Genome-wide characterization of the WRKY gene family and the role of LsfWRKY29 in regulating somatic embryogenesis in hybrid sweetgum (Liquidambar styraciflua × L. formosana).

Hybrid sweetgum (Liquidambar styraciflua × L. formosana) is a globally significant forest tree resource, exhibiting significant economic, ornamental, ecological and medicinal values. Somatic embryogenesis (SE) is an effective reproductive strategy, having great application potential and economic value in large-scale propagation, artificial seed production, genetic transformation, germplasm preservation and biotechnology. It's an essential technical approach for the industrial application of hybrid sweetgum. The WRKY gene family, one of the largest groups in plants, is crucial for regulating responses related to plant growth, while its function and molecular mechanism in SE are unknown. Here, we identified 61 members of the WRKY gene family from the genome of hybrid sweetgum and characterized their amino acid sequence characteristics, phylogenetic relationships, conserved motifs, cis-acting elements and gene structures at the genome-wide level. LsfWRKY29, identified as a key gene in Weighted Gene Co-expression Network Analysis (WGCNA), is significantly expressed during the morphogenesis stage of SE. Subcellular localization studies indicate that WRKY29 is located in the nucleus. Functional assays indicate that overexpression of LsfWRKY29 leads to more abnormal embryos and subsequent developmental arrest, suggesting its negative role during SE. Yeast two-hybrid (Y2H) and luciferase complementation imaging (LCI) assays validate the interaction between LsfWRKY29 and the growth regulator LsfGRF2, a member of the growth regulators that has been extensively proven to boost plant regeneration. This interaction implies that the WRKY29-GRF2 might be an important regulatory module in SE of hybrid sweetgum. These findings provide new insights into the role of WRKY genes in regulating SE in plants.