Functional study of grape DREB4 in response to alkaline salt stress

【Objective】DREB (Dehydration-Responsive Element Binding) transcription factors represent a crucial class of regulatory proteins that mediate plant adaptive responses to various abiotic stresses. Although their general role in stress signaling is established, the specific functions of individual DREB members, particularly in perennial fruit crops like grapevine, remain incompletely characterized. This study aims to systematically investigate the functional role of the grapevine VvDREB4 gene under saline-alkali stress conditions and to elucidate the underlying physiological and molecular mechanisms through which it enhances stress tolerance. Our working hypothesis posits that VvDREB4 operates as a central regulator that coordinates both oxidative stress responses and structural modifications to the cell wall, thereby conferring enhanced resilience to saline- alkaline environments.【Methods】Our experimental approach integrated computational biology with rigorous laboratory validation. We commenced with a comprehensive bioinformatic analysis of the VvDREB4 protein sequence, meticulously examining its physicochemical properties, secondary structure predictions, and evolutionary relationships through phylogenetic reconstruction with orthologs from other plant species. Subsequently, the fulllength coding sequence (CDS) of VvDREB4 was isolated and cloned from grapevine (Vitis vinifera L.) cDNA. This CDS was then ligated into a plant overexpression vector under the control of the constitutive CaMV 35S promoter. Transgenic grape calli were generated via Agrobacterium tumefaciens-mediated genetic transformation, and positive transformants were selected and confirmed using qRT-PCR. To rigorously assess gene function, both transgenic (overexpression, OE) and empty- vector (control, CT) calli were subjected to a controlled Na2CO3 treatment (concentration optimized at 50 mmol · L- 1 , pH - 7.8) to accurately simulate saline-alkaline stress over a 7-day period. We conducted detailed phenotypic observations and quantitative measurements of callus growth and biomass. A suite of physiological parameters was systematically analyzed at multiple time points: Antioxidant enzyme activities—including superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT)—were assayed spectrophotometrically; the extent of membrane lipid peroxidation was quantified by measuring malondialdehyde (MDA) content; key cell wall components—namely total sugar, cellulose, hemicellulose, and lignin—were quantified using established biochemical protocols. To bridge physiology with molecular events, we employed quantitative real-time PCR (qRT-PCR) to profile the dynamic expression patterns of genes central to cell wall metabolism and lignin biosynthesis, such as cellulose synthase-like (CSL) genes and specific peroxidase (POD) isoforms known to be involved in the oxidative polymerization of monolignols. 【Results】Our bioinformatic characterization identified VvDREB4 as a nuclear-localized, unstable hydrophilic protein, sharing highest sequence similarity and closest phylogenetic affinity with DREB proteins from citrus (Citrus sinensis). Under Na2CO3 stress, a clear phenotypic divergence was observed: wild-type calli exhibited severe browning and a -60% reduction in fresh weight, whereas VvDREB4- overexpressing calli maintained significantly healthier appearance and showed only -25% growth inhibition, demonstrating the gene's role in mitigating stress-induced growth retardation. Physiological profiling revealed a profoundly enhanced antioxidant capacity in OE lines. SOD activity increased by 2.1- fold, POD by 2.8-fold, and CAT by 1.9-fold compared to stressed WT controls. Consequently, MDA accumulation—a key indicator of oxidative damage—was reduced by 45% in OE calli, indicating significantly less membrane damage. Furthermore, we observed a remarkable reconfiguration of the cell wall architecture in transgenic lines. The content of total sugars increased by 1.7-fold, cellulose by 1.9-fold, hemicellulose by 1.5- fold, and most strikingly, lignin content surged to 3.16 times that of the stressed WT control, suggesting a substantial reinforcement of the cell wall matrix. At the molecular level, qRTPCR analysis provided a mechanistic link to these physiological observations. The expression of several cellulose synthase-like (CSL) genes was significantly upregulated (2.5 to 4.0- fold) in OE lines under stress. Concurrently, the transcript levels of specific peroxidase (POD) isoforms implicated in the final steps of lignin polymerization were markedly induced (up to 5.2-fold). Additionally, VvDREB4 overexpression modulated the expression of key genes involved in pectin metabolism, indicating a broader role in remodeling the cell wall polysaccharide network under adverse conditions.【Conclusion】Our findings demonstrate that VvDREB4 significantly enhances grapevine tolerance to saline- alkali stress through a sophisticated, and dual-pronged mechanism. Firstly, it acts as a potent activator of the cellular antioxidant defense system, boosting the activities of SOD, POD, and CAT to effectively scavenge reactive oxygen species (ROS) and minimize oxidative damage, as evidenced by reduced MDA levels. Secondly, it orchestrates a substantial re- inforcement of the cell wall, primarily by promoting the pronounced accumulation of structural polysaccharides—most notably lignin. This lignification enhances the mechanical strength and likely the rigidity of the cell wall, providing a more robust physical barrier against ion toxicity and osmotic stress. This study provides a comprehensive and systematic insight into the multi-faceted regulatory role of VvDREB4, integrating physiological responses with transcriptional reprogramming. It not only advances our fundamental understanding of DREB transcription factor-mediated stress resistance mechanisms in woody perennials but also offers valuable theoretical and experimental evidence for future biotechnological applications. Targeting VvDREB4 or its downstream path-ways holds promising potential for the genetic improvement of stress resilience in grapevines and potentially other economically important fruit crops facing increasing soil salinization.