Genome-wide identification and expression analysis of Aspartic proteases in Hordeum vulagare L. reveals candidates involved in salt and drought tolerance.

Aspartic proteases (APs) are abundant plant hydrolases implicated in storage-protein mobilization, membrane remodeling, and stress signaling. Here, we systematically identified AP genes in barley (Hordeum vulgare L.) by a genome-wide search and verified A1/AP domains, then resolved family relationships using maximum-likelihood phylogeny. We analyzed gene structure and conserved motifs, predicted subcellular localization, and scanned 1.5-kb promoters for cis-elements to infer regulatory programs. Functional context was assessed by GO enrichment and protein-protein interaction (PPI) inference, and transcriptional behavior was profiled by RNA-seq meta-analysis across eight conditions, with qRT-PCR time-courses (0, 12, 24, 48 h) validating stress responses in contrasting genotypes under salt and drought. The survey uncovered 55 HvAP genes that cluster into four robust clades, with Group I and II retaining multi-exon, phytepsin-like architectures and Group III and IV showing exon compaction and frequent loss of the C-terminal plant-specific insert. Localization predictions indicate a family largely routed to secretory/vacuolar compartments, with a small subset showing plastid/peroxisome signals. Promoters display clade-specific codes: Group I and II are enriched in light/GA motifs, whereas Group III and IV accumulate dense ABA/JA/MYB (drought) arrays. GO terms center on aspartic-type peptidase activity, and PPI modeling reveals a cohesive secretory hub with smaller satellites. Expression partitions into drought-induced, cold/salt-repressed, and photoperiod-responsive modules; qRT-PCR shows modest but consistent, earlier, and more sustained induction of several HvAPs in the tolerant line under stress. These results prioritize promising HvAP candidates for subcellular validation, promoter-reporter assays, and loss/gain-of-function tests, informing future breeding and engineering for stress resilience in barley.