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, best grow pots 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. In almost all viral diseases occurring in the vineyard, the virus is distributed systemically throughout the grapevine. Once introduced in the host, viral particles move rapidly within the vascular tissue towards sink tissues and establish infections . Although we detected the presence of GRBaV in vegetative and berry tissues during growing and harvest seasons, symptoms of red blotch were only evident after véraison, which suggests that the disease onset is mostly dependent on grapevine phenology and not necessarily linked to viral accumulation. Similar observations have been made during grapevine leaf roll disease, supporting the hypothesis that the appearance of viral disease symptoms in the vineyard may result from the interaction between pathogen and host cellular factors at specific phenological stages . Whether GRBaV is able to modulate its infection strategy as a function of plant development and/ or grapevines have distinct responses to red blotch throughout the season remains to be resolved. GRBaV shares several similarities with geminiviruses, including a small single stranded DNA genome that encodes six potential proteins . Because of their limited protein-coding capacities, geminiviruses rely heavily on host cellular machinery and interact with an assortment of plant proteins and pathways to promote infection . We confirmed the expression of five GRBaV genes in ripening red-skinned berries. Although three of these genes have been assigned putative functions in viral DNA replication and coat formation , their specific functions in pathogenesis are yet to be elucidated.
Our results indicated that the developmental stage of the berries may not influence the expression of GRBaV viral genes and that host factors could play a more critical role in the establishment of successful infections. Red blotch symptoms in grape berries include abnormal chemical composition and asynchronous ripening in the clusters , both reflecting alterations in host metabolic homeostasis and developmental processes. Interestingly, we determined that GRBaV infections induced some processes associated with early fruit development in berries at late stages of ripening, while repressing pathways involved in fruit ripening . Abiotic stress responses to hypoxia and temperature were also suppressed by red blotch in grape berries. These observations may imply that the virus, as a biotrophic pathogen, could redirect host metabolic processes to sustain higher energy demands due to viral replication and at the same time suppressing ripening related events and responses to stress , potentially counterproductive to viral infections. However, further evidence using infected berries from different grape cultivars and environmental conditions will be necessary to understand how both grapevine genotype and environment may influence the disease outcome. It is also important to consider that the effects of GRBaV infections on berries may not be comparable with those occurring in vegetative tissues; for instance, photosynthetic pathways are generally inhibited during viral infections of grape leaves . GRBaV infections restricted the biosynthesis and accumulation of phenylpropanoids and derivatives, which preferentially accumulate after véraison in red-skinned berries. These secondary metabolites function as antioxidants and phytoalexins to protect the berries against a variety of stresses, and are important contributors of berry quality parameters, such as color, flavor, and aroma . The inhibition of phenylpropanoid metabolism appears to bea hallmark of viral infections in red-skinned berries, as has previously been reported in leaf roll-affected berries . In particular, the anthocyanin bio-synthetic pathway was greatly impaired as a result of leaf roll and red blotch, correlating with the reduced coloration observed in GRBaV infected berries from several red-skinned cultivars . In this study, we demonstrated that GRBaV infections compromised the regulation of ripening by: suppressing specific ripening events; altering the expression patterns of transcription factors that control the transition from the growth to ripening phases and the activation of ripening pathways ; and causing hormonal imbalances. Most of the alterations in the ripening regulatory networks occurred in infected berries at véraison, the same developmental time when red blotch symptoms initiated, suggesting a link between mechanisms involved in the control of grapevine responses to viruses and berry development. Similar observations were made in leaf roll-affected berries, which presented lower expression levels of MYB transcription factors at late stages of ripening . Plant hormones modulate ripening processes and stress responses in grape berries. Red blotch had a profound impact on ABA, ethylene, and auxin pathways. ABA is considered the triggering signal of berry ripening, since its accumulation coincides with véraison and ABA responsive transcription factors have been implicated in the activation of ripening processes . Particularly, the induction of anthocyanin biosynthesis in a variety of grape cultivars has been linked to ABA-mediated signaling pathways . Our results indicate that alterations of ripening events in the GRBaV-infected berries, including the reduced anthocyanin content at late stages of ripening, plants in pots ideas could be a direct result of lower ABA levels at véraison. ABA has been shown to stimulate ethylene-mediated pathways in berries, and together both hormones appear to regulate the progression of ripening . GRBaV infections also affected genes involved in ethylene biosynthesis and responses , which could further account for the antagonistic effect of red blotch on ripening events and immune responses. In contrast, red blotch promoted auxin-mediated pathways, known to suppress berry ripening. Auxins play crucial roles in the early development of grape berries and are known to inhibit ripening processes by delaying ABA-triggered processes . The mechanisms by which grapevine viruses cause disease in ripening berries need to be investigated. GRBaV may actively interfere with the regulation of berry ripening by hijacking the plant post-transcriptional control.
