Viruses disrupt the plant cell cycle, inhibit cell death pathways, restrict macromolecular trafficking, alter cell signaling, protein turnover, and transcriptional regulation, and suppress defense mechanisms. The interference with these processes in the host leads to a wide range of plant developmental and physiological defects . Cultivated grapevines are highly susceptible to a variety of viruses and viroids, which cause significant crop losses and shorten the productive life of vineyards. More than 65 different viral species classified in at least 15 families have been reported to infect grapevines, which represents the highest number of viruses so far detected in a single cultivated plant species . Although these viruses are generally transmitted by plant-feeding insects or soilborne nematodes, they can also be spread through infected propagation material . Grapevine red blotch is a viral disease discovered in northern California in 2008 that has become a major economic problem for the wine industry in the USA . This disease is caused by the Grapevine red blotch-associated virus , a circular ssDNA virus with resemblance to geminiviruses, which infects wine grape cultivars with significant detrimental effects on productivity . The incidence and severity of the red blotch symptoms vary depending on the grape cultivar, environmental conditions, and cultural practices . In red-skinned varieties, GRBaV infections result in the appearance of red patches on the leaf blades, veins, and petioles; in white-skinned varieties, hydroponic bucket they manifest as irregular chlorotic regions on the leaf blades.
GRBaV also affects berry physiology, causing uneven ripening, higher titratable acidity, and lower sugar and anthocyanin content, among others . Consequently, must and wine produced from infected berries present altered flavor and aroma. To date, there is limited information on how GRBaV infections affect grape metabolism. Comprehensive analyses to study specific cellular processes that GRBaV exploits to promote infections in berries are still needed, in particular those that relate to changes in berry chemical composition during fruit development. Grape berry development exhibits a double sigmoid growth pattern with three distinct phases: early fruit development, lag phase, and berry ripening. Most metabolic pathways that promote desired quality traits in grape berries are induced during ripening. The onset of ripening is accompanied by significant changes in berry physiology and metabolism, including softening, sugar accumulation, decrease in organic acids, and synthesis of anthocyanins and other secondary metabolites that define the sensory properties of the fruit . Berry ripening is controlled by multiple regulatory pathways, and occurs in an organized and developmentally timed manner. Interactions between transcriptional regulators and plant hormones regulate the initiation and progression of ripening processes . Like other non-climacteric fruit, grape berries do not display a strong induction of ethylene production and respiration rate at véraison, and the activation of ripening events does not depend primarily on ethylene signaling.
Even though the hormonal control of grape berry development is not completely understood, it is established that abscisic acid , brassinosteroids, and ethylene are positive regulators of ripening processes, while auxin delays the initiation of ripening . In the context of virus–grape berry interactions, dissecting the mechanisms that regulate ripening and plant defenses may provide new opportunities to develop vineyard management strategies to control viral diseases and ameliorate the negative effects on berry quality. In this study, we integrated genome-wide transcriptional profiling, targeted chemical and biochemical analyses, and demonstrated that grapevine red blotch disrupts ripening and metabolism of red-skinned berries. We sampled berries at different ripening stages from vines infected with GRBaV and healthy vines in two vineyards. We identified grape metabolic pathways that were altered in ripening berries because of the viral infection. We determined that GRBaV-induced pathways that are normally associated with early fruit development in berries at late stages of ripening, and suppressed secondary metabolic pathways that occur during normal berry ripening and/ or in response to stress. Using targeted metabolite profiling and enzyme activity analyses, we confirmed the impact of GRBaV on phenylpropanoid metabolism. We identified specific ripening-related processes that were disturbed in GRBaV-infected berries. Remarkably, these processes included alterations in ripening regulatory networks mediated by transcriptional factors, post-transcriptional control, and plant hormones, which lead to berry developmental defects caused by red blotch.
To determine the impact of grapevine red blotch on berry physiology, we studied naturally occurring GRBaV infections in distinct wine grape-growing regions in northern California . We sampled red-skinned grape berries from two different vineyards, one in Oakville and one in Healdsburg . We used multiple vineyard sites to focus on observations consistently made across environments and, thus, to exclude factors associated with specific environmental or cultural conditions. Prior to sampling, vines were screened for the presence of GRBaV and other common grapevine viruses. The appearance of red blotch symptoms on leaves of GRBaV-positive vines and not on those of healthy controls confirmed the initial viral testing. We sampled grape berries from vines that tested positive for GRBaV and negative for other common grapevine viruses. At the same time, we also collected berries from vines that tested negative for all viruses and included them in the study as healthy controls. In order to determine the impact of the disease on berry development and metabolism, we collected GRBaV-positive and control berries at comparable developmental stages: pre-véraison , véraison , post-véraison , and harvest . This sampling strategy also aimed to limit confounding effects due to differences in the progression of ripening between berry clusters of GRBaV-positive and healthy vines. In some cases, we observed that GRBaV-positive vines presented grape clusters with evident uneven ripening . Comparisons between berries from GRBaV-positive vines and healthy controls indicated that, at equivalent stages of development, berries affected by red blotch had reduced soluble solids and total anthocyanins in agreement with previous reports on red-skinned wine grapes . Sampled berries were used for genome-wide transcriptional profiling of viral and grape genes. RNAseq was performed using 3–4 biological replicates of each ripening stage, infection status, and vineyard. We first confirmed the presence of the virus in the berries of GRBaV-positive vines by qPCR amplification of viral DNA . Viral activity in the berries was also assessed by quantifying plant-derived mRNA transcripts of GRBaV genes in the RNAseq data. Plant expression of five out of the six predicted genes in the GRBaV genome was detected in all berry samples obtained from GRBaV-positive vines but not in berries collected from the control vines . The most expressed GRBaV genes in the berries corresponded to V1, which encodes a coat protein, and V3 with unknown function. Expression levels of the GRBaV genes appeared to change as berries ripened. However, we could not determine to what extent the progression of ripening or other environmental factors influenced the plant’s transcription of viral genes because their pattern of variation between ripening stages differed in the two vineyards . Expression of 25 994 grape genes was detected by RNAseq across all berry samples. Principal component analysis was carried out with the normalized read counts of all detected genes. The two major PCs, which together accounted for 42.97% of the total variability, clearly separated the samples based on ripening stage, regardless of the vineyard of origin or their infection status . These results indicated that the inter vineyard variation was smaller than the ripening effect, stackable planters and the overall progression of ripening was similar between berries from GRBaV-positive and control vines. Therefore, we hypothesized that GRBaV infections in berries have altered the expression of particular grape genes and/or molecular pathways, which could subsequently have led to developmental and metabolic defects.While the PCA described above indicated that overall transcriptome dynamics associated with berry ripening were not perturbed by the infection, the lower levels of soluble solids and anthocyanins in GRBaV-positive berries, particularly later in development, suggested that red blotch may affect specific primary and secondary metabolic processes. We therefore focused the RNAseq analyses to identify grape molecular pathways that were differentially regulated as a result of GRBaV infections. We identified grape genes with significant differential expression due to red blotch by comparing GRBaV-positive and GRBaV-negative berries at each ripening stage and independently for each vineyard.
