Heat-responsive microRNAs participate in regulating the pollen fertility stability of CMS-D2 restorer line under high-temperature stress.

Anther development and pollen fertility of cytoplasmic male sterility (CMS) conditioned by Gossypium harknessii cytoplasm (CMS-D2) restorer lines are susceptible to continuous high-temperature (HT) stress in summer, which seriously hinders the large-scale application of "three-line" hybrids in production. Here, integrated small RNA, transcriptome, degradome, and hormone profiling was performed to explore the roles of microRNAs (miRNAs) in regulating fertility stability in mature pollens of isonuclear alloplasmic near-isogenic restorer lines NH and SH under HT stress at two environments. A total of 211 known and 248 novel miRNAs were identified, of which 159 were differentially expressed miRNAs (DEMs). Additionally, 45 DEMs in 39 miRNA clusters (PmCs) were also identified, and most highly expressed miRNAs were significantly induced in SH under extreme HT, especially four MIR482 and six MIR6300 family miRNAs. PmC28 was located in the fine-mapped interval of the Rf1 gene and contained two DEMs, gra-miR482_L-2R + 2 and gma-miR2118a-3p_R + 1_1ss18TG. Transcriptome sequencing identified 6281 differentially expressed genes, of which heat shock protein (HSP)-related genes, such as HSP70, HSP22, HSP18.5-C, HSP18.2 and HSP17.3-B, presented significantly reduced expression levels in SH under HT stress. Through integrating multi-omics data, we constructed a comprehensive molecular network of miRNA-mRNA-gene-KEGG containing 35 pairs of miRNA/target genes involved in regulating the pollen development in response to HT, among which the mtr-miR167a_R + 1, tcc-miR167c and ghr-miR390a, tcc-miR396c_L-1 and ghr-MIR169b-p3_1ss6AG regulated the pollen fertility by influencing ARF8 responsible for the auxin signal transduction, ascorbate and aldarate metabolism, and the sugar and lipid metabolism and transport pathways, respectively. Further combination with hormone analysis revealed that HT-induced jasmonic acid signaling could activate the expression of downstream auxin synthesis-related genes and cause excessive auxin accumulation, followed by a cascade of auxin signal transduction, ultimately resulting in pollen abortion. The results provide a new understanding of how heat-responsive miRNAs regulate the stability of fertility restoration for CMS-D2 cotton under heat stress.

Integrated Methylome and Transcriptome Analysis between the CMS-D2 Line ZBA and Its Maintainer Line ZB in Upland Cotton.

DNA methylation is an important epigenetic modification involved in multiple biological processes. Altered methylation patterns have been reported to be associated with male sterility in some plants, but their role in cotton cytoplasmic male sterility (CMS) remains unclear. Here, integrated methylome and transcriptome analyses were conducted between the CMS-D2 line ZBA and its near-isogenic maintainer line ZB in upland cotton. More methylated cytosine sites (mCs) and higher methylation levels (MLs) were found among the three sequence contexts in ZB compared to ZBA. A total of 4568 differentially methylated regions (DMRs) and 2096 differentially methylated genes (DMGs) were identified. Among the differentially expressed genes (DEGs) associated with DMRs (DMEGs), 396 genes were upregulated and 281 genes were downregulated. A bioinformatics analysis of these DMEGs showed that hyper-DEGs were significantly enriched in the "oxidative phosphorylation" pathway. Further qRT-PCR validation indicated that these hypermethylated genes (encoding the subunits of mitochondrial electron transport chain (ETC) complexes I and V) were all significantly upregulated in ZB. Our biochemical data revealed a higher extent of H2O2 production but a lower level of adenosine triphosphate (ATP) synthesis in CMS-D2 line ZBA. On the basis of the above results, we propose that disrupted DNA methylation in ZBA may disrupt the homeostasis of reactive oxygen species (ROS) production and ATP synthesis in mitochondria, triggering a burst of ROS that is transferred to the nucleus to initiate programmed cell death (PCD) prematurely, ultimately leading to microspore abortion. This study illustrates the important role of DNA methylation in cotton CMS.