Evolutionary and functional characterization of BBX genes in the barley pangenome.

backgroundB-box (BBX) transcription factors are central regulators of plant development, light signaling, and stress responses. Despite their well-established roles, the pangenomic organization, evolutionary dynamics, and regulatory functions of BBX genes in barley (Hordeum vulgare) remain largely unexplored, particularly in the context of abiotic stress. In this study, we performed a comprehensive characterization of the BBX gene family across genetically diverse barley genomes and investigated their potential involvement in salt stress responses.

resultsA pangenome wide analysis of 20 barley accessions identified 360 HvBBXs, which were clustered into 20 orthologous gene groups and classified into core, soft core, shell, and lineage specific categories. Most HvBBXs were highly conserved, showing stable copy numbers, conserved exon intron structures, and strong signatures of purifying selection. Nevertheless, limited presence and absence variation and copy number variation were detected, particularly at the HvBBX.CR11 locus, which exhibited expression divergence associated with presence and absence variation. Phylogenetic and conserved motif analyses revealed subgroup specific structural features, indicating functional diversification within the family. Analysis of transposable elements showed preferential enrichment of DNA transposons in regulatory regions flanking HvBBXs, suggesting a contribution to regulatory variation. Promoter analyses revealed abundant cis regulatory elements associated with light signaling, hormone responses, and stress responsiveness, while codon usage analyses indicated moderate bias shaped by both mutational pressure and natural selection. Transcriptome profiling across five tissues revealed pronounced tissue specific expression patterns. In addition, weighted gene co expression network analysis under salt stress identified four HvBBXs, HvBBX.CR2, HvBBX.SC3, HvBBX.CR11, and HvBBX.CR14, as hub genes associated with chloroplast related processes and salt tolerance. These findings were further supported by quantitative real time PCR validation.

conclusionsOur results demonstrate that the HvBBX gene family is largely conserved in barley while exhibiting substantial regulatory diversification driven by pangenomic variation and differential gene expression. The identification of salt stress–associated HvBBX hub genes provides new insights into BBXmediated regulatory networks and highlights promising candidate genes for improving stress tolerance in barley.