Genome-wide identification of cytochrome b5 gene family reveals their potential roles in nitrate response in Xanthium strumarium

Cytochrome b₅ protein (CB5) is a key physiological component in electron transport, playing a significant role in oxidative reactions, plant growth, and stress response mechanisms. In invasive plants, the CB5 gene family may potentiate invasion competitiveness by orchestrating oxidative stress homeostasis, thereby conferring adaptive advantages under novel environmental regimes. This study focused on the CB5 gene family in Xanthium strumarium, an invasive species. We conducted RNA-sequencing following treatments with 0.5 mM nitrate, 5 mM nitrate, and Gibberellins (GA), as these concentrations mimic varying nitrogen availability and hormonal responses that the plant may encounter in novel environments. 27 putative XstCB5 proteins were isolated and clustered them into four clades in X. strumarium. The 27 XstCB5 genes exhibited distinct gene structures encoding a wide range of physiological traits through their corresponding proteins. All the XstCB5s promoters harbored numerous cis-elements related to stress and phytohormones. RNA-sequencing results revealed significant upregulation of 26, 24, and 24 CB5 genes in response to the respective treatments. Real-time quantitative PCR and nitrate reductase (NR) activity further demonstrated that XstNR1 and XstNR2, the key nitrogen assimilation-related CB5 genes, exhibited distinct functions when subjected to conditions of low nitrate stress. The overexpression of XstNR2 in Arabidopsis thaliana resulted in an increased biomass following treatment with 0.5 mM nitrate. Our results provide a systematic overview of the XstCB5 gene family and emphasize their roles in varying nitrate conditions.