Subsequently, the biochemical data from both V7-berries and V9-berries were correlated to the WGCNA modules, and only 2 modules, M21 and M30, displayed substantial correlations with berry polyphenols, containing 5349 and 4559 genes, respectively . The M21 module was positively linked with TAC , but negatively associated with tannins, catechin, and quercetin glycosides . On the contrary, the M30 module exhibited a positive correlation with tannins, catechin, and quercetin glycosides , but was negatively linked with TAC . The DEGs obtained from the two pipelines were assigned to both M21 and M30, yielding 604 and 1362 genes, respectively . Interestingly, the number of DEGs in each module, M21 and M30, was roughly equal to the down- and upregulated genes, respectively . To identify flavonoids/tannins-related genes that might result in such astringency diversity between V7-berries and V9-berries, hub genes were searched in the DEGs list of both modules . Only 8 hub genes were identified based on their transcript abundances in V9- berries and predicted functions. However, based on our previous work , 30 litre plant pots we found another 11 genes that are significantly expressed but with a log2FoldChange less than 1.5, and they were included in our further analysis .
The enrichment analysis of GO showed considerable enrichment in the BP GO terms for secondary metabolite biosynthetic process , flavonoid biosynthetic/metabolic process , L-phenylalanine metabolic/ catabolic process , phenylpropanoid metabolic process , phenylpropanoid biosynthetic process , chorismate biosynthetic/metabolic process , cinnamic acid biosynthetic/ metabolic process , anthocyanincontaining compound biosynthetic/metabolic process . The KEGG pathway analysis confirmed the BP GO terms, exhibiting enrichment for the biosynthesis of secondary metabolites , phenylpropanoid biosynthesis , flavonoid biosynthesis , and glutathione metabolism .To precisely elucidate their significance in the tannins/astringency diversity between V7-berries and V9-berries, we studied the expression levels of the 19 hub genes associated with the shikimic and flavonoids pathway. Except for the PAL1_1 gene , the analysis of their relative expression by real-time quantitative PCR showed a significant correlation with the Transcripts Per Million values for genes of interest, validating the transcriptomic data from both V7- and V9-berries . In general, all genes showed higher expression levels in V9-berries compared to V7-berries, but with different degrees of induction. Forinstance, the two genes involved in the shikimic acid pathway, chorismate synthase , and chorismate mutase , showed visibly higher accumulation abundance in V9-berries at the third harvesting time with approximately 6-fold and 3-fold increases, respectively, compared to V7-berries.
Similarly, the upstream structural genes in the phenylpropanoids pathway, including phenylalanine ammonia lyase , trans-4- coumarate biosynthesis , and 4-coumaroyl:CoAligase 2 , were significantly induced by approximately 2- to 9-fold in V9-berries. Regarding flavonoids/PAs biosynthesis, chalcone synthase is considered a key enzyme in this pathway, converting p-coumaroyl-CoA to naringenin chalcone, which is later turned into naringenin by chalcone isomerase . Both genes were highly expressed in V9-berries . Naringenin is subsequently converted by flavonoid 3’-monooxygenase to dihydromyricetin and dihydroquercetin, which are further transformed by dihydroflavonol 4-reductase into leucodelphinidin and leucocyanidin, respectively . The expression levels of F3H and DFR also showed a commensurate induction with the upstream genes in V9-berries relative to V7-berries. Subsequently, leucoanthocyanidin dioxygenase and leucoanthocyanidin reductase catalyse the conversion of leucodelphinidin to delphinidin and -gallocatechin, respectively, as well as leucocyanidin to cyanidin and catechin, respectively. These three genes also exhibited a significant increase in V9-berries. Finally, the expression of genes encoding glutathione Stransferases , one of the most essential anthocyanin transporters, was significantly higher in V9-berries compared to V7-berries, with approximately 3- to 9.2-fold changes .
