Comprehensive analysis of WRKY gene family in yellowhorn (Xanthoceras sorbifolium Bunge) and functional characterisation of XsWRKY30 in salt stress

Abstract

WRKY transcription factors, as evolutionarily conserved regulators in higher plants, orchestrate transcriptional reprogramming that maintains metabolic homeostasis and facilitates adaptive responses under salinity stress. However, a comprehensive identification, characterisation, and functional analysis of XsWRKY transcription factors (TFs) in response to salt stress remains lacking in yellowhorn (Xanthoceras sorbifolium), a perennial woody oilseed species renowned for its remarkable salt tolerance. We employed a genome-wide mining approach to investigate the expression profiles and biological functions of XsWRKY TFs in response to salt stress. This analysis identified 43 XsWRKY TFs, all possessing a highly conserved domain. Phylogenetic analysis classified these TFs into three distinct subgroups, with genes within the same subgroup displaying similar motif compositions. Collinearity analysis revealed that some XsWRKY genes originated from segmental duplications, highlighting the conservation of their domains. Promoter cis-element analysis of XsWRKY genes revealed regulatory elements associated with hormonal signalling, abiotic stress responses, and developmental processes. Transcriptomic profiling under salt stress conditions highlighted distinct expression patterns, with XsWRKY30 emerging as a key regulator in the salt stress response. Experimental validation confirmed that XsWRKY30 exhibited transcriptional activation activity and was localised to the nucleus. Transient expression of XsWRKY30 in tobacco significantly reduced malondialdehyde (MDA) accumulation, while stable overexpression in Arabidopsis seedlings and yellowhorn calli enhanced antioxidant enzyme activities and proline levels, accompanied by a marked decrease in MDA content compared to wild-type controls. In addition, XsWRKY30-overexpressing Arabidopsis seedlings and yellowhorn calli exhibited a reduced Na⁺/K⁺ ratio, reflecting improved ion homeostasis under salt stress. Several salt stress-responsive genes were also upregulated in the transgenic lines and calli, suggesting that XsWRKY30 played a positive regulatory role in salt tolerance. Overall, these findings demonstrated that XsWRKY30 overexpression alleviated oxidative damage and Na⁺ toxicity under osmotic stress, providing new insights into the evolutionary conservation and functional significance of XsWRKY TFs in yellowhorn salt tolerance.