Artificial microRNAs against hypoxia-responsive small protein encoding genes reveal HRSP41 and 42 as regulators of...

Small proteins remain poorly characterized in Arabidopsis thaliana, partly because of genetic redundancy and the difficulty of identifying loss-of-function mutants. To address this gap, we investigated 58 hypoxia-responsive small protein genes (HRSPs) encoding proteins of 80-100 amino acids using a homology-based artificial microRNA (amiRNA) strategy.

Homozygous amiRNA lines were screened for developmental traits and stress-related phenotypes. Two closely related paralogs, HRSP41 and HRSP42, emerged as key regulators with both shared and distinct functions. HRSP41 promoted flowering under short-day conditions, whereas HRSP42 delayed flowering and specifically increased seed tolerance to oxidative stress. Both genes acted together to reduce the inhibitory effect of abscisic acid on seed germination. Transcriptome profiling revealed distinct downstream effects: HRSP42 silencing was associated with repression of glutathione metabolism, whereas HRSP41 silencing affected glycerolipid metabolism and cell wall-related pathways. Under hypoxia, simultaneous downregulation of HRSP41 and HRSP42 altered transcriptional, post-transcriptional, and translational responses and intensified the repression of photosynthesis-related genes.

Despite their high sequence similarity, HRSP41 and HRSP42 control plant development and stress adaptation through partially overlapping but clearly distinct molecular mechanisms. Our results identify these paralogs as previously uncharacterized regulators of flowering, oxidative stress responses, ABA sensitivity, and hypoxia-responsive gene expression.