Metabolic Responses of Grapevine Leaves to Grapevine Leafroll-Associated Virus 3 Infection

Background: Grapevine leafroll-associated virus 3 (GLRaV-3) is a severe phloem-limited grapevine virus, meaning it is restricted to sugar-transporting vascular tissue, which helps explain its strong effects on leaf physiology and carbon transport. However, its impact on leaf oxidative status, phenolic composition, and volatile organic compounds (VOCs) remains insufficiently characterized. Methods: Virus-free and GLRaV-3-infected grapevine leaves were analyzed for photosynthetic pigments, oxidative stress markers, phenolic compounds, and VOCs using spectrophotometric assays, HPLC-DAD/FLD, and SPME-Arrow-GC/MS. Data were evaluated using one-way ANOVA, multiple testing correction, principal component analysis (PCA), and exploratory partial least squares-discriminant analysis (PLS-DA). Results: GLRaV-3-infected leaves showed lower chlorophyll a (576.75 vs. 657.85 mg 100 g^-1 DW), chlorophyll b (282.96 vs. 314.05 mg 100 g^-1 DW), and total carotenoids (125.89 vs. 154.65 mg 100 g^-1 DW), but higher malondialdehyde (11.91 vs. 8.73 nmol g^-1 DW), H₂O₂ (0.36 vs. 0.25 μmol g^-1 DW), and proline (8.83 vs. 7.98 μmol g^-1 DW). Phenolic profiling showed increased levels of several flavonols and hydroxycinnamic acids, including kaempferol-3-O-glucuronide (2.81-fold), myricetin-3-O-glucoside (1.75-fold), quercetin-3-O-glucuronide (1.48-fold), and caffeic acid (1.30-fold). VOC profiling revealed higher relative abundances of several green leaf volatile-related compounds and methyl salicylate, including 1-methoxy-2-propanol (1.85-fold), 1-penten-3-ol (1.58-fold), hexanal (1.42-fold), and methyl salicylate (1.37-fold). PCA summarized treatment-related differences, with the first two components explaining 63.73% of phenolic and 74.09% of VOC variability, while exploratory PLS-DA/VIP analysis further supported the identification of treatment-associated discriminant metabolic features. Conclusions: GLRaV-3 infection is associated with reduced pigment content, increased oxidative stress markers, and coordinated changes in phenolic and VOC profiles. These metabolite changes provide insight into grapevine responses to viral infection and highlight GLRaV-3-associated metabolic features for future targeted studies of grapevine leafroll disease.