BACKGROUND
the grapevine fruit is a characteristic berry consisting of an external skin surrounding a fleshy pulp that encloses seeds.
grape biochemical composition is crucial for the different uses of grapes such as winemaking, production of juice and liqueur, fresh consumption or elaboration of raisins, and the final composition is mostly achieved during the ripening phase  <cit> .
grape ripening is triggered once the seeds have developed and radically changes fruit features from frugivore-repulsive to -attractive.
this shift comprises sugar accumulation in the vacuoles of mesocarp cells accompanied by organic acid metabolisation and titratable acidity reduction  <cit> .
mechanisms to protect seeds from biotic and abiotic stress sources are also activated, mainly in the berry skin  <cit> .
phenolic compounds are accumulated, including phytoalexins, damaging light-absorbing compounds and animal-attractive anthocyanin pigments  <cit> .
changes in terpenoid composition result in attractive aromatic profiles, while other compounds of the same family accumulate to accomplish protective functions  <cit> .
although grapevine ripening is regulated by an intrinsic program that is partially triggered by hormonal signals  <cit> , the process is also strongly modulated by external factors that influence the final berry composition and its commercial quality.
temperature is a major factor altering grape ripening with high temperatures hastening organic acid metabolisation and inhibiting anthocyanin accumulation  <cit> .
irradiation intensity and quality perceived in the berry skin produces changes in secondary metabolism.
light promotes flavonols and terpenoids accumulation with some effects being more specifically related with ultraviolet radiation, which is also able to enhance stilbenoids accumulation  <cit> .
moreover, temperature and light conditions cyclically fluctuate under field environments due to the earth's rotation and, in fact, the strength of their daily oscillation has been shown to affect grape ripening.
for instance, reduction in temperature fluctuation intensity hastens berry ripening and alters flavonoid partitioning  <cit>  and high temperatures applied during night-times are also able to reduce anthocyanin accumulation  <cit> .
in contrast, light pulses applied during the night-time enhance anthocyanin accumulation  <cit> .
these effects of environmental variation on grape ripening rely, at least in part, on changes at the level of gene expression  <cit> .
thus, it can be hypothesised that daily oscillations in factors such as temperature, light or humidity could influence grape ripening progression and consequently on its final composition by means of conditioning circadian fluctuations on the grape transcriptome.
an internal molecular clock is another daily cycling element modulating plant physiology.
core clock components are transcription factors that reciprocally regulate their expression resulting in characteristic circadian expression profiles.
altogether, they determine daily rhythms of expression in a great proportion of plant transcriptomes  <cit> .
in addition, the circadian clock integrates inputs from fluctuating conditions like light and temperature, which allows for plants to discriminate daily and seasonal changes  <cit> .
in this way, the circadian clock together with environmental factors control several plant physiological and developmental outputs to be triggered at the proper time of day or season such as photosynthesis, starch and nitrogen metabolism, growth, cold acclimation, bud dormancy, flowering time, tuber formation or stomata and organ movements  <cit> .
since grape ripening is modulated by light and temperature inputs, it is possible that part of this modulation could be integrated by circadian clock components as shown in other plant developmental processes.
however, the role of circadian clock on fruit ripening control has scarcely been considered before  <cit> .
in this study, nimblegen grapevine whole genome microarrays were used to follow transcriptome variations in ripening grapes throughout a 24 h daily cycle.
tempranillo and verdejo cultivars were analysed as genotypes appreciated in spanish viticulture for the production of red and white quality wines, respectively.
verdejo is a cultivar with different berry features than tempranillo bearing white skin colour and a characteristic aromatic profile, giving rise to wines distinguishable from other white wines  <cit> .
given that berry flesh and skin ripening transcriptional programs significantly differ  <cit> , and assuming that this divergence could be more pronounced in cultivars bearing black-skinned berries accordingly with the presumably higher gene expression activity related to the phenylpropanoid pathway in its berry skin, both pericarp tissues were separately analysed in tempranillo to be more precise.
this experiment was carried out using potted plants grown under controlled greenhouse conditions that allowed for identifying tissue-shared as well as tissue-specific cyclic expression profiles under monitored variation of environmental parameters.
part of this response was confirmed in a different experiment in verdejo berries grown under field environmental conditions despite divergences in genotype, berry colour, berry total soluble solids  or sampling day photocycles and thermocycles.
