Comprehensive genome-wide mining and characterization of the NAC transcription factor family in Gynostemma pentaphyllum identifies GpNAC68 as a positive regulator of drought tolerance in transgenic Arabidopsis

BACKGROUND: NAC (NAM, ATAF1/2, CUC2) transcription factors (TFs) serve as pivotal regulators in plant growth, development, and abiotic stress response, exhibiting great application potential in the stress-tolerant breeding of crops. Gynostemma pentaphyllum (Thunb.) Makino is a medicinally important species in the Cucurbitaceae family, whose medicinal quality and yield are directly determined by its stress resistance capacity. However, the genome-wide systematic characterization and functional validation of the NAC TF family has not yet been reported in G. pentaphyllum, which severely limits the understanding of its stress resistance regulatory mechanism and the development of molecular breeding. RESULTS: In this study, 86 full-length GpNAC genes were identified from the G. pentaphyllum genome, which were phylogenetically divided into 21 conserved subgroups. The reliability of this classification was verified by subsequent gene structure analysis and conserved motif characterization. Collinearity analysis revealed that segmental duplication, rather than tandem duplication, was the core driving force for the expansion of the GpNAC family, and all duplicated gene pairs were subjected to strong purifying selection during evolution. Tissue-specific expression profiling showed that GpNAC genes were widely involved in the growth and development of G. pentaphyllum, with most genes exhibiting preferential expression patterns in roots, flowers, and fruits. Phytohormone responsiveness analysis demonstrated that the majority of GpNAC genes were responsive to abscisic acid (ABA), methyl jasmonate (MeJA), ethylene (ETH), and melatonin (MT), among which eight genes were simultaneously induced by all four tested hormones. Furthermore, heterologous overexpression of GpNAC68 significantly enhanced drought tolerance in transgenic Arabidopsis. This functional phenotype was achieved by suppressing reactive oxygen species (ROS) accumulation, preserving cell membrane integrity, and upregulating the expression of core drought-responsive genes. CONCLUSIONS: In conclusion, this study provides the first comprehensive genome-wide characterization of the NAC TF family in G. pentaphyllum, screens out key candidate genes for the stress resistance breeding of this medicinal species, and offers novel insights into the evolutionary dynamics of the NAC family in Cucurbitaceae plants.