Tissue differences were more revealing at the stage when flavors peak; 4865 transcripts that were significantly higher in skins compared to pulp at 23.2 °Brix were tested for over represented GO functional categories . Some of the top GO categories included photosynthesis, isoprenoid biosynthesis, and pigment biosynthesis . Some of the transcripts with the largest differences between skin and pulp at 23.2 °Brix are β-ketoacyl-CoA synthase , taxane 10-β-hydroxylase , wax synthase, a lipase, an ABC transporter, and phenylalanine ammonia-lyase . The abundance of 5716 transcripts was significantly higher in pulp than skin at 23.2 °Brix . Some of the top GO categories over represented were a variety of transport processes and small GTPase mediated signal transduction . Some of the transcripts with the largest differences in abundance with pulp greater than skin at 23.2 °Brix were polygalacturonase , flavonol synthase, stachyose synthase, an amino acid transporter, a potassium channel , and HRE2 . The transcript abundance of 2053 genes had significantly differential expression across °Brix levels and tissues . The top GO categories over represented in this set involved photosynthesis and phenylpropanoid metabolism, both associated with the berry skin .
Other flavorcentric categories of the 57 categories over represented include aromatic compound biosynthesis, 10 liter drainage pot fatty acid metabolism and alcohol catabolism. This transcript set was further analyzed by dividing into 10 clusters using k-means clustering . The over represented GO categories were determined for each cluster . Eight of the 10 clusters had distinct over represented GO categories; two clusters did not have any over represented GO categories, meaning that the genes in these two clusters were assigned to GO categories of expected proportions when compared to the entire NimbleGen array. Clusters 1, 8, 9 and 10 had a large number of over represented categories. Many GO categories within a cluster are subsets of others in that cluster and were grouped together. For example, cluster 4 had four over represented GO categories, oxygen transport, gas transport, heat acclimation and response to heat. The four categories could be grouped into two, as two are subsets of the others; this is how they were listed in Table 1.It would be impossible to discuss here all the transcript abundance changes detected in these berries. As we were interested in compounds associated with berry flavors as they develop or change in the late stages of berry ripening, we took a more targeted approach for analysis with this in mind.
Berries at 24° Brix are known to be near-optimal for flavor, thus we took a simple approach to look for genes that were peaking around this stage. We found some significant and large increases in transcript abundance between the 22.6 and 23.2 °Brix levels. A group of VviERF6 transcription factor paralogs represented 6of the top 10 transcripts increasing in transcript abundance from 22.6 to 23.2 °Brix in the skin, but not in the pulp . These VviERF6 TFs were also found in Cluster 8 . This is very interesting since many flavor compounds are derived from the skin and ERF TFs are known to be responsive to ethylene, a known fruit-ripening hormone. These VviERF TFs were named ERF105 in the annotation by Grimplet et al. , however they are more orthologous with AtERF6 as determined by a more comprehensive phylogenetic method using many plant species at Gramene . Annotation details of the V1 gene models of the VviAP2/ERF superfamily can be found in Additional file 8 including updated Vvi symbols according to its closest Arabidopsis ortholog as instructed by the Grapevine Gene Nomenclature System developed by the International Grape Genome Program Supernomenclature committee. This renaming of the AP2/ERF super family should facilitate comparative analyses and functions with other species, particularly Arabidopsis. To properly annotate the AP2/ERF super family of Vitis vinifera according to the IGGP Supernomenclature committee instructions, a phylogenetic tree was generated for the AP2/ERF super family of Arabidopsis thaliana and Vitis vinifera using the TAIR 10 and V1 gene models, respectively .
