The DIR Gene Family in Watermelon: Evolution, Stress Expression Profiles, and Functional Exploration of ClDIR8

Dirigent proteins (DIR) are involved in lignan biosynthesis, stress responses, and disease resistance in plants. However, systematic characterization of the DIR gene family in watermelon (<i>Citrullus lanatus</i>) remains limited. Here, we identified 22 <i>ClDIR</i> genes in watermelon using bioinformatics methods, designated <i>ClDIR1</i> to <i>ClDIR22</i>, which were unevenly distributed across eight chromosomes and classified into three subfamilies (DIR-a, DIR-b/d, DIR-e) based on phylogenetic analysis, with DIR-b/d being the largest. Synteny analysis revealed that tandem duplication primarily drove <i>ClDIR</i> family expansion, and collinear relationships with <i>Arabidopsis</i>, rice, and cucurbit species indicated evolutionary conservation. <i>Cis</i>-acting element analysis showed abundant stress- and hormone-responsive elements in <i>ClDIR</i> promoters, suggesting roles in stress regulation. Tissue-specific expression analysis demonstrated distinct patterns, with most genes highly expressed in roots. Expression profiling under 16 abiotic and biotic stresses showed 18 <i>ClDIR</i> genes responded to stress, with <i>ClDIR8</i> differentially expressed across all conditions. qRT-PCR validation of six key genes (<i>ClDIR5</i>, <i>ClDIR8</i>, <i>ClDIR9</i>, <i>ClDIR12</i>, <i>ClDIR16</i>, <i>ClDIR22</i>) confirmed their expression patterns under high-temperature, drought, salt, and low-temperature stresses, showing a high degree of consistency with transcriptome data. Subcellular localization indicated ClDIR8 is peroxisome-localized. Yeast two-hybrid (Y2H) and bimolecular fluorescence complementation (BiFC) assays validated two ClDIR8-interacting proteins, Cla97C02G049920 (encoding peroxidase) and Cla97C08G152180 (encoding catalase). These findings provide insights into <i>ClDIR</i> genes in watermelon, highlighting <i>ClDIR8</i> as a key stress-responsive candidate for further functional studies and breeding.