Genome-wide analysis of the CHX family in Paulownia fortunei and functional characterization of PfCHX16 in salt stress tolerance mediated by a PfCHX16-PfNHX2 module

Abstract

Paulownia fortunei is a globally important fast-growing timber tree, whose productivity is severely constrained by multiple abiotic stresses. Ion homeostasis transporters represent ideal targets for stress-resilient breeding. Cation/H⁺ exchangers (CHXs) are pivotal for plant ion homeostasis and stress adaptation. However, the CHX gene family has not been systematically characterized in P. fortunei . Here, we identified 20 PfCHX genes through genome-wide analysis and classified them into five phylogenetic subfamilies. Structural and motif analyses revealed a conserved core alongside member-specific variations, suggesting functional diversification. Promoter regions were enriched with stress and hormone responsive cis -elements. Transcriptome and RT-qPCR analyses demonstrated that PfCHX genes exhibit distinct, and overlapping expression patterns under drought, salinity, and phytoplasma infection. Among them, PfCHX16 showed the marked and consistent upregulation across all stress conditions. Subcellular localization analysis demonstrated that both PfCHX16 and PfNHX2 were localized to the plasma membrane. Transient overexpression and physiological analyses in tobacco revealed that PfCHX16 may respond to salt stress adaptation by maintaining K⁺ homeostasis, modulating Na⁺ accumulation, and regulating redox balance. Protein interaction assays, including yeast two-hybrid and bimolecular fluorescence complementation provided preliminary evidence for a potential physical interaction between PfCHX16 and the Na⁺/H⁺ exchanger PfNHX2 at the plasma membrane, a finding further supported by molecular docking. This work provides the first comprehensive genomic overview of the CHX family in P. fortunei and suggests that PfCHX16 may act as a potential regulator of multi-stress responses. The putative PfCHX16–PfNHX2 module may contribute to ion homeostasis regulation under combined stresses. These findings provide potential genetic resources for molecular breeding of stress-resilient forest trees.