The slight difference in mapping rate between the two references can be explained by either the absence of some low-expression transcripts in the ISNT or the residual error rate in isoform sequences. Gene expression analysis using the ISNT as reference showed similar results compared to the Cabernet Sauvignon genome assembly, with a very high correlation of expression level and differential gene expression, and with similar global transcriptomic changes. However, we observed differences in the number of expressed and differentially expressed features that depend on the reference used. Those differences could be explained by the diploid phasing of the Cabernet Sauvignon genome assembly and that multiple ISNT transcripts might correspond to a single gene locus. Nonetheless, similar relative amounts of Biological Process GO terms were found among the differentially expressed genes, confirming that the transcriptome obtained using Iso-Seq captured the transcriptional reprogramming underlying the main physiological and biochemical changes during grape berry development. In addition, gene expression analysis revealed that some private isoforms are significantly modulated during berry development, square pots for plants indicating that in addition to identifying the private gene space, the ISNT reference makesit possible to observe its expression.
In conclusion, this study demonstrates that Iso-Seq data can be used to create and refine a comprehensive reference transcriptome that represents most genes expressed in a tissue undergoing extensive transcriptional reprogramming during development. In grapes, this approach can aid developing transcriptome references and is particularly valuable given diverse cultivars with private transcripts and accessions that are genetically distant from available genome references, like the non-vinifera Vitis species used as rootstocks or for breeding. The pipeline described here can be useful in efforts to reconstruct the gene space in plant species with large and complex genomes still unresolved.Grapevines are susceptible to the highest number of pathogens to infect a single crop, with over 70 viruses detected. In 2008, a new virus was first observed in Napa County, California, which economically threatened grapevines: grapevine red blotch virus. This virus is the causative agent of grapevine red blotch disease, which has been identified in vineyards across the United States, Canada, Argentina, Mexico, South Korea, and India . Reports indicate GRBV primarily spreads through propagation material and secondarily through an insect vector. Spissistilus festinus was shown to successfully transmit GRBV in greenhouse settings, yet this has not been replicated in vineyards to date.
GRBV has been identified as a virus from the Geminiviridae family containing a circular single-stranded DNA genome similar to other geminiviruses. GRBD expresses symptoms of reddening of leaf blades and margins, with reddening of the primary, secondary, and tertiary veins in red grape cultivars. GRBV affects grapevines in various ways. For example, leaves on infected vines show increased levels of sugar, phenolics, particular amino acids, and enzymatic activity related to plant defense, as well as a reduction in carbon fixation. However, the most damaging are the effects on grape composition which has been shown to be detrimental to final wine quality. GRBV delays ripening by decreasing the accumulation ofsugar and anthocyanin in berries, potentially due to the impairment of translocation mechanisms. The virus has variable impacts on primary and secondary metabolites, specifically phenolic and aroma compounds. In summary, detrimental economic impacts to vineyards in the United States could reach $68,548/ha with vine removal being the only current method of alleviation. Consequently, recent research has strived to understand the effects and functioning of GRBV to establish mitigation strategies to alleviate the impact on grape composition and wine quality. The first phase is characterized by cell division and production of seeds, as well as synthesis of tannins and organic acids. The second phase is characterized with the onset of veraison, which is when the grape berry begins to soften and change color. The final and third phase is berry engustment/ripening, where berries increase in size, sugar accumulates, acidity declines, and secondary metabolites such as anthocyanins and aromatic compounds are synthesized inside the berry.
Studies have shown that volatile compounds such as terpenoids and C6 compounds, begin to accumulate in berries after veraison, and are controlled by numerous factors. The synthesis of these compounds in berries is also altered by external factors such as light exposure or pathogens. In addition, these secondary metabolites are crucial to grape growers and winemakers due to their importance in the quality of a final wine. Grape maturity has shown to be a key driver in the composition of a final wine, where later harvested fruit produces wines with lower concentrations of C6 alcohols and higher in concentration of esters. However, the impacts of GRBV on volatile compound abundance in harvested grapes has not been investigated. A plant’s genetic material may influence susceptibility to viral infections. Additionally, rootstocks can impact grapevine physiology and impact the overall composition of a grape berry. For instance, rootstock 110R causes high vigor and high drought tolerance in grapevines; whereas 420A is a rootstock of low to moderate vigor and low drought resistance. Vigor, resulting in greater shoot length and hence leaf area, may impact net carbon assimilation and the translocation of metabolites into the berry, consequently affecting the final wine composition. These hydric differences affecting carbon metabolism in rootstocks can also impact the plant-pathogen interactions. Therefore, it is plausible that severity of GRBD symptoms will be dependent on the interaction between scion cultivar-rootstock. However, this has not been fully investigated. Macro and micro climate fluctuations may also be a factor in pathogen-plant interactions, and should be considered. This study investigated the impact of GRBV on the biosynthesis and accumulation of primary and secondary metabolites in grape berries throughout ripening and at harvest. Additionally, the influence of seasonal and genotypic factors on disease expression within grapevines were studied.Figures 1–3 depict sugar accumulation, anthocyanin levels, TA, and pH through ripening. Sugar accumulation was determined by converting ◦Brix to mg of sugar per berry. Anthocyanin content was lower in RB. grapes when compared to RB grapes for both years and rootstocks during ripening . However, the degree of impact varied depending on season and rootstock. In 2016, both rootstocks were equally impacted throughout ripening regarding sugar accumulation and anthocyanins levels. However, in 2017 sugar accumulation was generally not significantly impacted by disease status. In 2017, grape anthocyanin levels were more significantly impacted for infected vines on 110R rootstock than 420A rootstock, whereas rootstock impact was less apparent in 2016. At harvest in 2016 , CS 110R and 420A rootstocks, respectively, had a 2% and 11% decrease in anthocyanin content and a 12% and 18% decrease in sugar content in RB grapes when compared to RB grapes. In 2017, atharvest , anthocyanin content was 35% and 11% lower in RB when compared to RB , and sugar content was 9% and 7% lower, for 110R and 420A, respectively. By plotting ◦Brix over ripening and fitting a linear trendline, it is possible to compare the rate of ripening for RB and RB grapevines .