RESULTS
circadian oscillatory gene expression profiles in tempranillo berries grown under controlled conditions
the question of whether sub-daily changes in environmental conditions produce specific transcriptome shifts that could influence grapevine berry ripening was addressed.
alterations putatively related with changes in metabolism taking place at the last steps of berry ripening, in the absence of earlier processes like berry softening that are more related to the veraison stage, were analysed.
for this purpose, pre-ripe berries of tempranillo  were collected at six different time points throughout a 24 h period under a controlled oscillation of light, temperature and humidity conditions.
sampling time points were selected to include times of maximum and minimum temperatures and the end of light and dark periods  in order to maximize differences in the environmental conditions .
global transcriptome changes were first separately followed in berry skin and flesh.
after applying a 5% false discovery rate  in limma and 2-fold change cut-offs over normalized expression data on each tissue , a higher number of differentially expressed genes  was identified in the berry skin  than in the flesh .
four main expression profiles were observed in both analysed tissues, which involved oscillatory patterns following inflexion points determined by temperature or light presence variations.
they included transcripts up-regulated at 12:45 pm , transcripts down-regulated at 12:45 pm , transcripts up-regulated during the daytime , and transcripts up-regulated during the night-time .
night responses were maintained until the early morning  in flesh clusters f <dig> and f <dig> .
in both berry tissues, responses to heat were activated at 12:45 pm as indicated by the significant over-representation of ‘hsp-mediated protein folding’ and ‘hsf family transcription factor ’ functional categories in clusters s <dig> and f <dig> .
other functional categories were enriched only in specific flesh or skin clusters.
noticeably, among them, is the enrichment of ‘ethylene-mediated signalling pathway’, ‘circadian clock signalling pathway’, ‘biotic stress response’, and ‘mapk cascade’ categories as well as of ap <dig>  constans-like, wrky and gras families of tfs in skin cluster s <dig>  gene silencing by mirna was specifically enriched in cluster f <dig>  thus, daily light and temperature variations are related with the detection of gene expression oscillations in the skin and the flesh of pre-ripe berries, whereas genes showing oscillating expression were more frequent in the skin than in the flesh.
in order to search for those transcripts showing similar expression fluctuation in both pericarp tissues, a similar analysis  was run considering skin and flesh samples in the same time point as replicates.
in that way,  <dig> deg were identified, however, this analysis could underestimate transcripts similarly oscillating in expression in both tissues but involving differences in absolute expression levels.
this result indicates that a great proportion of daily oscillating transcripts were shared between both pericarp tissues .
in fact, four expression profiles were identified for pericarp deg  that resembled those identified in individual tissues and shared with them enriched functional categories .
cluster sf <dig> grouping transcripts up-regulated at 12:45 pm in both pericarp tissues was enriched in ‘hsp-mediated protein folding’ and ‘hsf family tf’ and moreover in ‘vacuolar malate transport’ due to the presence of two transcripts putatively coding for tonoplast dicarboxylate transporters .
on the other hand, cluster sf <dig> grouping transcripts down-regulated at 12:45 pm in both pericarp tissues was enriched in ‘jaz family tf’; while transcripts in cluster sf <dig>  up-regulated in the pericarp during the night-time, included an over-representation of ‘circadian clock signalling pathway’ as well as in kip <dig> santa, gras and constans-like families of tfs .
thus, a considerable number of transcripts showed similar circadian expression oscillation in grape skin and flesh, including responses to temperature and light cycles as well as circadian clock-related genes.