Our data showed that the expression of flavonoids/PAs related-genes was highly increased in V9-berries at the third harvest time compared to V7- berries, resulting in the accumulation of more PAs in V9-berries.Developing and producing table grapes with high quality is of utmost importance for the success of grapevine breeding programs. Scarlet Royal table grape , variety , is one such success story, producing premium fruit quality and becoming one of the major red varieties in California. However, under certain unknown circumstances, the berry quality of Scarlet Royal grapes can be affected by undesirable astringent taste, which can negatively impact marketability and consumer acceptance. Research on the relationship between astringency and phenolic composition in table grapes is still scarce, especially on the transcriptomic level. In this study, we aimed to understand the molecular events involved in the development of berry astringency, which is a complex set of sensations resulting from the shrinking, drying, drawing, or puckering of the mouth epithelium . We focused on Scarlet Royal berries from two different vineyards with contrasting astringency and analyzed the changes in phenolic-related compounds at six different time points from veraison until the last harvesting time. Our panel test revealed that the V9-berries were perceived as more astringent, a characteristic that could be attributed to their elevated levels of tannins . Differences observed between the two vineyards under study indicate that V7 vines yield more compared to V9. This difference may be attributed to the lower cluster count in V9, a factor known to potentially contribute to astringency, as suggested by Cañon et al., 2014 in wine grapes. Additionally, petiole analysis revealed higher levels of nitrogen and potassium in V9 vines compared to V7. Vine nutrient levels contribute to the final berry quality at harvest . These factors may also contribute to the higher levels of tannins detected in V9 berries; however, further research is needed to confirm this theory. It’s worth noting that weather conditions can play a role in inducing astringency. Nevertheless, the two vineyards are located in close proximity to each other, and weather data collected from the same station in the Delano area indicates similar conditions. Therefore, it is unlikely that astringency or higher phenolic compounds are induced by weather factors. In fact, several studies, mainly in wine, have pointed to PAs as a determining factor for astringency intensity . For example, Vidal et al. reported that the total amount of tannins is the most plausible factor for sensory astringency, with flavan-3-ols dimers, trimers, and non-galloylated tetramers contributing to the astringency sensation. The PAs are a group of oligomers and polymers of flavan-3-ols and are the naturally occurring and predominant type of tannins in grapes and wine . Another study on aronia berry juice confirmed PAs as the key astringent compounds using sensory evaluation and phenolic profile approaches along with in-vitro models . The study found that PAs with higher degrees of polymerization were responsible for the strong astringent mouthfeel . The composition of phenolic substances, especially PAs, 25 liter pot plastic seems to play a crucial role in determining berry astringency, and further exploration of this relationship is warranted in fresh fruits of different species and cultivars. Understanding the molecular basis of astringency development in Scarlet Royal berries can provide valuable insights for improving grape breeding programs and enhancing the overall quality of table grapes.The relationship between astringency and the berry polyphenols content has not been explored yet in table grape at the molecular level. To the best of our knowledge, the present study provides the first transcriptome profiling along with the changes of polyphenols in grape berries of the same variety but having astringency diversity .
