A cyclic nucleotide-gated channel gene HcCNGC11 positively regulates salt stress responses in kenaf (Hibiscus cannabinus L.).

Abstract

Cyclic nucleotide-gated channels (CNGCs) are evolutionarily conserved calcium-permeable non-selective cation channels that play critical regulatory roles in plant abiotic stress responses. This study characterizes HcCNGC11 in kenaf (Hibiscus cannabinus L.) through integrated genomic and functional analyses. Subcellular localization analysis using GFP-fusion constructs confirmed plasma membrane-specific targeting of HcCNGC11. Tissue-specific expression profiling revealed that HcCNGC11 transcripts accumulate predominantly in roots (2.8-fold higher than leaves), followed by leaves, stems, flowers, and seeds. Notably, HcCNGC11 demonstrated rapid transcriptional upregulation under 150 mM NaCl stress, reaching maximum induction at 3 h post-treatment. Virus-induced gene silencing of HcCNGC11 significantly inhibited kenaf growth under salt stress. Biochemical analyses of the silenced lines showed 5-66% reduced activities of antioxidant enzymes (SOD, POD, CAT), decreased osmoregulatory substances (soluble protein, proline), reduced chlorophyll content, and elevated ROS (H₂O₂, O₂·⁻) accumulation. Under salt stress, HcCNGC11-silenced plants displayed significant downregulation of antioxidant enzyme genes (HcSOD, HcPOD, HcCAT) as well as stress-responsive genes (HcP5CS, HcLTP, HcNCED). Conversely, Arabidopsis lines overexpressing HcCNGC11 exhibited 20-47% higher antioxidant enzyme activities, increased osmoregulatory substances, enhanced chlorophyll content, and markedly reduced ROS accumulation compared to WT under salt stress. Molecular analysis of these transgenic lines showed upregulated expression of antioxidant genes (AtSOD1, AtPOD1, AtCAT1) and stress-responsive genes (AtSOS1, AtNHX1, AtCOR15). Protein-protein interaction studies, employing both yeast two-hybrid (Y2H) and bimolecular fluorescence complementation (BiFC) assays, identified multiple HcCNGC11 binding partners, including HcCaM7, HcCNGC21, HcTHI1, and HcTCP14. Collectively, these results demonstrate that HcCNGC11 positively regulates plant salt tolerance through modulation of antioxidant systems and stress-responsive pathways.