transcripts differentially oscillating in expression between tempranillo berry flesh and skin
once shown that berry skin and flesh shared daily expression changes in tempranillo, tissue specific oscillations in expression in berry flesh or skin were searched by a direct comparison of both tissues.
the circadian series on each tempranillo berry tissue were compared in a two-class masigpro time series analysis, identifying  <dig> deg between tissues .
among them, more transcripts were specifically oscillating in expression in the skin than in the flesh  confirming results of analyses performed on each tissue .
four different skin-specific  and two flesh-specific  oscillatory expression profiles were identified.
moreover, transcripts that were up-regulated during the night-time in both tissues but showing inductive profile maintained until the early morning only in the flesh were grouped in cluster svsf <dig>  finally, transcripts showing an increased expression throughout the 24 h sampling cycle specifically in the skin were grouped in cluster svsf <dig>  the most abundant expression profiles were those including transcripts up-regulated specifically in the berry skin either during the daytime  or the night-time .
cluster svsf <dig> was significantly enriched in ‘wounding’ functional category .
in addition, this cluster included transcripts encoding phenylpropanoid biosynthetic enzymes like a ferulate 5-hydroxylase  and an anthocyanidin glycosylase ; terpenoid biosynthetic enzymes including a vinorine synthase  and two β-amyrin synthase ; and myb tfs .
cluster svsf <dig> was enriched in ‘protein kinase’, ‘wrky family tf’ and ‘ap <dig> family tf’ functional categories.
cluster svsf <dig> included transcripts whose expression was down-regulated specifically in the skin at time points in the middle of dark and light periods.
notably this cluster is enriched for ‘r proteins from plant-pathogen interaction’ and ‘auxin transport’ categories, the last one resulting from the presence of five transcripts encoding putative auxin transporters.
heavy metal responsive transcripts and c2c2-dof family tfs were also enriched within this cluster.
interestingly, the group of transcripts up-regulated during the daytime only in the flesh  was enriched in ‘secondary metabolism’ classified transcripts, mainly related to the presence of flavonoid biosynthetic transcripts including one phenylalanine ammonia-lyase , three chalcone synthase ,  <dig> flavonoid hydroxylases  and two flavonol synthases .
other phenylpropanoid biosynthetic genes and two monoterpenoid biosynthetic transcripts coding for 10-geraniol hydroxylase  were also found in cluster svsf <dig>  together with vvmyba <dig>  and vvmyba <dig>  tfs; the former being a key regulator promoting anthocyanin biosynthesis in grapevine  <cit> .
some of these transcripts underwent a net increase in expression in the flesh during the studied 24 h period .
‘stilbenoid biosynthesis’ was prominently over-represented in both clusters that involved profiles with expression peaking in the flesh at the early morning.
this was due to the presence of a set of  <dig> stilbenoid biosynthetic transcripts among transcripts more specifically up-regulated in the flesh  and another set of  <dig> transcripts within the group of transcripts that peaked earlier in the skin at the end of the night-time .
in this manner,  <dig> out of  <dig> stilbenoid biosynthetic genes annotated in the microarray were differentially involved in these two expression profiles and all of them were located in two gene clusters at chromosomes  <dig> and  <dig> , which suggest a functional diversification of grapevine stilbene synthase  promoters before the extensive duplication of these genes.
cluster svsf <dig> was also enriched in ‘nitrogen metabolism’ and ‘auxin signalling’ resulting both from the presence of seven nitrilase encoding transcripts.
an iaa31-like  and an auxin-responsive d <dig>  encoding transcript were also included within this profile.
the enrichment of ‘oxidative stress response’ in cluster svsf <dig> was determined by the presence of six laccases, one lactoylglutathione lyase and one glutathione s-transferase.
