Genome-Wide Characterization of the WOX Gene Family and Identification of Key PmWOX4 Gene Associated with Adventitious Root Formation in Pinus massoniana

Pinus massoniana Lamb. is an important native economic and ecological tree species in southern China. However, adventitious root regeneration severely restricts the asexual propagation of elite traits. The WOX gene family, a plant-specific transcription factor family, plays critical roles in various aspects of plant growth and development, particularly in root development, including the maintenance of the root apical meristem, root elongation, and the initiation and formation of lateral roots. In this study, a total of 21 WOX family proteins were identified from the genome of P. massoniana and designated as PmWOX1 to PmWOX21 based on their chromosomal locations. These 21 members were unevenly distributed across nine chromosomes, with a clustered distribution observed on chr7. Collinearity analysis suggested that gene duplication and purifying selection may serve as key driving forces in the evolution of WOX genes. Conserved motif analysis revealed divergence among different PmWOX clades, implying distinct functions in growth and development. Cis-element analysis indicated that PmWOX genes may be involved in the regulation of stress responses, hormone signaling, and developmental processes. Expression pattern analysis based on transcriptome data from root tips and shoot tips identified nine PmWOX genes with significantly higher expression in root tips. RT-qPCR further screened PmWOX4, which exhibited the highest expression in root primordia. Functional experiments verified that PmWOX4 genes encoded nuclear-localized proteins with transcriptional activity. The PPI results indicate that PmWOX4 may interact with CLE41/44, LBD, ATHB8 and CLV3/CLV1 to regulate adventitious root formation. Collectively, our findings suggest that PmWOX4 possesses functional potential in adventitious rooting, which could be further exploited to improve the efficiency of asexual propagation in P. massoniana.