We then looked at the intersection of differentially expressed genes between the two vineyards to identify common responses to red blotch. A total of 932 grape DE genes were found to be consistently down- or up-regulated in infected berries in both vineyards at a given ripening stage, and were classified as GRBaV-responsive genes . On average these GRBaV-responsive genes showed 0.49 ± 0.22-fold changes compared with the healthy controls. Comparing berries at similar ripening stages may have contributed to exclude more dramatic changes in gene expression associated with more pronounced ripening delay due to GRBaV. Key metabolic processes that were suppressed or induced as a consequence of red blotch in ripening berries were identified by enrichment analyses of the functional categories defined by Grimplet et al. in the set of GRBaV-responsive genes . Amino acid biosynthetic pathways were repressed in GRBaV-positive berries, while amino acid catabolic pathways were induced. Changes in carbohydrate metabolism were also observed; in particular, genes involved in glycolysis/gluconeogenesis and starch metabolism had reduced expression in GRBaV-infected berries. Most enzymes involved in phenylpropanoid metabolism are encoded by large gene families. There is also high redundancy among these genes, which ensures the functional integrity and plasticity of the phenylpropanoid-related pathways . Therefore, to test the hypothesis that the red blotch-induced transcriptional changes had an actual impact on phenylpropanoid metabolism, we measured the activity of key enzymes and the abundance of compounds involved in these pathways . We detected significant reductions in activity of seven enzymes that catalyze important steps in the core phenylpropanoid, stilbene, flavonoid, and anthocyanin biosynthetic pathways due to GRBaV infections of berries at three ripening stages . In addition, the first enzyme committed to flavone and flavonol biosynthesis, flavonol synthase , had significantly lower activity at post-véraison and harvest stages . Red blotch altered the accumulation of 17 compounds that result from the phenylpropanoid metabolism and two compounds upstream of this pathway, shikimic acid and gallic acid . Most of these compounds showed significantly lower abundance in the GRBaV-positive berries compared with the controls at later stages of ripening. The main anthocyanins present in grape berries: malvidin-3-O-glucoside, petunidin-3-O-glucoside, delphinidin-3-O-glucoside, pelargodin-3-O-glucoside, and cyanidin-3-O-glucoside, were significantly reduced by red blotch at harvest. Gallic acid, sinapic acid, and quercetin also showed lower abundance in infected berries. Few exceptions to this general suppression of phenolic accumulation were the accumulation of the precursor shikimic acid, which significantly increased in infected berries at harvest, and of resveratrol that showed significantly greater accumulation at véraison and postvéraison. Additional experiments are necessary to understand the accumulation of these two metabolites in the presence of GRBaV: preliminarily, we can hypothesize that the higher abundance of resveratrol is due either to a restriction of subsequent enzymatic steps in stilbene metabolism for which this compound is a substrate, or to the enzymatic hydrolysis of resveratrol glycosides or stilbenoid dimers previously synthesized. The integrated analysis of transcriptomic, metabolite, and enzyme activity data supported a general repression of the core and peripheral phenylpropanoid pathways, which are normally triggered in red-skinned berries throughout ripening and in response to stress . These results suggest that GRBaV infections disrupt secondary metabolic pathways by altering the regulation of berry ripening processes and/or signaling mechanisms related to plant defense. Interestingly, GRBaV infections seemed to have a more a pronounced impact on enzymatic activities and metabolite accumulation than on the expression levels of the genes in the pathway, which in general displayed small fold change differences between healthy and infected samples. This observation further confirms the importance of evaluating metabolic perturbations at multiple regulatory levels.Understanding how plants respond to external stimuli in the field is crucial to improve agricultural traits under naturally fluctuating conditions. Most studies on plant–pathogen interactions are performed with model organisms in the greenhouse or laboratory, which reduce the confounding effects of the environment, but also challenge the reproducibility of the results in the field . Compatible plant–virus interactions in perennial woody crops are complex due to the presence of multiple and systemic infections, tissue and developmental stage-specific responses, differences between species and cultivars, and the combination of biotic and abiotic factors during the crop season . The application of a system biology approach to study red blotch under multiple vineyard conditions allowed us to explore grapevine responses to GRBaV infections in real agronomic settings and to characterize the influence of viral activity on berry physiology.