concerning cluster svsf <dig>  it was enriched in ‘wrky family tf’ and ‘ammonium transport’ and included other phenylpropanoid biosynthetic transcripts in addition to stss and the vvmyb <dig> tf  .
finally, cluster svsf <dig> was enriched in transcripts belonging to the ‘oxidative stress response’ category.
therefore, circadian gene expression patterns in the flesh and the skin of pre-ripe berries include both tissue-shared and tissue-specific oscillatory genes that in several cases might be related to specific tissue functions.
circadian oscillatory gene expression profiles in verdejo berries grown under field conditions
a parallel circadian experiment was set up on field grown cultivar verdejo plants to test whether comparable daily transcriptome changes would take place in different cultivars and under vineyard conditions.
environmental conditions were slightly different during the sampling day at the verdejo vineyard.
temperature showed a wider oscillation , dark periods were slightly longer  and relative humidity was lower .
therefore, although differential expression detected between tempranillo and verdejo could not be undoubtedly explained, at least those shared expression profiles could be considered as consistent cycling responses.
as an important proportion of circadian regulated genes showed the same oscillatory expression profile either in tempranillo berry skin or flesh, the analysis of verdejo whole pericarp from pre-ripe berries  was considered suitable to be compared.
a total of  <dig> genes significantly oscillating in expression were identified in verdejo berry pericarps .
almost one third of verdejo deg  was also significant in any of the comparisons carried out in tempranillo  indicating that an important part of this response was shared between experiments regardless of genotypic, environmental and developmental differences.
when verdejo deg were clustered, only three main expression profile groups were detected because profiles of transcripts up-regulated around noontime could not be distinguished from those up-regulated during the daytime contrasting with the tempranillo experiment.
such discrepancy could result from differential oscillation in environmental conditions as berries experienced the maximum temperature near noontime in the tempranillo experiment and close to the end of the daytime in the verdejo one .
moreover, putative responses to night conditions were maintained until the early morning in verdejo pericarp as observed for tempranillo berry flesh .
the number of transcripts up-regulated during the daytime in verdejo was almost half to that of transcripts up-regulated during the night-time.
one expression profile included all daytime up-regulated transcripts .
similarly to tempranillo transcripts up-regulated at 12:45 pm, cluster v <dig> was enriched in ‘hsp-mediated protein folding’; however it also showed enrichment in ‘erf subfamily tf’ and ‘plant photosystem i supercomplex’ among other categories .
noticeably, cluster v <dig> included a hy5-like bzip tf .
concerning night up-regulated transcripts in verdejo pericarp, the absence of light in the first time point of this experiment made it possible to discriminate between transcripts putatively up-regulated by low temperatures  or by darkness .
functional categories over-represented among transcripts up-regulated in tempranillo berries during the night-time were also enriched in these two verdejo clusters such as ‘gras family tf’ in cluster v <dig> and ‘constans-like family tf’ in cluster v <dig>  additionally, cluster v <dig> was enriched in ‘biotic stress response’ and ‘abc multidrug transport’.
two putative 1-deoxy-d-xylulose 5-phosphate synthase  encoding transcripts were present within cluster v <dig> , while the first one was also induced during the night-time in tempranillo berry skin  together with another putative dxs .
verdejo cluster v <dig> was enriched in ‘xyloglucan modification’ because it contained eight xyloglucan endotransglucosylase  encoding transcripts .
‘cytokinin-mediated signalling pathway’ was also over-represented in cluster v <dig>  which included three transcripts coding for authentic-response regulator  type-a  and one for histidine kinase ahk2-like cytokinin receptor , while ‘response regulator family tf’ was enriched in both v <dig> and v <dig> clusters of night-time up-regulated transcripts also due to the presence of several arr genes.
noticeably, vvgai <dig> , encoding a repressor of gibberellin responses  <cit> , was also grouped within the v <dig> cluster.
although differential expression profiles between tempranillo and verdejo could not be clearly attributable to differences in genotype or growth conditions, time series in both cultivars were explicitly compared.
