Genome-Wide Identification and Pollen-Specific Promoter Analysis of the DIR Gene Family in Rosa chinensis

Dirigent proteins (DIRs) are pivotal regulators of lignin/lignan biosynthesis and play multifaceted roles in plant development and stress adaptation. Despite their functional significance, DIR genes remain unexplored in Rosa chinensis, a globally important woody ornamental species. This study identified 33 RcDIRs through whole-genome analysis, including their chromosomal distribution, phylogenetic relationships, collinearity, protein and gene structure, conserved motifs, and cis-acting element distribution, and classified them into three phylogenetically independent subgroups (DIR-a, DIR-b/d, and DIR-e). Notably, the DIR-e subgroup includes an exclusive tandem cluster comprising RcDIR7-RcDIR12, representing the largest lineage-specific RcDIR expansion in R. chinensis. Structural characterization revealed that most RcDIRs exhibit a conserved single-exon architecture. Promoter cis-element analysis uncovered abundant stress-/hormone-responsive elements and three pollen-specific motifs (AAATGA, POLLEN1LELAT52, GTGANTG10), with RcDIR12 from the DIR-e cluster showing high pollen-specific regulatory potential. Experimental validation included cloning the RcDIR12 promoter from R. chinensis ‘Old Blush’, constructing proRcDIR12::GUS vectors, and conducting histochemical GUS assays with pollen viability/DAPI staining in transgenic Arabidopsis. Histochemical assays demonstrated GUS activity localization in mature trinucleate pollen grains, marking the first experimental evidence of pollen-specific DIRs in rose. Our findings not only elucidate the DIR family’s genomic organization and evolutionary innovations in R. chinensis but also establish proRcDIR12 as a molecular tool for manipulating pollen development in plants.