BackgroundSunflowers are often grown in arid or semi-arid areas, where their growth is frequently limited by water availability, leading to thin seedlings and reduced yields. In this study, two sunflower genotypes with contrasting drought tolerance were selected to investigate their physiological responses and transcriptomic differences under well-watered (0 h) and drought-stressed (48 h) conditions, followed by functional validation of key genes.ResultsAnalysis of physiological and biochemical parameters revealed that ABA, antioxidant enzyme activities (SOD, POD, CAT), and the contents of osmoregulatory substances (Pro, SS) in both K55 and K58 were significantly higher under drought stress compared to the control. Transcriptome analysis identified 4,106 and 1,336 differentially expressed genes (DEGs) in the leaves of K55 and K58, respectively, while 2,717 and 5,171 DEGs were found in their roots. Weighted gene co-expression network analysis (WGCNA) identified four key modules, among which two modules (darkstateblue modules and brown4 modules) showed positive correlation with physiological traits, and the other two (lightsteelblue1 modules and orangered4 modules) showed negative correlation. KEGG enrichment analysis of genes within the significantly correlated modules indicated that "plant hormone signal transduction" plays a critical role in the drought response of sunflower. Notably, a gene named HaSAPK3.8 was identified from the ABA signaling pathway, and bioinformatic analysis revealed that its promoter region contains multiple drought-responsive and hormone-responsive cis-elements. Subcellular localization showed that HaSAPK3.8 is localized in the cytoplasm and nucleus. Heterologous expression in tobacco demonstrated that, after 7 days of drought treatment, plants overexpressing HaSAPK3.8 exhibited average increases of 85.18%, 25.03%, and 23.68% in SOD, POD, and CAT activities, respectively, an average reduction of 31.34% in MDA content, and average increases of 66.54%, 25.07%, and 21.76% in ABA, Pro, and SS levels, respectively, compared to wild-type plants. Taken together, our findings demonstrate that this gene confers drought tolerance to tobacco by regulating osmotic homeostasis and reactive oxygen species (ROS) scavenging capacity.ConclusionsIn conclusion, these findings provide insights into the molecular mechanisms of drought resistance in sunflowers and support the breeding of drought-resistant varieties.