Effects of drought and copper stress on the physiological characteristics and ADH gene family in Zaoheibao grape seedlings

【Objective】Grape (Vitis vinifera L.) is an important economic crop worldwide. Drought and heavy metal stress are major constraints on the sustainable development of the grape industry. Various strategies, such as applying exogenous substances, promoting ecological agriculture, and adopting water- and fertilizer- saving practices, have been explored to alleviate the adverse effects of drought and heavy metal stress on grapevine growth. However, the combined impact of drought and heavy metal stress on grape seedling growth remains poorly understood. Zaoheibao is a self- bred cultivar from Shanxi Province that is widely grown in the region. This study investigated the effects of combined drought and copper stress on grape seedling growth and identified the ADH gene family in grapevines through bioinformatic analysis.【Methods】Zaoheibao grape cuttings served as the experimental materi-als. Group A was irrigated with 1/2 Hoagland nutrient solution, while Group B received the same solution supplemented with 200 mg/kg CuSO4 · 5H2O to simulate copper stress. Each group was subdivided into four drought stress levels: control (CK, 80%±5% soil moisture), mild (T1, 60%±5% ), moderate (T2, 40%±5%), and severe (T3, 20%±5%), totalling for eight treatments. Chlorophyll content, photosynthetic parameters, and chlorophyll fluorescence were measured every five days after stress initiation. Stomatal characteristics were assessed on 45th d. Based on principal component and correlation analyses, four representative treatments were selected: CKA (control group), CKB (copper stress), T3A (severe drought stress), and T3B (compound stress) group. Antioxidant enzyme activities, acetaldehyde dehydrogenase (ADH) and malondialdehyde (MDA) levels were determined, and the ADH gene family was analyzed using bioinformatic methods.【Results】Photosynthesis in grape seedlings was markedly inhibited under stress. The net photosynthetic rate (Pn) declined significantly over time under both drought and combined stress conditions. On 15th d, Pn in the T3A/B group showed the greatest decline, decreasing by 55.49% and 44.5% relative to CKA/B under drought and combined stress, respectively. The transpiration rate (Tr) initially rose and then declined under drought stress, but remained lower than that in the CKA group. Under combined stress, the pattern of Tr was similar to drought; however, Tr in the CKB and T1B groups gradually increased. By the end of the stress period, the T3A/B group exhibited the largest reduction in Tr, with decreases of 68.99% and 64.86% under drought and combined stress, respectively. Under the two stress conditions, the stomatal conductance (Gs) and intercellular CO2 concentration (Ci) of grape seedlings initially increased and then decreased. Notably, the Gs in the T3A/B group gradually declined. On 15th d, Gs in T3A group showed a significant reduction of 76.28% compared to the CKA group, while the T2B group showed a significant reduction of 84.63% compared to the CKB group. On 45th d, the stomatal opening length (SOL) on the lower leaf epidermis varied across treatments, whereas stomatal density (SD) and stomatal length (SL) consistently decreased with increasing drought severity. Maximum photochemical efficiency (Fv/Fm) remained largely unchanged under stress. Under drought stress, the minimum initial fluorescence (F0) decreased over time in CKA, T1A, and T2A, while in T3A it first increased and then declined. Under combined stress, F0 in T1B, T2B, and T3B similarly increased initially and then declined. By the end of the stress period, F0 in T3A/B had significantly decreased by 18.92% and 11.34% , respectively. The photochemical quenching coefficient (qP) remained relatively stable throughout the stress period. Non-photochemical quenching coefficient (NPQ) increased with prolonged stress and decreasing soil moisture content. At the end of the stress period, NPQ in T3A/B exhibited the greatest increase, rising by 78.65% and 58.58%, respectively. Chlorophyll a (Chl a), chlorophyll b (Chl b), and total chlorophyll (Chl a+b) contents gradually declined under both drought and combined stress. At the end of drought stress, Chl a exhibited the largest decrease (31.88%), while Chl b declined most under combined stress (39.75%). Total chlorophyll content significantly dropped by 27.6% under drought stress. Carotenoid (Car) content also declined progressively with increasing stress duration. Correlation analysis of 12 physiological parameters revealed highly significant positive correlations (P ＜ 0.01) among Pn, Tr, and Gs, and also among Chl a+b, Chl a, Chl b, and Car. These pigment-related indices were also strongly and positively correlated with Pn (P＜0.01), but showed significant negative correlations with NPQ (P＜0.01) and Ci (P＜0.05). Principal component analysis indicated that combined drought and copper stress imposed more severe damage on grape seedlings compared to drought stress alone. To further assess the effects of drought and copper stress on the antioxidant capacity of grape seedlings, four treatments (CKA, CKB, T3A and T3B) were analyzed. The antioxidant enzymes, ADH activities and MDA contents of grape seedling leaves on 15th, 30th and45th d were measured under 4 conditions. Superoxide dismutase (SOD) activity in leaves increased with prolonged stress exposure. Initially, SOD activity remained relatively stable, but gradually increased as stress persisted. After 45 d of stress, the SOD and POD activities in the leaves of grape seedlings from the CKB, T3A, and T3B groups were significantly higher than those of the CKA group. Among these, the T3B group exhibited the highest SOD and POD activities, which were 1.05 and 1.73 times greater than those of the CKA group, respectively. The MDA content followed a trend similar to that of SOD and POD activities, gradually increasing in the CKB, T3A, and T3B groups. On 45th d, the MDA content in the T3A and T3B groups was significantly higher than that in the CKA/B group. Notably, the T3B group had the highest MDA content, which was 4.35 times higher than that of the CKA group. With prolonged stress duration, the ADH activity in the leaves of grape seedlings from the CKB, T3A, and T3B groups first decreased and then increased. On the 30th day, ADH activity in the T3A/B groups was significantly higher than that in the CKA group, with the T3B group exhibiting the highest activity, 25.62% higher than that of the CKA group. By the end of the stress period (on the 45th day), ADH activity in the CKB, T3A, and T3B groups did not differ significantly from that in the CKA group, with the T3B group still showing the highest activity, 18.42% greater than that of the CKA group. A total of 53 ADH genes were identified from grape genome data using bioinformatics approaches. These ADH proteins had amino acid lengths ranging from 300 to 771 residues, molecular weights from 32.37 to 83.14 kDa, and theoretical isoelectric points (pI) from 5.49 to 9.63. Most ADH proteins were rich in acidic amino acids and were predicted to function in acidic subcellular compartments. ADH genes displayed substantial variation across subgroups, whereas gene structures within the same subgroup remained relatively conserved and stable.【Conclusion】Under drought and copper stress, the photosynthetic system of Zaoheibao grape seedlings was damaged, with significant increases in the activities of SOD, POD, and ADH, as well as the content of MDA. Compared with drought stress alone, combined stress caused greater harm to grapevine growth. A total of 53 ADH genes were identified using bioinformatics tools and classified into four subgroups. There were significant differences between the ADH genes of each subfamily, but the structure within each subgroup remained relatively conserved and stable. This study provides a theoretical foundation for understanding the drought resistance mechanism in grapevines and for breeding stress-resistant varieties.