Xiaohan Tang

Background/Objectives: Ziziphus jujuba var. spinosa (sour jujube) is a traditional medicinal plant with remarkable tolerance to abiotic stresses, particularly salinity. However, the regulatory mechanisms underlying its salt stress tolerance remain unclear. NHX genes play a crucial role in plant adaptation to salt stress by mediating Na⁺/K⁺ transport to maintain intracellular ion homeostasis and pH balance. Although the NHX gene family has been characterized in many plant species, its functional characteristics in sour jujube have not yet been systematically investigated. Methods: In this study, using Arabidopsis thaliana as a reference, we identified NHX genes in sour jujube through genome-wide analysis and molecular approaches, and systematically analyzed their phylogenetic relationships, chromosomal locations, conserved motifs, gene structures, cis-regulatory elements in promoter regions, and expression patterns under abiotic stresses, particularly salt stress. Results: The results revealed the presence of eight NHX genes distributed across six chromosomes in sour jujube, which were classified into three subfamilies: Vac-class, Endo-class, and PM-class. Members within the same evolutionary clade exhibited high structural conservation in motif composition and gene architecture. Except for the PM-class, all other clades contained amiloride-binding sites (FF(I/L)(Y/F)LFLLPPI). Analysis of cis-regulatory elements indicated that the promoter regions of these genes were enriched with elements related to defense responses, stress adaptation, and phytohormone signaling, further supporting their role in plant environmental adaptation. Additionally, the qRT-PCR analysis showed that most of the ZjNHX genes in both roots and leaves are up-regulated by salt. Notably, ZjNHX1 expression in roots increased approximately 40-fold within 3 h, whereas ZjNHX2 and ZjNHX3 were strongly induced in leaves under prolonged salt exposure. Conclusions: Taken together, this work gives a detailed overview of the ZjNHX genes and their important roles in response to salt stress in sour jujube. Our findings also provide a foundation for further functional characterization of this gene family.

Terpenoids are critical components of plant environmental adaptation mechanisms. They also exhibit significant therapeutic potential in herbal medicine. 3-Hydroxy-3-methylglutaryl coenzyme A reductase (HMGR), a pivotal rate-limiting enzyme governing the initial stage of the mevalonate (MVA) pathway in triterpene saponin biosynthesis, remains uncharacterized in Ziziphus jujuba var. spinosa. Through genome-wide and molecular analysis, we systematically identified ZjHMGR isoforms and revealed differential tissue-specific expression patterns and significant salt stress-responsive regulation across identified isoforms. Our findings reveal three evolutionarily conserved ZjHMGR isoforms with a complete HMG-CoA reductase domain and closely related to Populus trichocarpa. Collinearity analysis revealed two collinear gene pairs, and purifying selection was identified as the primary evolutionary force acting on the ZjHMGR gene family. Cis-acting element analysis revealed that ZjHMGR gene family enriched MYB-related, TC-rich repeats, light- and hormone-responsive elements, suggesting transcriptional regulation by environmental stimuli and phytohormones. Spatiotemporal expression analysis via qRT-PCR revealed differential transcriptional patterns of ZjHMGR members, with pronounced upregulation under ABA, MeJA, and light induction. Saline stress disrupted the growth of wild jujube seedlings while activating ZjHMGR expression alongside other MVA pathway genes. Overexpression of ZjHMGR enhances salt stress resistance in Arabidopsis thaliana. This study lays the foundation for further investigations into the molecular mechanisms of the ZjHMGR gene family concerning saponin biosynthesis, phytohormone interactions, and salt tolerance in wild jujube.