We identified elements of the host post-transcriptional machinery that were misregulated in GRBaV-infected berries. Post-transcriptional regulation intersects plant immune responses, developmental transitions, and hormone signaling . Plant viruses, in particular geminiviruses, encode multiple silencing suppressors that interfere with host siRNA production and alter plant DNA methylation and miRNA pathways, causing developmental defects . A previous study on leaf roll indicated that the viral infection caused up-regulation of Dicer-like genes in ripening berries . Therefore, the relationship between the viral-induced repression of host RNA silencing processes and the post-transcriptional regulation of ripening should be addressed from the perspective of compatible grapevine– virus interactions. They usually occur in only a small proportion of a vineyard’s fruit , but in particular vineyards and years, shriveling can affect more than half of the crop . Most shrivel disorders make the fruit less desirable for wine making, with subsequent yield and production losses. Before taking steps to reduce the incidence of fruit shriveling in vineyards, it is necessary to differentiate between shrivel disorders. We describe four common causes of fruit shriveling and detail compositional differences between normally developing fruit and that affected by sugar accumulation disorder .Fruit exposed to direct sunlight for all or part of the day, especially in the heat of the afternoon, can be damaged by sunburn, which may be caused by high temperature, ultraviolet radiation or a combination of the two . The physical appearance of sunburned fruit depends on the grape variety and stage of development — white grapes and red grapes exposed before pigment accumulation begins develop brown discoloration, which varies depending on severity.Veraison and early post veraison red varieties with sunburn often exhibit poor color development, and may remain pink for the remainder of the season. Post-veraison sunburn leads to fruit with less color and a shiny appearance. Sunburned berries often crack, presumably due to damaged epidermal tissues. Extreme sunburn leads to complete berry desiccation and the formation of raisins in both red and white varieties. Sunburn only affects berries that are directly exposed to sunlight. The non-exposed side of a sunburned cluster often develops normally. If a cluster is fully exposed to the sun on both sides, or if the rachis itself is damaged, then the cluster may be completely affected. Obvious signs of sunburn may only occur on the exposed portions of individual berries. Sunburn can be avoided by reducing the fruit’s exposure to direct sunlight, especially in the afternoon. While leaves are removed in the fruit zone in many growing regions to increase cluster exposure to indirect light, in north-south row orientations leaves are removed on the east side of the canopy to reduce direct exposure in the afternoon and the probability of sunburn. This practice does not completely eliminate the risk of sunburn, however, because morning sun can also cause damage.Natural dehydration is another type of shrivel that may affect berries late in ripening but prior to commercial harvest. These berries appear similar to fruit with bunchstem necrosis, but the rachis look green and healthy. For this type of shrivel, which is especially pronounced in Syrah , berries lose weight due to water loss, and sugars are concentrated . Both increased transpiration and decreased phloem influx have been suggested as causes for late-season dehydration, but recent studies provide evidence that several varieties of grape berries remain hydraulically connected to the parent vine and therefore may lose water back to the parent plant late in ripening as well as to dry, ambient air .Clusters affected by bunchstem necrosis are identified by necrotic rachis tissue, with shriveled berries distal to the necrotic tissue. The visible symptoms of bunchstem necrosis begin as small black spots on pedicels , and progress to the lateral stem structure and rachis . Usually, necrosis symptoms are not noted until the rachis is affected. Bunchstem necrosis may affect an entire cluster as well as the wings and tips of otherwise healthy clusters .