The signature raspberry flavor comes from aromatic volatiles, mostly composed of a mixture of ketones and terpenes . α-Ionone and β-ionone are carotenoid-derived aromatic volatiles that are mostly responsible for floral notes in fruit ; these compounds usually intensify as raspberries ripen. Also, α-terpineol, which has a sweet, flowery aroma was found to have a positive correlation with sweetness in our study. Aromatic volatiles become prominent during fruit ripening and tend to increase towards senescence, ultimately developing the aroma and flavor for the fruit . Guichard reported that in raspberries all the terpenes and sesquiterpenes concentrations significantly increased during ripening with an increase in α -ionone followed by a slight increase in β-ionone. In our research, α-ionone concentration increased over time in air and 5 kPa atmosphere storage, but decreased in raspberries stored in 15 kPa atmosphere, perhaps due to slowing of further ripening. α-Terpineol, limonene, linalool and hexanoic acid also decreased 3-4-fold with increasing CO2 concentration in storage. Linalool, α-terpineol, limonene and hexanoic acid showed increases in concentration with time in air storage. In our study, the aromatic volatiles were mostly associated with raspberries stored in lower CO2 atmospheres . This may be largely due to little or no inhibitory effects on further ripening in these atmospheres. Ripening of fruit is usually accompanied by softening and production of flavor and aroma volatiles Some fruity/floral volatiles are known to enhance the perception of sweetness . Volatiles such α-ionone, linalool, square pot and α-terpineol have a sweet floral aroma . This may explain why we observed a positive correlation of sweetness with these particular volatiles in our study.
While raspberries held in higher CO2 atmospheres had lower concentrations of aromatic volatiles, most, but not all, of the differences can be explained by ripening inhibition. High CO2/low O2 atmospheres also restrict enzyme activity, diminishing generation of certain organic volatiles, and reducing the effects of ethylene on CA-stored produce . Off-flavor’s association with low CO2 atmosphere storage may be related to the concentration of limonene which was higher in low CO2 stored raspberries and positively correlated with off-flavor. Elmaci et al. also reported an association of off-flavor with increasing percentage of limonene during storage of mandarins. It is possible that off-flavor was also linked to development of decay or leakiness because the rate of decay and leakiness was higher in fruit stored in low CO2 atmospheres due to the lack of fungistatic conditions or inhibition of metabolism. However, raspberries stored in 8 kPa atmosphere performed better in sensory evaluations in terms of raspberry flavor, juiciness, and sweetness. Raspberries stored in air or 5 kPa atmosphere lost almost all their sensory quality by 10 days. Selection of modified atmospheres for raspberries should be based on the storage time and desired quality. While 15 kPa atmosphere prolonged shelf life the longest, 8 kPa atmosphere prolonged shelf life to 10 days while maintaining sensory quality. Based on these findings, modified atmosphere conditions can be formulated and applied during transportation to further investigate the impacts on quality under commercial conditions. Also, synthesis of volatile compounds and associated gene expression as effected by high CO2 atmospheres would be an interesting area for further exploration.
Grapevine leaf roll-associated viruses are among the most consequential pathogens affecting grapevine and have considerable economic impact . GLRaVs are diverse and belong to the family Closteroviridae, with six species and numerous strains in three genera . Grapevines are often infected with several of these viruses simultaneously . Given their impact and global distribution, efforts to manage the spread of GLRaVs, characterize their effects, and understand the interaction between the vine and GLRaVs have been undertaken. Generally, plant responses to viruses include numerous changes in gene expression, gene regulation, and metabolism . Pathogens and stresses elicit conserved responses from their hosts . Infections with GLRaVs have been associated with poorer fruit quality, lower yield, and leaves that curl, redden, and become brittle. Gene expression studies that implicate regulatory systems in the leaf roll disease phenotype are few in number and have focused on the impact of GLRaV-3, highlighting changes in the expression of senescence-associated and flavonoid bio-synthetic pathway genes . Additional transcriptomic study could help generate novel hypotheses concerning the controls that are fundamental to GLRaV responses . Though common responses might be expected in infected plants given the relatedness of GLRaVs, there is considerable variability in the severity of GLRaV infections. Some GLRaV infections appear without symptoms or are mild , but others cause significant changes in photosynthesis, metabolism, and gas exchange in leaves . Changes in fruit yield, organic and amino acids, titratable acidity, potassium, sugars, and flavonoids are also observed .