The labeled family classifications were derived from the Arabidopsis naming scheme by Nakano et al.. There are 130 members in the VitisAP2/ERF superfamily in the Pinot Noir reference genome. However, the six paralogs of ERF6 discussed above belong to a Vitis vinifera clade in subfamily IX and are distinctly different or separate from any Arabidopsis subfamily IX ERF TFs . All of these TFs in this clade are orthologs of AtERF6. VviERF6L1 [UniProt: F6I2N8; VIT_16s0013g00900] had one of the most interesting profiles of the 12 members of this clade because its transcript abundance peaked at 23.2 °Brix . Using k-means clustering, VviERF6L1 fell within Cluster 8 with 369 transcripts, including five additional VviERF6 paralogs. The top GO categories associated with Cluster 8 were genes associated with terpenoid metabolism and pigment biosynthesis . Other interesting flavor associated categories included fatty acid and alcohol metabolism . Representative transcripts from Cluster 8 that were correlated with the transcript abundance profile of VviERF6L1 can be seen in Figure 4. These are ACC oxidase, which is involved in ethylene biosynthesis; a lipoxygenase, part of a fatty acid degradation pathway giving rise to flavor alcohols such as hexenol; α-expansin 1, a cell wall loosening enzyme involved in fruit softening, and two terpene synthases, which produce important terpenes that contribute to Cabernet Sauvignon flavor and aroma. The high similarity of these transcript profiles indicates that ethylene biosynthesis and signaling may be involved in the production of grape aroma. Supporting this argument, two recent studies have shown that a tomato ERF TF , falling in the same ERF IX subfamily, has a strong effect on ethylene signaling and fruit ripening. The transcript abundance of AtERF6 in Arabidopsis is strongly increased by ethylene, which is triggered by the MKK9/MPK3/MPK6 pathway. The transcript abundance of VviMKK9 in the Cabernet Sauvignon berries was higher in the skin than the pulp, but there were no significant differences for VviMPK3 or VviMPK6 . This is not too surprising since AtMKK9 activates AtMPK3 and AtMPK6 by phosphorylation. In addition, the transcript abundance of AtERF6 in Arabidopsis increases with ROS, SA, cold, pathogens, and water deficit. There were no visible signs of pathogen infection in these berries. Additional circumstantial evidence for ethylene signaling in the late stages of berry ripening was that the transcript abundance of many VviERF TFs was significantly affected by berry ripening and/or tissue . The transcript abundance of 129 members from the berries was determined to be above background noise levels on the microarray . The expression profiles of the 92 significantly affected AP2/ERF superfamily members were separated into six distinct clusters by hierarchical clustering and indicated that this super family had a complex response during berry ripening . The 12 members of Cluster 1 responded similarly in both the skin and pulp, gradually decreasing with increasing °Brix with a large decrease in transcript abundance at the 36.7 °Brix level. Cluster 2 with 14 members, including 8 members of the VviERF6 clade, had much higher transcript abundance in the skin with a sharp peak at 23.2 °Brix. Cluster 3 had similar profiles in both the skin and pulp with a peak abundance at 25° Brix. Cluster 4 with 7 members was a near mirror image of cluster 2, 25 liter pot with a sharp valley for transcript abundance in the skin between 23 and 25 °Brix. Cluster 5 had 36 members with a steady increase in transcript abundance in the pulp but no substantial increase in the skin until 36.7 °Brix. Finally, in Cluster 6, there were 13 members with a higher transcript abundance in skins compared to pulp. Their transcript abundance increased with increasing °Brix level, but decreased in the skin. The transcript abundance of important components of the ethylene signaling pathway characterized in Arabidopsis and presumed to be functional in grape were also affected by °Brix level and tissue .
