A Genome-Wide Analysis of the BAM Gene Family and Identification of the Cold-Responsive Genes in Pomegranate (Punica granatum L.)

Beta-amylases (BAMs, EC 3.2.1.2), belonging to a multigene family, play a pivotal role in starch breakdown and are also involved in hormonal and stress responses, notably to cold stress. Pomegranate trees (Punica granatum L.) are adapted to warm climates and are sensitive to cold temperatures. In this study, we analyzed eight PgBAM genes from the pomegranate genome dataset. These members unevenly distributed across chromosomes and were categorized into four groups based on their orthologous members. The motif composition was highly consistent among most members. In contrast, exon numbers and arrangements were conserved within groups or subgroups, whereas significant diversity was observed between different groups. A syntenic analysis revealed that three PgBAM members (PgBAM1/4/5) showed a total of 11 syntenic relationships with the BAM members from Arabidopsis, kiwifruit, and Chinese white pear, respectively. Promoter binding motif prediction suggested potential roles for PgBAMs&rsquo: genes in light, stress, hormones, and development signaling. Gene expression indicated that PgBAM4 was predominantly expressed in leaves, PgBAM7 in flowers, and PgBAM8 in roots and leaves and during fruit ripening, particularly in pericarp development. A transcriptome analysis identified the starch and sucrose metabolism pathway (map00500) as a key factor in the cold stress response of cold-sensitive cultivar &lsquo:Tunisia&rsquo: seedlings. PgBAM4 exhibited remarkable expression and was closely associated with the cold-responsive BAM genes, characterized by a closer phylogenetic relationship, conserved catalytic residues, and similar secondary and tertiary structures. Moreover, the differences in soluble sugar levels and PgBAM4 expression were closely associated with the varying cold stress resistance observed between &lsquo:Tunisia&rsquo: and &lsquo:Sanbai&rsquo: seedlings. Furthermore, yeast one-hybrid assays confirmed that PgCBF7, a critical transcription factor for enhancing freezing tolerance, binds to the promoter region of PgBAM4. Our findings provide a systematic overview of the PgBAM gene family and shed new light on the regulatory mechanisms underlying cold stress tolerance in pomegranate.