Differential involvement of WRKY genes in abiotic stress tolerance of Dendrobium huoshanense

Extreme environments have adversely affected the habitats of medicinal Dendrobium species. Dendrobium huoshanense, a well-known traditional Chinese medicinal plant, is highly susceptible to abiotic stress. Evidence suggests that WRKY transcription factors play versatile roles in responding to both biotic and abiotic stressors experienced by terrestrial plants. The regulatory mechanisms underlying the stress responses of WRKY genes have been characterized for several model plants. In this study, 64 WRKY genes were identified in the D. huoshanense genome. Chromosome localization analysis showed that all the WRKY genes were anchored on the 15 pseudochromosomes, among which chromosome 5 was occupied by the highest number of WRKY genes (from DhWRKY7 to DhWRKY19), followed by chromosome 17 (from DhWRKY45 to DhWRKY53). Subcellular localization predictions indicated that most of the WRKY genes were located in the nucleus, with only a few WRKY genes localized in peroxisome, plastid, and mitochondria. Analysis of the conserved motifs and gene structure demonstrated that most genes, except for a few truncated genes, had more than two exons, and this was likely to be due to transposon insertion. A comparative genomics investigation based on the number of synteny blocks indicated the close evolutionary relationships of several orchid species, such as D. nobile, D. chrysotoxum, and G. elata, with D. huoshanense. Interspecies gene duplication events suggested that Dendrobium experienced a large-scale duplication event that included whole genome duplication and segmental duplication, and a limited number of WRKY paralogs were produced by tandem duplication. Based on the construction of a phylogenetic tree with AtWRKY nomenclature, DhWRKY genes could be categorized into three primary subfamilies. Group II could be further subdivided into five subclades. Group IIc had the highest number of WRKY genes, followed by Groups I and III. Multiple alignment of the amino acid sequences indicated that all DhWRKYs encoded a zinc-finger domain in addition to the highly conserved WRKYGQK domain, indicating their potential roles in transcriptional regulation and protein–protein interactions. Expression of most of the WRKY genes could be induced by light signals, phytohormones, and diverse abiotic stresses. Quantitative RT-PCR (qRT-PCR) and expression profiling results confirmed that the expression of WRKY genes could be induced by abscisic acid, salicylic acid, methyl jasmonate, gibberellin, and cold stress. Overall, the results of the cis-acting element analysis, expression profiling, and qRT-PCR highlighted DhWRKY42 as a key regulator involved in the regulation of jasmonic-acid-responsive genes. This study can provide valuable references for the study of WRKY genes in other Dendrobium species.