considering that tempranillo berry skin and flesh shared circadian oscillatory gene expression  and that a considerable portion of the ripening transcriptional program is common to both pericarp tissues  <cit> , tempranillo skin and flesh samples on each time point were considered as replicates to be compared to verdejo pericarp samples.
in this manner,  <dig> deg were identified .
more transcripts specifically oscillating in expression were identified in verdejo than in tempranillo.
however, they were enriched in similar functional categories as those regulated in tempranillo.
they involved ‘hsp-mediated protein folding’ and ‘response regulator family tf’ among transcripts up-regulated only in verdejo during the daytime or during the night-time, respectively .
apart from these similarities, differences in secondary metabolism were observed.
three caffeoyl-coa o-methyltransferase  and one secoisolariciresinol dehydrogenase  phenylpropanoid biosynthetic transcripts were up-regulated during the daytime only in tempranillo .
another secoisolariciresinol dehydrogenase  and a flavonoid hydroxylase  were induced during the night-time specifically in tempranillo .
in contrast, terpenoid biosynthetic transcripts specifically oscillated in expression in verdejo including a linalool synthase , a myrcene synthase , two β-amyrin synthase  and three lupeol synthase  that were up-regulated during the daytime; while an isoprene synthase , a β-ocimene synthase  and a germacrene synthase  were up-regulated during the night-time.
interestingly, vvmybf <dig> , a light inducible tf  <cit> , was up-regulated during the daytime only in the verdejo experiment .
these results indicate that circadian expression changes in grapes are mostly consistent among different genotypes.
divergences putatively related with genotypic or environmental differences were also found.
circadian clock homologous genes oscillating in expression in grapevine berries
given that expression profiles enriched in circadian clock signalling-related functional categories were identified in all analysed datasets, homologs to core clock components within the grapevine 12× v <dig> genome gene predictions were searched for, in order to analyse their expression profiles throughout the studied 24 h cycles .
vvlhy  was the only identified grapevine ortholog to arabidopsis thaliana cca <dig> and lhy partially redundant morning core clock myb genes.
this indicates that the lhy duplication observed in other angiosperms took place after the separation of clades giving rise to the vitaceae as has previously been reported  <cit> .
vvlhy did not show any significant oscillation in expression in the experiments contrasting to the results obtained for another homolog to morning myb tfs, vvrve <dig> .
this grapevine gene was previously annotated as cir1/rve <dig> , despite it is the closest homolog to arabidopsis reveille <dig>  circadian clock gene  <cit> .
transcription of vvrve <dig> was up-regulated during the night-time and peaked in the morning in tempranillo and verdejo pericarps .
in addition, vvppr7_ <dig>  was up-regulated during the daytime in tempranillo pericarp, while vvelf3_ <dig>  expression peaked in the morning in verdejo berries.
several grapevine homologs to night core clock genes could be identified in the grapevine genome including vvtoc <dig> , although they did not significantly change in expression.
therefore, core circadian clock gene homologs are present in the grapevine genome and only some of them seem to oscillate in expression in berry tissues.
acoverage of the arabidopsis protein by the grapevine predicted protein sequence.
bclusters of significant transcripts in figures  <dig>   <dig> and  <dig> and additional files  <dig> and  <dig>
discussion
berry transcriptome plasticity in the adaptation to daily environmental changes
plants as sessile organisms have evolved physiological and developmental adaptations to changes in the surrounding environment.
grape transcriptome changes promoted by the soil composition and weather conditions of different years and locations have been described as examples of plasticity displayed by the berry ripening transcriptome  <cit> .
herein changes are shown in the berry ripening transcriptome taking place within the same plants as a response to sub-daily fluctuations in the surrounding environment.
after applying consistent statistical analyses and cut-offs, a total of  <dig> and  <dig> transcripts showing circadian oscillation in expression were respectively identified in tempranillo and verdejo experiments .
