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, cut flower bucket 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 , 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.Vitis vinifera grapevines originated approximately 65 million years ago from Eurasia and have been cultivated for at least the last 8000 years for its fruits that are crushed to make wine. Grapevines are now grown throughout the world in many kinds of environments. Grape berry development is a complex process involving three developmental phases and multiple hormones. It is in the latter ripening phase that many compounds involved in flavor and aromas are synthesized, flower display buckets conjugated or catabolized. Most of these compounds reside in the skin of the berry and seem to develop in the very last stages of berry development . Aroma and flavor are important sensory components of wine. They are derived from multiple classes of compounds in grapes including important volatile compounds from the grape and from yeast metabolism during grape fermentation. Each grape cultivar produces a unique set of volatile and flavor compounds at varying concentration that represents its wine typicity or typical cultivar characteristics. Esters and terpenes are volatile compound chemical classes largely responsible for the fruity and floral aromas in wines. Esters are largely produced during yeast fermentation from grape-derived products such as aliphatic alcohols and aldehydes. Grape lipoxygenases are thought to provide the six carbon precursors from fatty acids for the synthesis of the fruity aroma, hexyl acetate, in yeast during wine fermentation. Terpenes mostly originate from the grapes and are found in both the free and bound forms. Both plant fatty acid and terpenoid metabolism pathways are very sensitive to the environment. Climate has large effects on berry development and composition. Besides grape genetics other factors may influence metabolite composition including the local grape berry microbiome, the soil type and the rootstock. While there is evidence that rootstock can affect fruit composition and transcript abundance, this effect appears to be minor relative to other environmental factors. Many cultural practices used by the grape grower may directly or indirectly affect the environment sensed by the grapevine . Temperature and light are major contributors to “terroir”. Terroir refers to the environmental effects on grapes and how it contributes distinctive characteristics to the typicity of a wine. The terroir term includes biotic and abiotic factors, soil environments as well as the viticultural practices. In the present work, we will use the term “place” to address all of the above except for the viticultural practices. Recently, a transcriptomic approach was used to elucidate the common gene subnetworks of the late stages of berry development when grapes are normally harvested at their peak maturity. One of the major subnetworks associated with ripening involved autophagy, catabolism, RNA splicing, proteolysis, chromosome organization and the circadian clock. An integrated model was constructed to link light sensing with the circadian clock highlighting the importance of the light environment on berry development. In this report, in order to get a better understanding of how much of the gene expression in Cabernet Sauvignon berry skin could be attributed to environmental influences, we tested the hypothesis that there would be significant differences in gene expression during the late stages of Cabernet Sauvignon berry ripening between two widely different locations: one in Reno, NV, USA and the other in Bordeaux, France .
The analysis revealed a core set of genes that did not depend on location, climate, vineyard management, grafting and soil properties. Also, the analysis revealed key genes that are differentially expressed between the two locations. Some of these differences were linked to the effects of temperature and other environmental factors known to affect aromatic and other quality-trait-associated pathways. Many gene families were differentially expressed and may provide useful levers for the vine grower to adjust berry composition. Among others, these families encompassed genes involved in amino acid and phenylpropanoid metabolism, as well as aroma and flavor synthesis.To test the hypothesis that the transcript abundance of grape berries during the late stages of ripening differed in two locations with widely different environmental conditions, we compared the transcript abundance of grape berry skins in BOD and RNO. The vineyards were originally planted in RNO in 2004 and in BOD in 2009. The RNO vines were grown on their own roots, whereas the BOD vines were grafted on to SO4 rootstock. A vertical shoot positioning trellis design was used in both locations. There were a number environmental variables that differed between the two locations. BOD is located at a slightly more northern latitude than RNO. This resulted in slightly longer day lengths in BOD at the beginning of harvest and slightly shorter at the end of harvest . On the final harvest dates, the day length differed between RNO and BOD by about 30 min. RNO had warmer average monthly maximum temperatures than that in BOD, but minimum September temperatures were cooler in RNO . Thus, RNO had a larger average daily day/night temperature differential of 20 °C, whereas BOD had a smaller average daily day/ night temperature differential of 10 °C during the harvest periods. RNO had warmer day temperatures by about6 °C and cooler night temperatures by about 4 °C than that of BOD. The monthly precipitation totals for RNO in September were 2.03 mm whereas it was 65.5 mm in BOD; the average relative humidities were 34 and 74% for RNO and BOD, respectively. The soil at the RNO vineyard was a deep sandy loam with a pH of 6.7; the BOD vineyard was a gravelly soil with a pH of 6.2. No pathogens, nutrient deficiencies or toxicity symptoms were observed on or in the vines at either site.The analysis of transcript profiles of Cabernet Sauvignon grapes harvested in RNO in September of 2012 was previously described. Individual berry skins were separated immediately from the whole berry and the individual total soluble solids level of the berry, which is mostly composed of sugars, was determined. The Cabernet Sauvignon berry skins from BOD were harvested in a similar manner as the RNO berry skins. The berry skins in BOD were harvested from midway in September, 2013 until the first week of October . The berry skins were separated and the °Brix analyzed in the same manner as that in RNO. Grapes were harvested at a lower °Brix range in BOD than in RNO because fruit maturity for making wine is typically reached in the BOD region at a lower sugar level. Transcript abundance of the RNA-Seq reads from both RNO and BOD was estimated using Salmon software with the assembly and gene model annotation of Cabernet Sauvignon. The TPM were computed for each gene from each experimental replicate from berry skins at different sugar levels ranging from 19 to 26°Brix . Principal component analysis of the transcriptomic data showed clear grouping of experimental replicates with the largest separation by location = 51% variance and then °Brix = 22% variance of the berry skin samples . To get different perspectives of the data, three approaches were used to further analyze the transcriptomic data.