Three different ethylene receptors, VviETR1, VviETR2, and VviEIN4 decreased with °Brix level in the skin, however there was very little or no change in the pulp. Likewise, VviCTR1, another negative regulator of ethylene signaling that interacts with the ethylene receptors, decreased between 22.6 and 23.2 °Brix in both the skin and the pulp. The transcript abundance of the positive regulator, VviEIN2, peaked at 25 °Brix in both the skin and the pulp. AtEIN2 is negatively regulated by AtCTR1 and when it is released from repression, turns on AtEIN3 and the ethylene signaling pathway downstream. The transcript abundance of VviEIN3 increased with °Brix level, peaking at 25 °Brix in the skin, and was much higher than in the pulp. Although more subtle, its profile was very similar to VviERF6L1. Derepression of the negative regulators and the increase in positive regulators indicated that ethylene signaling was stimulated during this late stage of berry ripening.The transcript abundance of many of the genes involved in the isoprenoid biosynthesis pathway peaked between 23 and 25 °Brix level, particularly in the skin; this stimulation of transcript abundance continued in both the carotenoid and terpenoid biosynthesis pathways . DXP synthase is a key regulatory step in isoprenoid biosynthesis and its profile was similar to VviERF6L1; its transcript abundance was correlated with the transcript abundance of several terpene synthases in the terpenoid biosynthesis pathway . About 50% of the putative 69 functional terpene synthases in the Pinot Noir reference genome have been functionally characterized. Another 20 genes may be functional but need further functional validation or checking for sequencing and assembly errors. On the NimbleGen Grape Whole-Genome array there are 110 probe sets representing transcripts of functional, partial and psuedo terpene synthases in Pinot Noir . It is uncertain how many may be functional in Cabernet Sauvignon. There were 34 probe sets that significantly changed with °Brix or the °Brix and Tissue interaction effect; 20 of these are considered functional genes in Pinot Noir. Terpene synthases are separated into 4 subfamilies in the Pinot Noir reference genome; they use a variety of substrates and produce a variety of terpenes. Many of these enzymes produce more than one terpene. The top 8 transcripts that peaked in the skin at the 23.2 to 25 °Brix stages were also much higher in the skin relative to pulp . Five of the eight probesets match four functionally-classified genes in Pinot Noir ; these terpene synthases clustered very closely with VviTPS54, a functionally annotated – Linalool/- Nerolidol synthase. VviTPS58, a -geranyl linalool synthase, was also in the cluster. The other two probesets match partial terpene synthase sequences in the Pinot Noir reference genome. The transcript abundance of genes involved with carotenoid metabolism also changed at different °Brix levels and with tissue type . CCDs are carotenoid cleavage dioxgenases and are involved in norisoprenoid biosynthesis. The transcript abundance of VviCCD1 changed significantly with °Brix level and was higher in skin than pulp, except at 36.7 °Brix. Likewise, the transcript abundance of VviCCD4a and VviCCD4b changed significantly with °Brix level, but was higher in the pulp than the skin. The transcript abundance of VviCCD4c significantly increased with °Brix level, but there were no significant differences between tissues. VviCCD1 and VviCCD4 produce β- and α-ionone , geranylacetone , and 6-methyl-5-hepten-2-one in grapes. The transcript abundance of VviCCD8 significantly increased with°Brix level and was higher in pulp than skin. Phytoene synthase, which was also increased in the skin compared to the pulp , and VviCCD1, have been associated with β-ionone and β-damascenone biosynthesis. Other important grape flavors are derived from the fatty acid metabolism pathway and lead to the production of aromatic alcohols and esters. The transcript abundance of many genes associated with fatty acid biosynthesis and catabolism changed with °Brix level . In particular the transcript abundance of a number of genes were correlated with the transcript abundance of VviERF6L1 including VviACCase, Acetyl-CoA carboxylase; KAS III ; VviOAT, ; VviFAD8; ; VviLOX2 and VviHPL . The transcript abundance of alcohol dehydrogenases was affected by tissue and °Brix level . Some ADHs are associated with the production of hexenol and benzyl alcohol. Methoxypyrazines give herbaceous/bell pepper aromas. They are synthesized early in berry development and gradually diminish to very low levels at maturity. Nevertheless, humans can detect very low concentrations of these aroma compounds. Four enzymes, VviOMT1, VviOMT2, VviOMT3 and VviOMT4 , synthesize methoxypyrazines.