As indicated by the slope of the best fit line, the rate of ripening was always higher for RB data vines when compared to RB data vines, with the exception for CS 420A in 2017. In addition, large square plant pots the rate was also lower in 2016 than 2017 across virus status and rootstocks. Interestingly, the difference in the rate of ripening between RB and RB data vines was larger in 2016 than in 2017 which correlates to the larger differences in accumulated sugar at harvest. In 2017, the rate was lower for CS 420A than CS 110R across virus status.The volatile compound profiles of RB and RB grapes were determined in both 2016 and 2017 seasons . PCA was performed to plot the variability between RB and RB grape samples . Between 80.6- 94.5% of the variance is explained by the PCA in Figures 4 and 5. For CS 110R, only the significantly different volatile compounds between RB and RB are plotted. There were, respectively ten and nine significant volatile compounds that explained the difference between treatments for CS 110R in 2016 and 2017. For CS 420A, the volatile compounds that contributed most to the variance of the PCA were plotted, due to few volatile compounds being significantly different. This selection was based on the squared cosine which shows the importance of the volatile compounds to explain the variance in the data. For CS 420A, in 2016, only cis-3-hexen-1-ol was significantly different, and in 2017, only β-linalool and β-citronellol were significantly different with a α level of 0.95.After veraison, volatile compound accumulation begins in grapes and changes through ripening. However, the impacts of GRBV on grape volatile compounds have not been investigated. We found 35 different aromatic compounds in grapes from the two rootstocks over two seasons, of which 24 were similar between the two years studied . In 2016, across rootstock, RB grapes were generally lower in volatile compound levels than RB , except for C6 compounds such as 2-hexenal, hexanal, cis-3-hexen-1-ol, trans-2-hexen-1-ol, and hexanol . These C6 volatile compounds are synthesized in the grape skin through the lipoxygenase pathway, are generally responsible for green or grassy aromas and accumulate in CS grapes up to 18 ◦Brix . With the exception of hexanol, the levels of these compounds begin to significantly decrease thereafter, with a 67% decrease in grapes at 25 ◦Brix when compared to grapes at 18 ◦Brix. These observations correlate with the common finding that GRBV causes a delay in ripening, with green aromas being present and correlated with the lower sugar accumulation. On the other hand, RB grapes were highly correlated with monoterpenes such as limonene, ß-myrcene, α-terpinene, geranial and p-cymene , which are responsible for floral and fruity aromas. These compounds have been associated with CS grapes at harvest and are known to increase through grape ripening and may decrease at over ripeness. In addition, RB grapes were also highly correlated with esters such as ethyl acetate, ethyl hexanoate, hexyl acetate, and ethyl butyrate. Although ester formation is mainly related to yeast or bacteria metabolism during winemaking , grapes are also known to synthesize esters. Anthraniloyl-coenzyme A :methanol acyltransferase is known to be responsible for the formation of methyl anthranilate in grapes and it is also classified as an ester-forming acyltransferase, which could be responsible for the formation of esters in grapes. The esters found in the current work that in general related mostly to RB grapes, are known to produce red and black fruit aromas. Collectively, these results confirm that RB grapes underwent normal ripening processes and produced more fruity aromas, while RB grapes at harvest have aroma characteristics more related to early ripening stages.Results indicated that in 2016 GRBD had a larger impact regarding sugar accumulation, pH, TA, and final sugar content than in 2017. In addition, the harvest dates were two to three weeks later in 2017 than in 2016. These observations can potentially be explained by the difference in temperature between the two seasons. In 2017, Napa County experienced a heat wave from 26 August–11 September, where nine days were over 35 ◦C, and four days were over 40 ◦C. The cumulative growing degree days for both years can be seen in Figure 1e. Extreme heat conditions during grape maturation have been shown to inhibit enzymatic activity and halt the biosynthesis of metabolites inside the grape berry. Inhibition of these processes due to heat leads to decreases in sugar accumulation and increases in acidity in healthy fruit. This is thought to be caused from a decrease in rate of translocation of sugars from leaves to fruit, through the reduction in photosynthesis at temperatures greater than 30 ◦C. The rate of ripening in 2017 was faster than 2016prior to the heat spike . However, during the heat spike in late August to harvest, sugar accumulation plateaued resulting in extended harvest times in 2017. In addition, research has shown that temperature can alter virus-induced gene silencing which is triggered with the infection of a virus as a plant-derived defense mechanism to downregulate the genes of interest. Previous work on other plant species infected with a geminivirus has shown that the extent of gene silencing is related to temperature. Specifically, Chellappan et al. showed that temperatures over 30 ◦C induced gene silencing, which interfere with gene expression, resulting in decreases in viral DNA accumulation and decreases in symptoms.