Integrated in silico and experimental analysis of a stress-responsive DOXC21-A natural antisense transcript suggests small RNA-associated regulation in Arabidopsis thaliana

Abstract Background Natural antisense transcripts (NATs) represent an important regulatory layer that can modulate gene expression in plants. However, their functional relevance at specific stress-responsive loci and evolutionary emergence remain poorly characterized. The DOXC21 clade of 2-oxoglutarate–dependent dioxygenases (2OGDs) is associated with abiotic stress adaptation, and the NAT overlapping DOXC21-A ( AT3G19000 ), annotated as AT3G19002 , represents a candidate locus-specific regulatory element. Here, we combined in silico analyses and quantitative gene expression assays to investigate the evolutionary origin and potential regulatory role of this NAT at the DOXC21 locus in Arabidopsis thaliana . Results Phylogenetic reconstruction of 590 plant DOXC homologs revealed that DOXC21-A and its neighboring homologous gene, DOXC21-B, form a Brassicales-specific clade of stress-responsive dioxygenases distinct from canonical hormone biosynthesis enzymes. In silico analysis of AT3G19002 identified a conserved 29-nucleotide motif with partial similarity to plant microRNAs, thermodynamically stable stem-loop secondary structures, and predicted candidate targets associated with stress response, metabolism, and transcriptional regulation. Reanalysis of publicly available small RNA sequencing datasets confirmed persistent, low-abundance small RNA reads mapping to the AT3G19002 locus, supporting the presence of small RNA-associated signals. Quantitative PCR analysis in wild-type plants, two independent NAT-disrupted mutants ( nat1 , nat2 ), and a doxc21-a knockout line showed that osmotic stress significantly upregulated DOXC21-A expression in both nat mutants, whereas transcript levels remained stable in wild-type plants. NAT expression was highest in the doxc21-a knockout under control conditions and markedly decreased under osmotic stress, while remaining low in the nat mutants under all conditions. These reciprocal expression patterns support a regulatory relationship between DOXC21-A and its antisense transcript. Sequence alignment and motif analysis also identified conserved features potentially associated with small RNA biogenesis and predicted candidate targets, including genes involved in signaling, secondary metabolism, and transcriptional regulation. Conclusions Our combined computational and experimental analyses suggest that AT3G19002 contributes to the regulation of DOXC21-A under stress, potentially through transcriptional interference or small RNA-mediated mechanisms. Together with the reciprocal expression of the sense and antisense transcripts, these results identify the DOXC21-A locus as a candidate stress-responsive regulatory module and provide a foundation for future functional studies on NAT-mediated regulation in plant stress responses.