A non-specific lipid transfer protein gene HcnsLTP111 positively regulates drought and salt stress tolerance in kenaf (Hibiscus cannabinus L.)

Abstract

Kenaf (Hibiscus cannabinus L.), an important bast fiber crop with strong abiotic stress tolerance, holds significant significance in the utilization of saline-alkali land and other marginal lands. Non-specific lipid transfer protein (nsLTPs) are key regulators of plant stress responses, yet their roles in kenaf and the underlying molecular mechanisms remain uncharacterized. In this study, a genome-wide analysis was conducted and 111 nsLTP genes were identified in kenaf. The identified genes exhibited an uneven distribution across 18 chromosomes and were grouped into nine distinct subfamilies based on phylogenetic analysis. Cis-acting element analysis revealed that HcnsLTPs promoters are enriched with abiotic stress-responsive motifs, suggesting their pivotal role in stress adaptation. Expression profiling under drought and salt stress identified HcnsLTP111 as a key stress-inducible gene, with significantly upregulated transcription and plasma membrane localization. Functional validation in yeast demonstrated that heterologous expression of HcnsLTP111 enhanced survival rate under drought and salt conditions. Conversely, silencing of HcnsLTP111 in kenaf via virus-induced gene silencing (VIGS) resulted in pronounced growth inhibition, reduced activities of key antioxidant enzymes (SOD, POD, CAT), elevated accumulation of reactive oxygen species (ROS), and diminished concentrations of osmoregulatory compounds. The expression levels of ROS-scavenging related genes (HcSOD, HcPOD, and HcCAT) and stress tolerance-related genes (HcNHX1, HcSOS1, HcABF2, HcAAP2, HcIPCS1, and HcP5CS1) were significantly reduced in the silenced lines. Additionally, the interaction between HcnsLTP111 and HcIPCS1, a key enzyme involved in stress signaling pathways, was shown through yeast two-hybrid (Y2H) assays and bimolecular fluorescence complementation (BIFC) analyses. Collectively, HcnsLTP111 acts as a positive regulator of drought and salt tolerance in kenaf by modulating ROS homeostasis and interacting with stress-response proteins. This study provides novel insights into nsLTP-mediated stress adaptation and identifies HcnsLTP111 as a promising candidate for crop improvement strategies.