This is consistent with the higher NCED3, NCED5 and BAM1 transcript abundance in RNO berries . Thus, there are complex responses of ABA metabolism and signaling. It would appear that there may be two different ABA pathways affecting ABA concentrations and signaling: one involved with embryo development and one involved with the water status in the skins. Auxin is also involved with ABA signaling during the late stages of embryo development in the seeds. Auxin signaling responses are complex. ABF5 is an auxin receptor that degrades Aux/IAA proteins, which are repressors of ARF transcriptional activity. Thus, a rise in auxin concentration releases Aux/IAA repression of ARF transcription factors, activating auxin signaling. In the berry skins, there was a diversity of transcriptional responses of Aux/IAA and ARF genes in the two locations, some with increased transcript abundance and others with decreased transcript abundance. As with ABA signaling, there may be multiple auxin signaling pathways operating simultaneously. One pathway appears to involve seed dormancy. ARF2 had a higher transcript abundance in BOD berries. ARF2 promotes dormancy through the ABA signaling pathway. This is consistent with the hypothesis that BOD berries reach maturity at a lower sugar level than RNO berries.Grapevines have very dynamic gene expression responses to pathogens. The top 150 DEGs for BOD berries were highly enriched with biotic stress genes. The BOD vineyard site had a higher rainfall and higher relative humidity than RNO and these conditions are likely to be more suitable for fungi to grow. We detected a much higher transcript abundance of powdery mildew-responsive genes in BOD berries and this may be connected to a higher transcript abundance of ethylene and phenylpropanoid genes as part of a defense response.

The transcript abundance profiles of some of these genes are remarkably similar. Increased ethylene signaling in grapevines has been associated with powdery mildew infection and phenylpropanoid metabolism and appears to provide plant protection against the fungus. Genes involved with phenylpropanoid metabolism, blueberry pot size especially PAL and STS genes, appear to be quite sensitive to multiple stresses in the environment . In Arabidopsis there are four PAL genes. These PAL genes appear to be involved with flavonoid biosynthesis and pathogen resistance in Arabidopsis. Ten different PAL1 and two PAL2 orthologs had higher transcript abundance in BOD berry skins; many STS genes also had a higher transcript abundance in BOD berry skins . Stilbenes are phytoalexins and provide pathogen resistance in grapes and STS genes are strongly induced by pathogens. Thus, the higher transcript abundance of powdery mildew genes may be associated with the higher transcript abundance of genes in the ethylene and phenylpropanoid pathways.The transcript abundance of a number of iron homeostasis genes were significantly different in the two locations and there was a difference in soil available iron concentrations in the two locations. However, iron uptake and transport in plants is complicated depending on multiple factors, such as pH, soil redox state, organic matter composition, solubility in the phloem, etc. Thus, it is impossible to predict iron concentrations in the berry without direct measurements. The roles of these genes in iron homeostasis and plant physiological functions are diverse. Iron supply can affect anthocyanin concentrations and the transcript abundance of genes in the phenylpropanoid pathway in Cabernet Sauvignon berry skins. One of the DEGs, SIA1, is located in the chloroplast in Arabidopsis and appears to function in plastoglobule formation and iron homeostasis signaling in concert with ATH13. Another DEG, YSL3, is involved in iron transport. It acts in the SA signaling pathway and appears to be involved in defense responses to pathogens.

It also functions in iron transport into seeds. FER1 is one of a family of ferritins found in Arabidopsis . VIT1 and NRAMP3 are vacuolar iron transporters and are also involved in iron storage in seeds. Other DEGs are also responsive to iron supply. IREG3 appears to be involved in iron transport in plastids; its transcript abundance increases with increasing iron concentrations. ABCI8 is an iron-stimulated ATPase located in the chloroplast that functions in iron homeostasis. It is unclear what specific roles these iron homeostasis genes are playing in grape berry skins, but they appear to be involved in iron storage in seeds and protection against oxidative stress responses. One possible explanation for the transcript abundance profiles in the BOD and RNO berry skins is that ferritins are known to bind iron and are thought to reduce the free iron concentrations in the chloroplast, thus, reducing ROS production that is caused by the Fenton reaction. As chloroplasts senesce during berry ripening, iron concentrations mayrise as a result of the catabolism of iron-containing proteins in the thylakoid membranes; thus, berry skins may need higher concentrations of ferritins to keep free iron concentrations low. This might explain the increase in ferritin transcript abundance with increasing sugar levels. Most soils contain 2 to 5% iron including available and unavailable iron; soils with 15 and 25 μg g− 1 of available iron are considered moderate for grapevines, but soils with higher concentrations are not considered toxic. Therefore, for both soils in this study, iron concentrations can be considered to be very high but not toxic. The higher available iron concentrations in the BOD vineyard may be associated with the wetter conditions and the lower soil pH.Other researchers using Omics approaches have identified environmental factors that influence grape berry transcript abundance and metabolites. One study investigated the differences in transcript abundance in berries of Corvina in 11 different vineyards within the same region over 3 years. They determined that approximately 18% of the berry transcript abundance was affected by the environment.

