Endogenous Antioxidant Defense Responses in Grapevine Plants to the Infection of GLRaV-3

Abstract

Aim. The aim of this study was to investigate how viral infections, which severely impair plant growth and productivity, affect reactive oxygen species (ROS) accumulation, and to evaluate the roles of both enzymatic antioxidant defenses and non-enzymatic low-molecular weight antioxidants, including ascorbate (ASA), glutathione (-L-glutamyl-L-cysteinyl-glycine = GSH), and tocopherols (TOCs), in mitigating virus-induced oxidative stress in plants. Methods. In the present study, a total of 45 leaf samples were collected from mature 10-year-old vines of the cultivars 'Gara Shani' (red) and 'Ag Shani' (white). Sampling was conducted in three vineyards, where five vines were selected per vineyard, and three physiologically mature leaves were collected from each vine, resulting in 15 leaves per vineyard and 45 leaves in total (3 vineyards × 5 vines × 3 leaves). In each vineyard, one vine without visible symptoms was designated as a healthy control, while the remaining vines exhibited typical viral symptoms such as reddening of leaf margins and petioles, reduced vigor, leaf rolling, yellowing, and curling. Consequently, 36 leaves were obtained from symptomatic vines and 9 leaves from asymptomatic (healthy) vines. Grapevine leafroll-associated virus 3 (GLRaV-3) infection was confirmed in grapevine samples using AgriStrip rapid assay, double-antibody sandwich ELISA (DAS-ELISA), and reverse transcription-polymerase chain reaction (RT-PCR). Subsequently, virus-induced changes in non-enzymatic antioxidants were quantified by spectrophotometric determination of ascorbate and glutathione, tocopherols via the Emmerie-Engel reaction, and lipid peroxidation through MDA content using the thiobarbituric acid reactive substances (TBARS) assay. Enzymatic antioxidant activities, including ascorbate peroxidase and catalase, were measured spectrophotometrically, and soluble protein content was determined by the Sedmak method. Results. Viral screening using AgriStrip assays and DAS-ELISA, followed by RT-PCR targeting the coat protein gene, confirmed GLRaV-3 infection in 13 samples (approximately 36.1%), while symptomless vines tested negative, demonstrating consistency between visual assessment and molecular diagnostics. GLRaV-3 infection resulted in significant increases in non-enzymatic antioxidant levels compared with healthy controls. Tocopherol (TOC) content increased by 18-73% across infected samples 2-6, whereas sample 1 showed no significant change. Glutathione (GSH) levels were elevated by 20-60% in infected samples 1, 2, 4, 5, and 6, while sample 3 showing no significant difference. Ascorbic acid (ASA) content increased by 10-23% in infected samples 1, 3, 5, and 6, with sample 2 and 4 showing no significant difference. Lipid peroxidation, as indicated by malondialdehyde (MDA) content, was also significantly enhanced in infected leaves. MDA levels increased approximately 1.5-2.6-fold in samples 1, 2, 4, 5, and 6 compared with healthy controls, reaching absolute values of 0.85-1.75 µmol g-1 FW versus 0.45-0.9 µmol g-1 FW in controls. In parallel, the activities of key antioxidant enzymes were elevated: catalase (CAT) activity increased by 25-48%, and ascorbate peroxidase (APX) activity increased by 30-55% in infected leaves compared with corresponding controls. Conclusions. Surveys conducted in Salyan vineyards confirmed GLRaV-3 infection supporting the link between observed symptoms and biochemical oxidative stress responses. These biochemical changes demonstrate that GLRaV-3 infection triggers oxidative stress and concurrently activates both enzymatic and non-enzymatic antioxidant defense mechanisms in grapevine leaves. The coordinated increase in ASA, GSH, TOC, CAT, and APX, alongside elevated MDA, indicates a complex redox adjustment aimed at mitigating virus-induced oxidative damage. ```