although these daily oscillations could involve a smaller proportion of circadian cycling genes than in other species or tissues  <cit> , they are larger than those transcriptome changes observed between consecutive berry ripening stages near maturity, consistently described to be low under diverse experimental conditions and genotypes  <cit> .
thus, sub-daily variation of gene expression in the berry could be more decisive for the final berry composition than other developmental cues at late ripening stages.
most detected cycling profiles were apparently directed to adapt berry cells to changing environmental conditions such as temperature and light.
they included the over-expression of hsps, erf tfs and bag5-like bcl- <dig> associated athanogene  positively correlating with high temperatures as well as the down-regulation of abc transporters expression negatively correlating with them as described for thermotolerance responses in muscat hamburg berries  <cit> .
the identification of a higher number of hsps specifically induced during the daytime in verdejo berries, which experienced more extreme temperatures than the tempranillo ones, also suggests the existence of plasticity in berry thermotolerance responses.
nonetheless, some of these as well as other circadian transcriptional responses observed herein may not result in physiological responses as they might not always involve changes at the protein level  <cit> .
on the other hand, transcriptional changes related to biotic stress responses were activated during the night-time and more greatly in samples including berry skin.
induction of pathogen r-gene-mediated resistance before dawn, the time when the pathogen normally disperses the spores, has been described as a mechanism controlled by core clock genes in arabidopsis to anticipate infection  <cit> .
an analogous mechanism in the grape berry, an organ particularly susceptible to pathogen attacks when approaching ripeness, could have been evolutionarily selected to ensure seeds dispersal and species survival.
tfs of the wrky family were also induced around dawn in all analysed datasets suggesting that they could participate in the regulation of this defence response  <cit> .
stss were simultaneously induced only in tempranillo skin and flesh.
higher expression of sts transcripts has also been correlated with low temperatures in corvina berries grown under different environmental conditions  <cit> .
circadian regulation of sts genes could be partially controlled by vvmyb <dig>  which was also induced at dawn only in tempranillo berry skin, since this tf is able to activate sts promoters in grapevine  <cit> .
in contrast, activation of defence pathways against virus at warmest times of day could be characteristic of grape berries in view that rna silencing argonaute <dig> homologues  were induced around noontime in all experiments.
in fact, plant rna silencing-mediated defence pathways are generally less active under low temperatures and more active at high temperatures concomitantly with plant virus activity  <cit> .
constans-like genes participate in the integration of light and circadian clock signalling  <cit> , whereas transcripts within this family consistently peaked around dawn both in tempranillo and verdejo berries.
relative to circadian clock signalling, cca <dig> and lhy are considered the core morning loop genes in the arabidopsis molecular clock  <cit> .
vvlhy was the only ortholog to these genes found in the grapevine reference genome and did not oscillated in expression in our experiments .
in contrast, in all analysed grape samples, vvrve <dig> showed a cyclic expression profile paralleling these of lhy/cca <dig> genes in arabidopsis leaves.
this gene is the ortholog of rve <dig>  another myb cca <dig> subfamily circadian clock-related gene that, like lhy and cca <dig>  is able to bind to the ‘evening element’ in the promoter of evening-phased circadian rhythmic genes  <cit> .
taking into account these coincidences, it might be interesting to test whether vvrve <dig> could act as a core clock morning gene in grapevine pre-ripe fruits.
absence of significant regulation in other central clock homologs in the grape analyses may suggest that a simplified version of the clock would operate in grapevine late ripening fruits as shown in other non-leaf arabidopsis organs like roots  <cit> .
the biology of grapevine fruits is extremely different to that of plant tissues and species where the circadian clock signalling has been explained.
therefore, further research is required to confirm how distinct can this pathway be in particular grapevine tissues.
on the other hand, hormone signalling was enriched within several expression profiles peaking in the morning in tempranillo and verdejo berries.
