The amino acid metabolism functional GO category is highly enriched in the group of DEGs between BOD and RNO and more specifically in the top 400 BOD DEGs . Some examples of genes involved in amino acid metabolism that have a higher transcript abundance in BOD berries are phenylalanine ammonia lyase 1 , which catalyzes the first step in phenylpropanoid biosynthesis, branched-chainamino-acid aminotransferase 5 , which is involved in isoleucine, leucine and valine biosynthesis, 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase 1 , which catalyzes the first committed step in aromatic amino acid biosynthesis, and tyrosine aminotransferase 7 , which is involved in tyrosine and phenylalanine metabolism. Included in this group were 44 stilbene synthases , which are part of the phenylpropanoid pathway; these STSs had a higher transcript abundance in BOD berries as compared to RNO berries, with very similar transcript abundance profiles to PAL1 .In a previous analysis, WGCNA defined a circadian clock subnetwork that was highly connected to transcript abundance profiles in late ripening grapevine berries. To compare the response of the circadian clock in the two different locations, we plotted all of the genes of the model made earlier. Most core clock genes and light sensing and peripheral clock genes had significantly different transcript abundance in BOD berries than that in RNO berries at the same sugar level . All but one of these had higher transcript abundance in BOD berries relative to RNO berries. The transcript abundance of other genes had nearly identical profiles .

These data are summarized in a simplified clock model , black flower bucket which integrates PHYB as a key photoreceptor and temperature sensor that can regulate the entrainment and rhythmicity of the core circadian clock, although to be clear it is the protein activity of PHYB, not the transcript abundance that is regulating the clock.The common gene set for both locations represented approximately 25% of the genes differentially expressed with sugar level or location. Presumably these gene sets represent genes that were not influenced by location but were influenced by berry development or sugar level. This study is limited in that only two locations in one season were investigated. As more locations are compared in the future, these gene sets will likely be reduced in size even further. The processes involved in these gene sets or modules included the increase of catabolism and the decline of translation and photosynthesis. It is clear that these processes play important roles in berry ripening. Most of the genes in the genome varied in transcript abundance with increasing sugar levels and berry maturation and most of these varied with the vineyard site. Many of the DEGs were enriched with gene ontologies associated with environmental or hormonal stimuli.Plants are exposed to a multitude of factors that influence their physiology even in controlled agricultural fields such as vineyards. The vineyards in BOD and RNO are exposed to very different environments ; these environmental influences were reflected in some of the DEG sets with enriched gene ontologies. The results from this study are consistent with the hypothesis that the transcript abundance of berry skins in the late stages of berry ripening were sensitive to local environmental influences on the grapevine. While most transcript abundances in berries are largely influenced by genetics or genotype, environment also plays a large role.

It is impossible with the experimental design of this study to determine the amount that each of the environmental factors contributed to the amount of differential expression in these two locations. There were too many variables and too many potential interactions to determine anything conclusively. Replication in other seasons will not aid this analysis as climate is highly variable and will produce different results. All we can say is that these genes were differentially expressed between the two locations, which were likely due to known and unknown factors . As additional studies are conducted indifferent locations and seasons in the future, meta analyses can be employed to provide firmer conclusions. It is possible that some of the DEGs identified in this study resulted from genetic differences between the different Cabernet Sauvignon clones and root stock used in the two locations. Not knowing what these genes might be from previous studies prevents us from drawing any clues. These and other factors most certainly affected the berries to some degree. The data in this study indicated that the grape berry skins responded to multiple potential environmental factors in the two vineyard locations in addition to potential signals coming from the maturing seed. We say potential environmental factors because we did not control for these factors; we associated transcript abundance with the factors that were different in the two locations. The transcript abundance profiles along with functional annotation of the genes gave us clues to factors that were influencing the berries and then associations were made with the known environmental variables. Further experiments are required to follow up on these observations. We were able to associate differences in transcript abundance between the two locations. These DEGs could be associated with temperature, light, moisture, and biotic stress.