The transcriptome profile of both V7-berries and V9-berries underlined the remarkable transcriptional shift during berry ripening at different vineyards . Commonly, berry transcriptome profiles may widely vary based on many factors, including genotypic variations among varieties/species and developmental stages , as well as environmental circumstances. In our case of study, stemmed differences from the developmental stages and genotypic variations were eliminated, andhence the difference of the vineyard locations was the main source of variance with 97% of variance . The identified DEGs output of V9-berries compared to V7-berries is highly explanatory, including polyphenolic-related genes that are robustly expressed and co-regulated with astringency development, particularly in the V9-berries. The enrichment of the up-regulated genes with BP GO terms related to the biosynthesis of secondary metabolites, phenylpropanoid, and nitrogen compound metabolic process , commensurate with the higher amounts of N found in V9- berries . In fact, not only the levels of N fertilization but also its different forms highly affected the composition of phenolic compounds in leaves and wine . However, the synergistic/antagonistic effects of other macro- and micro-nutrients should also be considered. Our results highlighted the negative impact of above-normal amounts of macronutrients, mainly N, and to a lesser extent P and K, on the desirable attributes of grape berries. Otherwise, the positive effect of Ca, Mg, and Mn were achieved as their levels were maintained within the normal range . These data should be also seen in the light of the highly enriched BP GO terms in the down regulated genes . Particularly, those for hormonal signaling pathways such as auxin, abscisic acid, strigolactones, as well as the KEGG pathway for the carotenoid biosynthetic pathway .The transcriptome profiling identified the common and unique molecular events featuring the development of tannins/astringency in grape berries. It is well-documented that the synthesis of PAs in grapevines is achieved via three sequential pathways: the shikimate pathway, the phenylpropanoid pathway, and ultimately the flavonoid pathway . Our results revealed that the expression levels of flavonoids/PAs-related genes were highly induced in V9- berries at the third harvesting time compared to V7-berries. The 19 selected genes were involved in the three pathways: the shikimate pathway, phenylpropanoids pathway, and flavonoids pathway. The shikimate pathway is an alternative route to produce aromatic compounds, including phenylalanine, tyrosine, and tryptophan, which serve as precursors for various metabolites, such as phenolic compounds . The up-regulation of genes like chorismate synthase and chorismate mutase in V9-berries may lead to the accumulation of phenylalanine, which is a critical precursor for the phenylpropanoid pathway. The latter pathway is responsible for synthesizing several end products, including PAs, anthocyanins, lignin, lignans, hydroxycinnamic acid esters, and hydroxycinnamic acid amides . Under the conditions of the V9 vineyard, several PAs/flavonoids structural genes such as PAL, C4H, 4CL, CHS, CHI, F3H, LDOX, LAR, and ANR were induced in V9-berries, leading to the accumulation of PAs in the berries . This process is facilitated by GSTs and transported by multidrug and toxic compound extrusion transporters. The activation of the PAs biosynthetic pathway in V9- berries may lead to a reduction in the necessary substrates for anthocyanin synthesis, resulting in low red color intensity in V9- berries compared to V7-berries. Additionally, the accumulation of PAs is associated with the development of astringency taste in V9-berries. Our study provides valuable insights into the molecular events underlying astringency development in Scarlet Royal berries. By integrating transcriptome profiling with polyphenolic composition analysis, the research shed light on the co-regulation of genes involved in the shikimate, phenylpropanoid, and flavonoid pathways, leading to the synthesis of PAs and ultimately influencing astringency. The findings from this research have implications for grapevine breeding programs and the production of high-quality table grapes. Understanding the molecular mechanisms underlying astringency development can help breeders in selecting and developing grape varieties with desirable attributes. Additionally, the knowledge gained from this study can inform vineyard management practices, such as nutrient fertilization, to optimize polyphenolic composition and berry quality. In conclusion, the data presented in this study indicates that berry astringency is strongly correlated with a high tannin content, likely resulting from the activation of nineteen genes within the phenylpropanoid pathway. The activation of these genes shifts the flavonoid biosynthesis pathway towards proanthocyanins, leading to increased tannin accumulation in the berries. It appears that triggering these events is associated with nutritional imbalances and a lower number of clusters per vine, as confirmed by petiole nutrient levels and the observed lower berry soluble solids and higher titratable acidity levels. The identification of these genes holds significant value for table grape genetic improvement programs. The nutrient imbalance theory derived from this research could be applied worldwide to optimize grapevine fertilization programs. Furthermore, it paves the way for further research in this area, with a particular focus on vine nutrients, crop load management, and berry astringency, thereby contributing to advancements in the field of table grape cultivation.The names of the repository/repositories and accession number can be found in the article/Supplementary Material.The cultivated garden strawberry , an allo-octoploid , has a unique natural and domestication history, originating as an interspecific hybrid between wild octoploid progenitor species approximately 300 years before present.