Wheat VPE Family: Identification, Evolution and Expression Associated with Grain Colour

Vacuolar processing enzymes (VPEs) are legumain-type cysteine proteases regulating plant protein processing, programmed cell death, seed maturation and stress responses. Nevertheless, the VPE family remains poorly characterized in hexaploid wheat (Triticum aestivum L.), and its possible roles in grain pigmentation are unclear. Here, we genome-wide identified 27 TaVPE genes and performed multi-omics analyses. Phylogenetic classification separated TaVPE proteins into four clades, all possessing conserved Peptidase_C13 and legumain_C domains, with similar motif patterns within each clade. These 27 TaVPE genes were unevenly located across 12 chromosomes of homoeologous groups 2, 3, 5 and 6. Collinearity analysis uncovered 26 intra-wheat collinear blocks and 47 orthologous gene pairs between wheat and rice, maize and foxtail millet, while no collinear counterparts were found in Arabidopsis. All duplicated pairs had Ka/Ks values less than 1, suggesting strong purifying selection. Numerous cis-acting elements related to hormones (ABA, MeJA, SA), stresses, and MYB/bHLH transcription factor binding sites were detected in TaVPE promoters. Expression data from public databases and transcriptomes of purple-grained Donghei-1 and white-grained Jinmai-66 (17, 31, 36 and 41 days after flowering) demonstrated predominant TaVPE expression at anthesis and grain-filling stages. Five TaVPE genes were retained as candidate genes associated with purple grain colour: TaVPE6, TaVPE15, TaVPE18, TaVPE25 and TaVPE26. All five showed significantly higher expression in the purple-grained cultivar than in the white-grained one at one or more mid-to-late grain-filling stages. This work establishes the first systematic profile of wheat VPE family and identifies candidate genes potentially associated with anthocyanin accumulation, supporting future functional research and molecular breeding for anthocyanin-enriched wheat.