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, french flower bucket 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 super family 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, 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. There were no significant effects on the transcript abundance of VviCCD7. 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. The transcript abundance of VviOMT1 was higher in the pulp than the skin . In addition, bucket flower the transcript abundance of VviOMT1 decreased significantly with °Brix level in the pulp. There were no significant differences in the trancript abundance in the skin or pulp for VviOMT2, VviOMT3 or VviOMT4 . There was a high correlation of the transcript abundance of VviOMT1 in the pulp with 2-isobutyl-3-methoxypyrazine concentrations in the berries . The transcript abundance of VviOMT2, VviOMT3, or VviOMT4 in either skin or pulp was not correlated with IBMP concentrations . This is consistent with the suggestion that the pulp is the main contributor of IBMP in the berry. Our data indicated that VviOMT1 in the pulp may contribute to the IBMP concentration in these berries.Orthologs of RIN and SPL tomato transcription factors, which are known to be very important fruit ripening trancription factors, were at much higher transcript levels in the skin and decline with °Brix level .
The transcript abundance of the VviNOR ortholog in grape was higher in the pulp and increased slightly to peak at 25 °Brix. In addition, the transcript abundance of VviRAP2.3, an inhibitor of ripening in tomato , decreased in the skin with a valley at 23.2 °Brix; it belongs to Cluster 4 of the AP2/ERF super family . Of particular interest was VviWRKY53 [UniProt: F6I6B1], which had a very similar transcript profile as VviERF6L1 . AtWRKY53 is a TF that promotes leaf senescence and is induced by hydrogen peroxide. This is the first report we know of implicating WRKY53 in fruit ripening . AtERF4 induces AtWRKY53 and leaf senescence, so the interactions between WRKY and ERF TFs are complex. WRKY TFs bind to the WBOX elements in promoters and VviERF6L1 has a number of WBOX elements in its promoter . In addition, AtMEKK1 regulates AtWRKY53 and the transcript abundance of VviMEKK1 peaked at 23.2 °Brix in the skin as well. Interestingly, the transcript abundance of both VviERF4 and VviERF8, whose orthologs in Arabidopsis promote leaf senescence, were at their highest level of transcript abundance at the lowest °Brix levels examined in this study .This study focused on the very late stages of the mature Cabernet Sauvignon berry when fruit flavors are knownto develop. Cabernet Sauvignon is an important red wine cultivar, originating from the Bordeaux region of France. It is now grown in many countries. They also can contain herbaceous characters such as green bell pepper flavor that are particulary prevalent in under ripe grapes. Grape flavor is complex consisting not only of many different fruit descriptors, but descriptors that are frequently made up of a complex mixture of aromatic compounds. For example, black currant flavor, in part, can be attributed to 1,8-cineole, 3-methyl-1-butanol, ethyl hexanoate, 2- methoxy-3-isopropylpyrazine, linalool, 4-terpineol, and β- damascenone and major components of raspberry flavor can be attributed to α- and β-ionone, α- and β- phellandrene, linalool, β-damascenone, geraniol, nerol and raspberry ketone. Some common volatile compounds found in the aroma profiles of these dark fruits and berries include benzaldehyde, 1-hexanol, 2-heptanol, hexyl acetate, β-ionone, β-damascenone, linalool, and α-pinene. In a study of Cabernet Sauvignon grapes and wines in Australia, Cabernet Sauvignon berry aromas wereassociated with trans-geraniol and 2-pentyl furan and Cabernet Sauvignon flavor was associated with 3-hexenol, 2-heptanol, heptadienol and octanal. In another comprehensive study of 350 volatiles of Cabernet Sauvignon wines from all over Australia, the factors influencing sensory attributes were found to be complex; in part, norisoprenoids and δ − and γ-lactones were associated with sweet and fruity characteristics and red berry and dried fruit aromas were correlated with ethyl and acetate esters. In Cabernet Sauvignon wines from the USA, sensory attributes were complex also and significantly affected by alcohol level of the wine. Linalool and hexyl acetate were postitively associated with berry aroma and IBMP was positively correlated with green bell pepper aroma. In France, β-damascenone was found to contribute to Cabernet Sauvignon wine aroma. Thus, flavor development in berries and wines is very complex, being affected by a large number of factors including genetics, chemistry, time and environment. In this paper we begin to examine the changes in transcript abundance that may contribute to flavor development. We show that the transcript abundance of many genes involved in fatty acid, carotenoid, isoprenoid and terpenoid metabolism was increased in the skin and peaked at the °Brix levels known to have the highest fruit flavors . Many of these are involved in the production of dark fruit flavors such as linalool synthases, carotenoid dioxygenases and lipoxygenases. These genes serve as good candidates for berry development and flavor markers during ripening. A broader range of studies from different cultivars, locations and environments are needed to determine a common set of genes involved in berry and flavor development. A similar study was conducted on the production of volatile aromas in Cabernet Sauvignon berries across many developmental stages, including a detailed analysis of the °Brix levels that was surveyed in this study. They found that the production of alcohol volatiles from the lipoxygenase pathway dominated in the later stages of berry ripening and suggested that the activity of alcohol dehydrogenases also could play an important role.