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

## TL;DR

This study identifies molecular mechanisms that allow some pepper plants to maintain fertility in heat, offering targets for breeding heat-resistant varieties.

## Contribution

The study integrates multi-omics data to reveal conserved miRNA–target interactions and pathways that enable heat tolerance in pepper anthers.

## Key findings

- Heat-tolerant pepper line B021 maintains anther dehiscence at 39°C through coordinated stress and hormone signaling.
- miR397 targeting a laccase gene is linked to controlled lignification in heat-tolerant pepper anthers.
- B021 shows higher antioxidant enzyme activity and lower membrane damage under heat stress.

## Abstract

High temperatures during flowering can sharply reduce pepper yield because anthers (the organs that release pollen) may not open. We compared a heat-sensitive cultivar (DL) and a heat-tolerant line (B021) under elevated temperatures. DL showed abnormal anther wall development and early tissue breakdown, producing closed anthers and poor pollen release, while B021 largely maintained normal structure and opening. To uncover the controls behind these differences, we integrated messenger-RNA profiling with microRNA (small regulatory RNA) analysis and a cleavage-based assay that confirms microRNA targets, together with gene network analysis and antioxidant measurements. B021 sustained programs that strengthen and remodel the anther wall, coordinated stress and hormone signals, and limited harmful oxidants through higher antioxidant enzyme activities (SOD, CAT, and POD) and lower membrane damage. Several conserved microRNA–target pairs were linked to these protective responses. These findings provide practical molecular markers and candidate targets to breed pepper varieties that remain fertile during heat waves.

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.