Climate had an overwhelming effect but viticultural practices were also significant. Phenylpropanoid metabolism was very sensitive to the environment and PAL transcript abundance was associated with STS transcript abundance. In another study of a white grape cultivar, Garganega, berries were analyzed by transcriptomic and metabolomic approaches. Berries were selected from vineyards at different altitudes and soil types. Again, phenylpropanoid metabolism was strongly influenced by the environment. Carotenoid and terpenoid metabolism were influenced as well. Two studies investigated the grape berry transcriptomes during the ripening phase in two different regions of China, a dry region in Western China and a wet region in Eastern China. These two locations mirror some of the differences in our conditions in our study, namely moisture, light and elevation, plant raspberry in container although the dry China western region has higher night temperatures and more rainfall than the very dry RNO location. In the Cabernet Sauvignon study, they compared the berry transcriptomes from the two regions at three different stages: pea size, veraison and maturity. The TSS at maturity was slightly below 20°Brix. Similar to our study, the response to stimulus, phenylpropanoid and diterpenoid metabolism GO categories were highly enriched in mature berries between the two locations. Differences in the transcript abundance of NCED and PR proteins were also noted. Like in our study, the authors associated the transcript abundance of these proteins to the dry and wet locations, respectively. In the second study comparing these two regions in China , the effects of the environment on the metabolome and transcriptome of Muscat Blanc à Petits Grains berries were investigated over two seasons; specifically, terpenoid metabolism was targeted. Like in our study, the transcripts in terpenoid were in higher abundance in the wetter location. The transcript abundances were correlated with terpenoid concentrations and a coexpression network was constructed. A specific set of candidate regulatory genes were identified including some terpene synthases , glycosyl transferases and 1-hydroxy-2-methyl-2-butenyl 4-diphosphate reductase . We examined the transcript abundance of some of these candidate genes in our own data but did not find significant differences between our two locations. The contrasting results between our study and Wen et al. could be for a variety of reasons such as different cultivar responses, berry versus skin samples, or different environmental conditions that affect terpenoid production. Terpenoid metabolism is influenced by the microclimate and is involved in plant defense responses to pathogens and insects. Light exposure to Sauvignon Blanc grapes was manipulated by removing adjacent leaves without any detectable differences in berry temperatures. Increased light exposure increased specific carotenoid and terpene concentrations in the berry. Some effect of temperature was associated with carotenoid and terpenoid production, but to a lesser extent than light. Higher concentrations of rotundone, a sesquiterpene, have been associated with cooler temperatures. Water deficit can also alter carotenoid and terpenoid metabolism in grapes.

Terpenes can act as signals for insect attacks and attract insect predators. Thus, terpenoid metabolism is highly sensitive to the environment and influenced by many factors. In contrast to these studies, excess light and heat can affect transcript abundance and damage berry quality. In addition to a higher rate of malate catabolism, anthocyanin concentrations and some of the transcript abundances associated with them are decreased as well.BOD berries reached maturity at a lower °Brix level than RNO berries; the cause is likely to be the warmer days and cooler nights in RNO. Higher day temperature may increase photosynthesis and sugar transport and cooler night temperatures may reduce fruit respiration. °Brix or TSS approximates the % sugar in a berry and is a reliable marker of berry maturity in any given location ; however, TSS is an unreliable marker of berry maturity when comparing grapes from very different climates. The differences in TSS between BOD and RNO are consistent with other studies on the temperature effects on berry development. Indirect studies have associated gradual warming over the last century to accelerated phenology and increased sugar concentrations in the grape berries . Increasing temperature can accelerate metabolism, including sugar biosynthesis and transport, but the increase in metabolism is not uniform. For example, the increase in anthocyanin concentration during the ripening phase is not affected as much as the increase in sugar concentration. These responses vary with the cultivar, complicating this kind of analysis even further. Direct studies of temperature effects on Cabernet Sauvignon berry composition also are consistent with our data. In one study, the composition of Cabernet Sauvignon berries was altered substantially for vines grown in phytotrons at 20 or 30 °C temperatures. Cooler temperatures promoted anthocyanin development and malate concentrations and higher temperatures promoted TSS and proline concentrations. In a second study, vines were grown at 20 or 30 °C day temperatures with night temperatures 5 °C cooler than the day. In this study, higher temperatures increased berry volume and veraison started earlier by about 3 to 4 weeks. The authors concluded that warmer temperatures hastened berry development. In a third study, Cabernet Sauvignon berry composition was affected in a similar manner by soil temperatures that differed by 13 °C. TSS concentrations are also affected by light and the vine water status. Light is generally not a factor because there is usually a large enough leaf area and sufficient light levels to saturate this source to sink relationship. Sun-exposed Cabernet Sauvignon berries in the vineyard had higher TSS than shaded berries. This sunlight effect was attributed largely to an increase in berry temperature rather than an increase in the fluence rate per se. A higher grapevine water status results in larger berry size and lower sugar concentrations and water deficit is known to increase sugar concentrations in Cabernet Sauvignon. However, temperature is thought to have the largest effect on sugar concentrations. Other transcriptomic data in the present study indicated that BOD berries were more mature at a lower sugar level than RNO berries. These included the transcript abundance profiles of genes involved in autophagy, auxin and ABA signaling, iron homeostasis and seed development. Many of these DEGs had an accelerated rate of change in BOD berries. While these transcripts are in the skins, they may be influenced by signals coming from the seed. In addition, there was a higher transcript abundance for most genes involved with the circadian clock in BOD berries. PHYB can regulate the circadian clock and PHYB activity is very sensitive to night temperatures ; PHYB reversion is accelerated to the inactive form at warmer temperatures . The inactivity of phytochrome promotes the expression of RVE1, which promotes auxin concentrations and seed dormancy. Thus, all things considered, it is likely that temperature and/or the temperature differentials between day and night significantly contributed to the differences in the rate of berry development and sugar accumulation in the two locations.