To answer this question, RNA-seq data generated at one ripening timepoint was associated to the changes in polyphenolic levels using a systems biology approach, WGCNA . The module-trait association analysis positively correlated the key flavonoid/PAs biosynthetic genes with the accumulation of tannins, catechin, and quercetin glycosides exclusively in V9-berries. The modulation of the berry’s transcriptomic profile is concomitant with its polyphenols’ composition, which finally disturbs berry quality, including astringency levels.Five-year-old V. vinifera cv. Scarlet Royal grafted on Freedom rootstock was chosen for its berry astringency diversity at two commercial vineyards located in Delano, San Joaquin Valley, California, USA. Vineyards were located at a close distance of 10 km, and the local weather conditions during the two seasons were collected from the Delano CIMIS weather station . Both vineyards were planted at the spacing of 2.44 and 3.66 m in an open gable trellis supporting system with East-West row orientation.
Vines were pruned in a Quadrilateral cordon training with 7–8 spurs left on each cordon during the winter pruning. In addition, nft hydroponic general UC guidelines practices were applied in both vineyard. Random forty vines from different four rows from each vineyard were used in this study. Starting from veraison and until the end of the season, during two consecutive years . During the first year, sampling dates were July 8th , August 1st , August 10th , September 9th , September 15th , and October 19th ; and for the second year, sampling dates were: July 15th , August 10th , August 25th , September 10th , September 29th , and October 21st . Sampling dates varied from the first to the second year due to the vineyard’s accessibility. At each sampling point, two sets of fifty berries were collected periodically. The first set was used to measure the berry weight, and then these berries were macerated in an electric blender, filtered through a paper towel, and an aliquot of juice was used to determine soluble solids , pH, and titratable acidity . Soluble solids were determined using a tabletop Milwaukee MA871-BOX digital refractometer . The TA and pH were determined by titrating a 40 mL aliquot of juice with 0.1 N NaOH to a pH of 8.2 using an automatic titrator Excellence T5 .
Another random 50 berries from each replicate were collected for color, tannins, and phenolic compounds and sent immediately in a cooler to EST laboratories. At harvest, which was during the month of September, an extra set of samples was collected and promptly frozen in liquid nitrogen and stored at −80°C for subsequent analysis, including RNA extraction and gene expression studies. Harvest time was determined by the growers, and the marketable clusters were picked based on the color, and yield was determined from the three harvest dates.Scarlet Royal table grape is one of the major red varieties in California. Despite the premium fruit quality of the variety, in some cases, an undesirable taste was observed under certain unknown circumstances. To gain comprehensive insights into the development of the occasional berry astringency of Scarlet Royal and understand the underlying mechanism of this phenomenon, berries were investigated at two contrasting vineyards , both following the same commercial cultural practices. However,leaf petioles analysis of grapes from both vineyards showed considerable differences in nutrient levels, especially in the primary macronutrients . During both seasons, the amount of nitrogen in the form of nitrate in LP-V9 was roughly 2 to 3 times higher than the normal levels, in contrast to its counterpart in LP-V7, which slightly accumulated more or less N. Similarly, LP-V9 contained higher percentages of phosphorus and potassium compared to LP-V7 . Conversely, the amounts of secondary macronutrients, calcium and magnesium , in LP-V7 were within the normal range but greater than LP-V9, which showed Mg deficiency in the first year only.
Regarding the micronutrients, their levels were mainly within or around the normal range at both vineyards and during both seasons, with some differences . For example, zinc was slightly higher in LP-V9, especially in the first year. On the contrary, manganese and chlorine were roughly 2 times higher in V7 . Similarly, soil analysis shoed a higher level of nitrogen, potassium and magnesium . However, no significant difference was observed in all other soil macro and micronutrients. During the two seasons of the study, we determined the total marketable yield and the number of clusters in both vineyards. Our data revealed a higher yield in V7 compared to V9 in 2016 and 2017, respectively. The lower yield in V9 can likely be attributed to the smaller number of clusters in V9 compared to V7 during 2016 and 2017. To monitor the changes in the biochemical composition of Scarlet Royal berries, V7 and V9 berries were periodically sampled at six time points from veraison until the end of the season . The obtained data showed that berry polyphenols exhibited discernible patterns in both vineyards, most notably during the ripening stage . Of special interest were the tannin compounds, which widely affect organoleptic properties such as astringency and bitterness . Our data showed that berries from both V7 and V9 vineyards maintained lower levels of tannin from veraison up to the middle of August . Subsequently, a significant gradual increment of tannin took place. However, only V9-berries showed consistent accumulation of tannin over the two studied seasons compared to V7-berries, where the significant induction occurred only during the first season. It is worth noting that the levels of tannin were lower in both vineyards during the second year compared to the first season. Nevertheless, they were more pronounced in V9-berries compared to V7-berries, with roughly 2- to 4.5-fold increases by the end of the harvesting time during the two seasons, respectively . The patterns of catechin and quercetin glycosides were inconsistent during both seasons, particularly within V7-berries . During the first year, for instance, the levels of catechin were similar in both vineyards, showing a dramatic increase only by the end of the season . In contrast, during the second year, such induction of catechin was exclusively restricted to V9-berries, starting from time S3 . For quercetin glycosides, V7-berries exhibited significantly higher amounts at early stages during both seasons relative to V9-berries . However, subsequent amounts were comparable in both vineyards during the first season only , but not in the second one, where V7-berries showed a significant drop at the last sample S6 . Interestingly, the levels of quercetin glycosides were roughly equal at the last V9-berries sample between both seasons despite such inconsistency. For total anthocyanins , the levels in early samples were comparable in both vineyards and seasons . Afterwards, their pattern started to vary between V7 and V9 within the same season, as well as from the first season to the second, as the nutrient amounts fluctuated as well . Nevertheless, TAC accumulation was positively correlated with the progress of ripening in V7-berries, but not V9-berries. To further confirm our data, we measured these phenolic compounds for the third time in mid-September of the next year . Overall, the results showed that the patterns of tannins and TAC were reciprocally inverted between V7-berries and V9-berries as ripening advanced. In addition, both catechin and quercetin glycosides most likely followed the pattern of tannins despite their seasonal fluctuations. To further distinguish V7-berries and V9-berries and assess their astringency development, a panel test was performed using samples at three commercial harvest times . A group of 12 nontechnical panelists scored berry astringency on a scale from 1 to 7, where 1 is extremely low and 7 is extremely high. The panelists were trained using samples from contrasting standard varieties, including Flame Seedless and Crimson as non-astringent and Vintage Red known for its astringent taste . The results showed that V7-berries exhibited lower intensity of astringency compared to V9-berries . As ripening proceeded, nft system astringency levels increased in V9-berries, but decreased in V7-berries.
