Functional analysis and interaction networks of Rboh in poplar under abiotic stress

Jing Wang Xiaojiao Liu Yude Kang Aizhong Liu ^* Li Ping ^*

• Southwest Forestry University, Kunming, China

Plant respiratory burst oxidase homologs (Rbohs) are essential in the generation of reactive oxygen species (ROS) and play critical roles in plant stress responses. Despite their importance, Rbohs in poplar species remain under-explored, especially in terms of their characteristics and functional diversity across different species within the same genus. In this study, we employed bioinformatics methods to identify 62 Rboh genes across five poplar species, all of which consistently contained six conserved functional domains and were classified into four distinct groups (I-IV). The number of Rboh members across poplar species was consistent with evolutionary patterns. These Rbohs exhibited relatively conserved amino acid lengths (832-989) and shared basic protein characteristics, including cell membrane localization, which highlights their roles in oxidative bursts and stress responses. Furthermore, variations in gene structure and physical properties revealed functional and evolutionary differentiation among these genes. Chromosomal distribution analysis revealed an uneven distribution of PyRbohs (Populus yunnanensis Rbohs) across chromosomes, with abundant collinearity pairs among different plant species, indicating tandem segment duplications and a shared evolutionary origin within group members. Cis-element analysis identified stress-responsive and hormone signaling-related elements, suggesting the potential role of PyRbohs in stress adaptation. This was further validated through qRT-PCR, which demonstrated the upregulation of PyRbohs under salt, drought, PEG, and ABA treatments. Protein interaction predictions using the STING database identified potential functional mechanisms of PyRbohs, including interactions with calcium-dependent protein kinases (CPKs). The interaction between PyRbohs and CPKs was confirmed using yeast two-hybrid (Y2H) assays, suggesting that CPK binding might regulate PyRboh activity and ROS production. Overall, these findings provide a comprehensive understanding of the evolutionary, structural, and functional diversity of poplar Rbohs and highlight promising candidate genes for enhancing stress tolerance in poplar species. This study lays a foundation for future research on the molecular mechanisms underlying Rboh-mediated stress responses in poplar.