Additional factors were associated with transcript abundance involved with physiological responses and berry traits such as seed and embryo development, hormone signaling , phenylpropanoid metabolism, and the circadian clock. In the following sections we discuss in more detail some of the possible environmental factors that were reflected in the enriched gene ontologies found in the gene sets from this study.Light regulates the transcript abundance of many genes in plants. It has been estimated that 20% of the plant transcriptome is regulated by white light and this includes genes from most metabolic pathways. Light is sensed by a variety of photoreceptors in plants; there are red/far red, blue and UV light receptors. PHYB is a key light sensor, regulating most of the light sensitive genes and sensing the environment through red light to far-red light ratios and temperature. PHYB entrains the circadian clock affecting the rate of the daily cycle and the expression of many the circadian clock genes; PHYB induces morning phase genes and represses evening phase genes. Other photoreceptors can entrain the circadian clock as well. PHYB and the circadian clock are central regulators of many aspects of plant development including seed germination, seedling growth, and flowering. The circadian clock influences the daily transcript abundance of genes involved in photosynthesis, sugar transport and metabolism, biotic and abiotic stress, even iron homeostasis. Light signaling was very dynamic in the berry skin transcriptome in the late stages of berry ripening with a higher transcript abundance of many light signaling genes in BOD berries. Many photo receptors that interact with the circadian clock had a higher gene expression in BOD berries. In the circadian clock model, Circadian Clock Associated 1 is an early morning gene and has its highest expression at the beginning of the day. It is at the start of the circadian core clock progression through the day, square black flower bucket whereas the transcript abundance of Timing Of CAB Expression 1 is highest at the end of the day and finishes the core clock progression . In both of these cases, there is a higher transcript abundance of these genes in BOD than in RNO. The evening complex is a multi-protein complex composed of Early Flowering 3 , Early Flowering 4 and Phytoclock 1 that peaks at dusk. None of these proteins, had significant differences in transcript abundance between the two locations . The transcript abundance of ELF3 increased with sugar level and shortening of the day length . ELF3, as part of the evening complex , has direct physical interactions with PHYB, COP1 and TOC1 linking light and temperature signaling pathways directly with the circadian clock. It is interesting that most of the components of the clock showed significant differences in transcript abundance between BOD and RNO, except for the three proteins that make up the evening clock. The transcript abundance profile of PHYB was similar in both BOD and RNO berries , however the changes in transcript abundance with sugar level occurred in BOD berries at a lower sugar level. There was a gradual decline of PHYB transcript abundance with increasing sugar level until the last measurement at the fully mature stage, where there was a large increase in transcript abundance. A very similar profile is observed for Reveille 1 . RVE1 promotes seed dormancy in Arabidopsis and PHYB interacts with RVE1 by inhibiting its expression. PIF7 , interacts directly with PHYB to suppress PHYB protein levels.

Likewise, PIF7 activity is regulated by the circadian clock. PIF7 had higher transcript abundance in the BOD than that of RNO berries and generally increased with increasing sugar level. The transcript abundance of two of the other grape phytochromes did not vary significantly between the two locations or at different sugar levels. PHYC had a higher transcript abundance in RNO berries and did not change much with different sugar levels. Many other light receptors , FAR1 , FRS5 , etc. had higher transcript abundance in BOD berries . Thus, light sensing through the circadian clock is a complicated process with multiple inputs. RVE1 follows a circadian rhythm. It behaves like a morning-phased transcription factor and binds to the EE element, but it is not clear if it is affected directly by the core clock or through effects of PHYB or both. PHYB down regulates RVE1; RVE1 promotes auxin concentrations and decreases gibberellin concentrations. Warmer night temperatures cause more rapid reversion of the active form of PHYB to the inactive form and thus may promote a higher expression/activity of RVE1. Pr appears to accelerate the pace of the clock . It is unclear what role phytochromes might have in seed and fruit development in grapes. Very little is known about the effect of PHY on fruit development in general. In one tomato study, the fruit development of phy mutants was accelerated, suggesting that PHYB as a temperature/light sensor and a regulator of the circadian clock may influence fruit development. Carotenoid concentrations, but not sugar concentrations, also were affected in these mutants. Photoperiod affects the transcript abundance of PHYA and PHYB in grape leaves. In the present study, the transcript abundance of the majority of the photoreceptor genes in berry skins, including red, blue and UV light photoreceptors, had a higher transcript abundance in BOD berries . It is unclear what the effect of PHYB and the circadian clock have on grape berry development. However, there were clear differences between the two locations; it seems likely that PHYB and the circadian clock are key grape berry sensors of the environment, affecting fruit development and composition.The grape berry transcriptome is sensitive to temperature. The RNO berries were exposed to a much larger temperature differential between day and night than BOD berries and were also exposed to chilling temperatures in the early morning hours during the late stages of berry ripening . The transcript abundance of some cold-responsive genes was higher in RNO berry skins than in BOD berry skins , including CBF1. CBF1 transcript abundance is very sensitive to chilling temperatures; it is a master regulator of the cold regulon and improves plant cold tolerance. PIF7 binds to the promoter of CBF1, inhibiting CBF1 transcript abundance, linking phytochrome, the circadian clock and CBF1 expression. Our data are consistent with this model; transcript abundance of PIF7 was higher and CBF1 transcript abundance was lower in BOD berry skins than RNO berry skins .ABA concentrations in plants increase in response to dehydration and ABA triggers a major signaling pathway involved in osmotic stress responses and seed development. ABA concentrations only increase in the seed embryo near the end of seed development when the embryo dehydrates and goes into dormancy. ABA concentrations remain high to inhibit seed germination. The transcript abundance of ABA signaling genes such as ABF2 and SnRK2 kinases increase after application of ABA to cell culture and in response to dehydration in leaves of Cabernet Sauvignon. The data in this study are consistent with the hypothesis that BOD berries are riper at lower sugar levels. The ABA signaling genes in the berry skins had higher transcript abundance in BOD berries indicating that ABA concentrations were higher in BOD than RNO berries even though RNO berries were exposed to drier conditions .