data suggestthat GhSPL9may function as a transcriptional activator of GhDFR and GhF3H infibers.In developing fibers, anthocyanin levels were gradually reduced from5dpa to20dpa and slightly increased from20dpa to25dpa. Therefore, it is suggested thatGhmiR156, via silencingGhSPL9expression, may negatively regulate the expression ofGhDFR, GhF3H, and GhANS, which may led to a strong declineinanthocyaninsynthesis at20dpa. It was believed that the H2O2burst at20dpa may serve as atermination signal of fiber rapid elongation. However, it is unknown how the redox stateinside fiber cells is regulated. As an important reducing substance, anthocyaninmayregulatethe redox state in cells. Hence, its downregulation at20dpa byGhmiR156-targeted GhSPL9may partially contribute to the H2O2burst, which couldinducetermination of fiber elongation. When inhibitors of anthocyanin synthesis wereadded into the medium of in vitro ovule culture, cotton fiber development wasobviously disturbed, suggesting that inhibition of anthocyanin synthesis by GhmiR156mayalso greatly block early fiber development.