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 superfamily 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, growing blueberries in containers 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.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.This study focused on the very late stages of the mature Cabernet Sauvignon berry when fruit flavors are known to 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 underripe 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. The abundance of the transcript of VviOMT1 decreased in the pulp with increasing °Brix level and was correlated with IBMP concentrations in the late stages of berry development in this study. Both OMT1 and OMT3 have been shown to synthesize IBMP. Furthermore, the transcript abundance of each gene has been correlated with IBMP concentration, but the transcript abundance of each gene cannot fully account for the total IBMP present in all genotypes and conditions. OMT3 was found to be the major genetic determinant for this trait in two independent studies. Nevertheless, it is possible that OMT1 may contribute to the IBMP concentration, because OMT1 can synthesize IBMP and it is located at the edge of a QTL significantly contributing to this trait. Furthermore, the majority of IBHP , the precursor for the OMT1 and OMT3 biosynthesis of IBMP, is produced in the pulp of the berry complicating the factors that influence IBMP concentration. Our results raise questions that require additional research to clarify this relationship of transcript abundance to IBMP concentration, including determination of the rates of biosynthesis and catabolism, enzyme activities, volatilization of IBMP from the berry, as well as the concentrations of substrates for the enzymes involved. There are a number of other transcriptomic ripening studies in grapes and other fruit species. Many of these have compared broad developmental stages with partial genome microarrays. One study compared transcriptomic responses of the lates stages of ripening of whole berries of Chardonnay. This study used a different microarray platform with only about half of the genome represented on the array. In this study, 12 genes were found to be differentially expressed in each of the 3 different stages investigated. There were approximately another 50 genes that were differentially expressed at one stage versus another. Several genes were proposed as good candidates for markers of ripeness and these were also examined in Cabernet Sauvignon berries using qPCR. Several of these candidate genes are consistent with our results in the present study. They include CCD4a , square pots a late embryogenesis abundant protein , a dirigent-like protein , and an S-adenosyl-L-methionine:salicylic acid carboxyl methyltransferase . Of these, the transcript expression of SAMT was found to be temperature insensitive. Like the previous study, the present study focused on very close stages in the mature berry when fruit flavors are known to develop.
In contrast to the previous study on Chardonnay, there were massive changes in the transcript abundance in hundreds of GO categories over this narrow window of ripening. This may in part be due to using six biological replicates rather than the standard three, which probably improved the detection of significantly changing transcripts. In addition, we used a different threshold level for statistical significance and an improved microarray platform, which was able to detect double the number of transcripts. In the present study, many differences were found between the skin and the pulp, °Brix levels and the interaction of tissue and °Brix. Important fruit ripening processes were affected including ethylene signaling, senescence, volatile aroma production, lipid metabolism and cell wall softening. These data indicate that fruit ripening in the late stages of maturity is a very dynamic and active process.Ethylene is involved in climacteric fruit ripening with a CO2 burst preceding the rise in ethylene. In tomato, this occurs at the time the seeds become mature in the mature green fruit stage. At this stage, tomato fruits become sensitive to ethyene and can continue through the ripening stage. Prior to the mature breaker stage, ethylene cannot promote tomato ripening to full ripeness. In non-climacteric fruit, there is no respiratory burst of CO2 and the ripening of most non-climacteric fruits was thought not to respond significantly to an extra application of ethylene. However, recently some non-climacteric fruit such as strawberry, bell pepper and grape have been found to produce a small amount of ethylene and appear to have responses to ethylene at certain stages. In the study of grapes, this peak was observed just before the start of veraison, followed by decreases in ethylene concentrations for several weeks afterwards; the late mature stages of ripening were not examined. Ethylene action is dependent upon ethylene concentration and ethylene sensitivity or signaling. In this study, there were clear and significant changes in transcript abundance of genes involved in ethylene signaling and biosynthesis in the late stages of berry ripening. Seeds become fully mature at this time . Perhaps there is a signal from the seeds when they become mature that allows the fruit to ripen and senesce? Perhaps small amounts of ethylene are produced or there is a change in sensitivity to ethylene? Seymour et al. suggested the response of EIN3 might be a common signaling mechanism for both climacteric and non-climacteric fruit. The responses of VviEIN3 in this study and in a pepper fruit ripening study are consistent with this hypothesis. In addition, the transcript abundance of VviEIN3 in grape is very responsive to ethylene and the ethylene inhibitor, MCP . There are many other factors other than fruit development that can influence ethylene signaling. Could chilling of the fruit or other aspects of the processing of the grapes influence these responses? Could there be some influence of other abiotic or biotic stresses? These are questions that can only be addressed in future studies with additional experiments that are designed to answer these questions.Sexuality is a fundamental mechanism that acts for the maintenance of genetic diversity within a species. In contrast to animals, hermaphroditism is thought to be ancestral and most common in flowering plants. A minority of species have subsequently, and independently, evolved separated sexuality, with separate male and female individuals .These species represent up to 5% of angiosperm species. Plant biologists have thrived to understand the evolutionary steps associated with transitions into or out of dioecy since the first findings of genetic sex determination in flowering plants.Typical transitions out of dioecy have been associated with domestication events in some crops. For example, in papaya or grape, the loss of function of the Y chromosome-encoded female suppressor genes, resulting in hermaphroditism, was artificially selected, presumably for stable cultivation. On the other hand, polyploidization is also thought to be a main key player in the evolution of sexuality, often causing a transition out of dioecy.