Deciphering the role of small RNAs in the development of empurpled phenotypes in the Gossypium hirsutum mutant HS2

Abstract

Cotton fibers are among the most important natural fibers worldwide. Developing natural colored cotton (NCC) varieties is crucial for cotton fiber utilization due to their eco-friendly properties. Manipulation of anthocyanin synthesis is an effective strategy for creating novel NCCs. Our previous research revealed enhanced anthocyanin accumulation in an empurpled Gossypium hirsutum mutant, HS2, but the regulatory mechanism has not been fully elucidated. In this study, we employed an integrated analysis of RNA sequencing, small RNA (sRNA) sequencing, and degradome sequencing to assess the expression patterns and potential roles of sRNAs in the development of the empurpled phenotype in HS2. Our findings revealed that the expression profiles of sRNAs are highly similar between the wild type and the mutant, with only 11 miRNAs and 761 siRNAs showing significant expression variation. Eight miRNAs and one siRNA exhibited inverse regulation with their targets, which were not directly involved in regulating anthocyanin biosynthesis. Notably, degradome data analysis identified interactions between miR395 and ATP sulfurylase genes (APS1 and APS3). Further assessment determined down-regulation of miR395 and up-regulation of APS genes in HS2, as well as the enhanced biosynthesis of cysteine and glutathione. These results suggest that miRNA mediated post-transcriptional regulation might not be the primary mechanism driving anthocyanin enhancement in HS2. Instead, the miR395-APS modules are possibly involved in modulating cellular processes to cope with the increased anthocyanin levels. Overall, this study deepens our understanding of the molecular mechanisms underlying the empurpled phenotype in HS2 and facilitates its future use in NCC breeding.