AREB/ABF-mediated transcriptional regulatory network for salt stress response in Dendrobium officinale

Abstract Salt stress is a major abiotic constraint limiting the large-scale artificial cultivation of Dendrobium officinale , a traditional precious medicinal orchid in China. yet the molecular regulatory mechanisms underlying its salt stress response remain unclear. In this study, tissue-cultured seedlings of D. officinale were treated with 250 mM NaCl, and roots and leaves were sampled at 0 h, 4 h, and 12 h post-treatment for transcriptome sequencing. Combined with genome sequences from the NCBI database and homologous Arabidopsis sequences from the TAIR database, we performed genome re-annotation, differential expression analysis, WGCNA co-expression network construction, and genome-wide identification of the AREB/ABF gene family, followed by phylogenetic analysis, promoter cis-element analysis, tissue-specific expression analysis, and qRT-PCR validation. The results showed that roots are the core responsive organ to salt stress in D. officinale , with approximately twice as many differentially expressed genes as in leaves. A total of eight AREB/ABF family members were identified genome-wide, among which five Clade A members were confirmed as core regulators of salt stress response. Through multiple lines of evidence, the core regulatory gene Do07G001659 was identified. This gene exhibits extremely low basal expression under normal conditions but shows significant time-dependent upregulation in roots under salt stress, thereby regulating the expression of the downstream core target gene Do17G000728 , which is involved in osmotic regulation and antioxidant processes. This study elucidates the transcriptional regulatory characteristics of salt stress response in D. officinale and defines the ABA-dependent core regulatory pathway mediated by Do07G001659 , Furthermore, yeast one-hybrid assay confirmed direct binding of Do07G001659 to the Do17G000728 promoter, and transient gene silencing of Do17G000728 significantly compromised salt stress tolerance, providing experimental validation of this regulatory pathway. providing key targets and theoretical support for deciphering salt tolerance mechanisms and molecular breeding for stress resistance.