These are influenced by host genotype , which virus or combination of viruses is present , and environmental conditions . Leaf reddening, for example, is only observed in red-fruited grapevines . Evidence relating GLRaV responses to rootstock is mixed . In a study of Cabernet Franc vines grafted to different rootstocks, the effect of GLRaV infection on pruning weight depended on rootstock and the largest effects were observed in Kober 5BB-grafted vines . Similarly, fruit yield was influenced by both infection type and rootstock, with Kober 5BB-grafted vines most severely affected by a mixed infection with GLRaV-2, GLRaV-3, and grapevine fleck virus . In another report, Red Globe scion buds infected with a strain of GLRaV-2 were used to inoculate Cabernet Sauvignon plants grafted to 18 different rootstocks; the infection was lethal in plants grafted to several rootstock genotypes, including Kober 5BB . This study used Cabernet Franc grapevines infected with zero, one, or two GLRaVs and grafted to two different rootstocks to identify leaf roll effects in ripening berries that were conserved across experimental conditions, and determine whether or not GLRaV responses could be distinguished in berries from plants grafted to different rootstocks. Grapevines were grown in a single experimental vineyard and evaluated in four consecutive years. Vine growth and several measures of fruit composition were taken in the first two years. Total soluble solids were measured in all four years. RNA sequencing , hormone, and metabolite data were collected from Cabernet Franc berries at four stages during ripening in the third and fourth years. RNA-Seq reads were mapped to the Cabernet Franc genome, which was sequenced in long PacBio reads, assembled using the FALCON-Unzip pipeline, and scaffolded using Hi-C data. The same samples were used to measure the levels of stress and ripening-associated hormones and metabolites. Among these were abscisic acid , jasmonic acid , and salicylic acid . Though many of the GLRaV effects occurred in individual years, a subset of reproducible conserved responses and rootstock differentiating responses were discovered.Cabernet Franc grapevines infected with individual and pairs of GLRaVs and grafted to different rootstocks were studied during grape berry ripening in a dedicated experimental vineyard at the University of California, Davis. Typical grapevine leafroll disease symptoms were observed by mid-ripening . In addition, square plastic planter there was a visible, stark reduction in canopy density and cluster size in GLRaV-1,2 versus GLRaV in vines grafted to Kober 5BB that was not readily apparent in vines grafted to MGT 101-14 with the same infection status . Vine growth, cluster weight, and other measures were collected in 2015 and 2016 . The effect of GLRaV infection on dormant pruning weight, berry weight, pH, and tartaric acid content in 2015 and on moisture content, total anthocyanin content, and titratable acidity in 2016 differed significantly based on the rootstock present . This interaction was significant for malic acid in 2015 and 2016. Significant differences in dormant pruning weight, total cluster weight, and tartaric acid were observed in plants with different GLRaV infection status and rootstock . In contrast, few or no significant differences between GLRaV given the same rootstock were observed for total anthocyanins, moisture content, malic acid content, pH, titratable acidity, weight per berry, or yeast assimilable nitrogen and overwhelmingly in a single year if at all . Overall, GLRaV infection tended to reduce dormant pruning weight and cluster weight. The dormant pruning weights and cluster weights of GLRaV-1,2 was significantly lower than those of GLRaV and other GLRaV ; this was observed for both rootstock genotypes. Significant differences in fruit tartaric acid levels were observed only in 2015 and were between GLRaV-1,3 grafted to different rootstocks and between plants with different GLRaV infection status . In each year except 2015, there was a significant interaction between rootstock and GLRaV infection status in terms of TSS at harvest . This interaction was significant at each other developmental stage in 2017 and at prevéraison in 2018 . Significant differences in TSS at harvest were found between GLRaV and GLRaV in each year except 2017 . Overall, significant reductions in TSS relative to GLRaV were limited to the dual infections and GLRaV-3 . Significant differences were observed between rootstocks in GLRaV 1,2 at every developmental stage, albeit only in 2017 . These data provide limited evidence that different GLRaV infections may or may not affect various aspects of vine growth and fruit composition, some of these differences are rootstock specific, and although some of these effects are observed across years, year-to-year differences may impact whether or not effects Occur.
We used RNA-Seq to sequence the transcriptome of 384 Cabernet Franc berry samples collected from plants grafted to different rootstocks , with different GLRaV infection status, at four developmental stages , and in two consecutive years . Because of the remarkable structural and gene content variability among grape cultivars , we built a genome reference specificallyfor the analysis of these RNA-Seq data. The Cabernet Franc genome was assembled into 504 primary contigs for a total assembly size of 570 Mb. This is comparable to the size of the Zinfandel , Cabernet Sauvignon , Chardonnay , and Pinot Noir PN40024 genomes. In total, 3,085 additional haplotigs were assembled with an N50 of 184 kb . The primary assembly and haplotigs were annotated with 33,563 and 19,146 protein-coding genes, respectively . Ripening was associated with transcriptomically distinct developmental stages. Samples clustered primarily by developmental stage and secondarily by year, though samples at harvest clustered separately . Genes with comparable, significant responses in both years of the study were selected to identify reproducible responses to GLRaVs during ripening. Gene expression in GLRaV was compared to gene expression in GLRaV grafted to the same rootstock at the same developmental stage . In addition, the effects of each GLRaV infection on gene expression at each developmental stage were compared in plants grafted to different rootstocks . On average, 7.1% of the genes differentially expressed between GLRaV and GLRaV were reproduced in both years . This percentage was slightly above average for plants with dual, relatively more severe, infections and below average for individual infections . A subset of 32 genes significantly changed their expression level in two or more GLRaV infection conditions, in both rootstock conditions, and at least one developmental stage . These genes constitute the “conserved” responses to GLRaVs in Cabernet Franc berries during ripening. The majority of these differentially expressed genes are associated with defence, ABA signalling, and cytoskeleton organization and biogenesis . Six of these were genes encoding nucleotide-binding site and leucine-rich repeat-containing proteins; half of these were upregulated. Two F-box genes encoding SNIPER4 were upregulated , as was a gene encoding a hydroxyproline-rich glycoprotein . HRGP and NBS-LRR proteins are associated with pathogen detection . Genes encoding a respiratory burst oxidase protein D , a wall-associated kinase-like protein , and a β-glucosidase 3 were downregulated. RBOHD participates in the production of reactive oxygen species and hypersensitive responses to pathogens . RBOH family proteins are targeted by Snf1-related kinase 2 phosphorylation, a key component of the ABA signalling pathway. Likewise, a WAKL gene in citrus participates in JA and ROS signalling . Among the functions of β-glucosidases are the activation of ABA and SA by freeing them from the conjugates that render them inactive . Several ABA-related genes were among the conserved GLRaV responses, including an upregulated ABC transporter and two downregulated genes, AMP1 and RDA2. AMP1 negatively regulates ABA sensitivity . RDA2 participates in the inhibition of ABA signalling and the promotion of MAPK signalling . Five genes related to cytoskeleton organization were sensitive to GLRaV infection. Only one of these, a myosin VI motor proteincoding gene, was downregulated.