An Integrative Small RNA-Degradome-Transcriptome Analysis Reveals Mechanisms of Heat-Induced Anther Indehiscence in Pepper.

Heat threatens male fertility in crops, yet the regulatory basis of anther dehiscence under high temperatures remains unclear. We compared a heat-sensitive pepper cultivar (DL) with a heat-tolerant landrace (B021) across two anther stages using integrated transcriptome, small-RNA, degradome, co-expression, and enzymatic assays. DL showed a collapse of anther dehiscence above 34-38 °C, whereas B021 retained normal dehiscence at 39 °C, and histology revealed tapetal enlargement, premature degeneration, and locule contraction only in DL. RNA-seq indicated genotype- and stage-dependent reprogramming, with DL suppressing phenylpropanoid/cell-wall, transport, and proteostasis pathways, while B021 maintained reproductive and stress-integration programs. Small-RNA profiling and degradome sequencing identified conserved miRNA families with in vivo target cleavage, and notably, miR397 targeting a laccase gene showed stronger evidence in B021, which is consistent with controlled lignification. Functional organization of differentially expressed miRNA targets highlighted modules in respiration/redox, hormone and terpenoid metabolism, vascular-cell-wall programs, and proteostasis/osmotic buffering. WGCNA modules correlated with heat-tolerance traits converged on the same processes. Enzyme assays corroborated multi-omics predictions, with SOD, CAT, and POD activities consistently induced in B021 and limited MDA accumulation. Together, the data supports a model in which tolerant anthers sustain dehiscence under heat by coordinating secondary-wall formation, auxin/jasmonate/gibberellin crosstalk, respiratory and reactive oxygen species buffering, and protein/membrane quality control, providing tractable targets for breeding heat-resilient peppers.