Moreover, we collected samples from clusters with various astringent taste and measured its tannins content. We were able to determine that the threshold level of tannins that causes the Scarlet Royal astringency taste is around 400 mg/L . Taking into account the levels of polyphenol compounds and the taste panel data together , it is evident that astringency development is positively associated with tannins’ accumulation throughout the ripening process of V9-berries. Nevertheless, organoleptic analysis revealed a significant difference in the berries of the two vineyards, particularly in terms of total soluble solids and titratable acidity . Notably, V9 berries exhibited higher titratable acidity and lower total soluble solids, especially in the later stages . To better understand the molecular events associated with the induction of tannins and astringency upon ripening, the berry transcriptome profile was analyzed in both V7-berries and V9- berries at the late commercial harvest date . Following the quality and quantity check, extracted RNA from quadruplicate samples was deeply sequenced . Of the 19.7 to 24.4 million high-quality clean reads per replicate, 61.9% to 66.1% were mapped against the V. vinifera transcriptome . Hierarchical clustering of the RNAseq data showed explicit changes in the berry transcriptome profile between V7- berries and V9-berries . The Principal Component Analysis showed high consistency among biological replicates . Samples were mainly separated along the first component , which was responsible for 97% of the variance, and was definitely associated with the site of cultivation; V7 and V9. In contrast, the second component was trivial, accounting for only 1% of the variance and was probably attributed to experimental error. Such results were expected, as berry samples came from the same cultivar, Scarlet Royal , and the only difference between them was the vineyard locations. To identify the differentially expressed genes in V7- berries and V9-berries at this specific time within the ripening window, the RNAseq data were analyzed using two different Bioconductor packages, DESeq2, and EdgeR . Subsequently, the DEGs with FDR < 0.05 and log2fold change > 1.5 or < –1.5 generated by both pipelines were considered . The pairwise comparison between berry transcriptomes resulted in 2134 DEGs, with 1514 up-regulated and 620 down-regulated . The data manifested the impact of the cultivation site on the transcriptional reprogramming of a large number of genes that ultimately affect berry quality. Most apparently, at the V9 vineyard, where roughly 2.5-fold higher number of berry transcripts were upregulated compared to V7 . Subsequently, the enrichment of Gene Ontology terms and Kyoto Encyclopedia of Genes and Genomes pathways were analyzed among the up- and down-regulated DEGs using the Vitis vinifera Ensembl GeneID . Among the significantly enriched GO terms, the up-regulated transcripts in V9-berries exhibited high enrichment in the molecular function GO terms for quercetin 3-O-glucosyltransferase activity and quercetin 7-Oglucosyltransferase activity . Additionally, the V9-berries induced DEGs were highly enriched in the biological process GO terms for the jasmonic acid signaling pathway and cellular response , Lphenylalanine metabolic process , L-phenylalanine biosynthetic process , and nitrogen compound metabolic process . Similarly, these DEGs were highly enriched in the KEGG pathways for the biosynthesis of secondary metabolites and phenylpropanoid biosynthesis . On the other hand, the down-regulated transcripts in V9-berries showed substantial augmentation in the MF GO terms for hormone binding , abscisic acid binding , and potassium ion transmembrane transporter activity . Correspondingly, the BP GO terms for hormone-mediated signaling pathway and response , auxin-activated signaling, cellular response, and homeostasis , abscisic acid-activated signaling, response, and cellular response , response to strigolactone , potassium ion transmembrane transport , and potassium ion transport , as well as the KEGG pathways for plant hormone signal transduction , brassinosteroid biosynthesis , and carotenoid biosynthesis were highly enriched in the down-regulated genes of V9-berries . Overall, the transcriptome analysis pointed out the substantial changes in transcript abundance that coordinate and reflect the observed induction of tannins/astringency during the maturation and ripening of V9-berries compared to the V7-berries .To elucidate which fundamental processes were altered during tannins/astringency induction within berries, the Weighted Gene CoExpression Network Analysis was applied to construct coexpression networks. Forty-two modules were identified based on pairwise correlations among the 17553 non-lowly expressed genes . 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 .