hormonal responses coordinated by light- and circadian clock-mediated signalling were shown important for daily promotion of growth in arabidopsis  <cit> .
thus, it might be worth testing whether similar mechanisms could modulate grape growth and ripening processes that are also under the control of phytohormones  <cit> .
for instance, relative to gibberellins signalling, rhythmic expression of della genes peaking around dawn under circadian clock control has been described in arabidopsis seedlings  <cit>  and similar expression profile of vvgai <dig> was observed in verdejo pericarp.
berry composition features affected by daily changing conditions
final composition of mature grape berries is highly related with activity of secondary metabolism.
the differential daily cycling responses between berry tissues and cultivars included greater variations in the expression of phenylpropanoid biosynthetic transcripts in the tempranillo experiment despite these results could be underestimated in our approach.
vvmyba <dig> and vvmyba <dig> tfs were co-induced during the daytime only in tempranillo berry flesh together with a large group of flavonoid biosynthetic transcripts.
the hybridization signal for vvmyba <dig> and vvmyba <dig> microarray probe sets was constitutively higher in tempranillo berry skin and in verdejo pericarp when compared to tempranillo flesh samples .
this could indicate that cross hybridization between both tf probe sets and transcripts takes place in the microarray because, as known, vvmyba <dig> should be expressed in verdejo berries but not vvmyba <dig>  whose expression is not expected in white cultivars  <cit> .
indeed, vvgt <dig> , a direct target of vvmyba <dig> tf and considered to encode the anthocyanidin-glycosyltransferase catalysing the limiting step for anthocyanin accumulation in berry skin  <cit> , was constitutively more highly expressed in tempranillo berry skin than in flesh and very much less in verdejo pericarp .
further research would be required to show whether vvmyba <dig> or vvmyba <dig> positively regulate flavonoid biosynthesis in grape mesocarp cells in a circadian cycle-dependent manner.
the activation of terpenoid biosynthetic genes during the daytime, including monoterpenoid synthases, concurrently to that of light signalling components took place only in verdejo white and aromatic berries.
these light signalling genes included a homolog of hy <dig>  a key gene involved in light perception transduction in arabidopsis  <cit> , and vvmybf <dig>  which is induced by sunlight and promotes flavonol biosynthesis in grapes  <cit> .
absolute expression of vvmybf <dig> and this hy <dig> homolog  during the daytime was higher in verdejo than in tempranillo samples .
photosynthetic genes activated during the daytime only in verdejo could also be indicative of greater light responses in these berries as some of them are induced by hy <dig> and by the circadian clock during the daytime in arabidopsis  <cit> .
considering these results together with the fact that scent terpenoids accumulation in grapes is enhanced by light  <cit> , it would be interesting to test whether hy <dig> and vvmybf <dig> tfs regulate light induced terpenoid biosynthesis in grapes.
additionally, the extension of circadian gene expression analysis to other black and white cultivars under different cycles of controlled temperature and irradiation conditions would help to explain if some of these specific responses are related to the experimental conditions, the berry skin colour or to other cultivar-specific features.
concerning primary metabolism-related ripening processes, breakdown of malic acid in grapes is hastened by high temperatures  <cit> .
only a few genes related to this process cycled in expression despite great temperature oscillations took place during the sampling time courses.
interestingly, two dicarboxylate vacuolar transporters, with a likely function in organic acids import into the vacuole  <cit> , were induced at 12:45 pm in tempranillo berries opposing high temperature effects on malic acid metabolisation.
in general, results suggest that circadian changes of expression in grapes at late ripening stages affect more deeply secondary than primary metabolism pathways.
part of these expression changes may have important consequences on the final berry composition including berry features depending either on the plant genotype or on the circadian fluctuations in the surrounding environment.
CONCLUSIONS
our study demonstrates the existence of daily oscillatory changes in the grapevine berry transcriptome at late ripening stages.
these expression profiles involved responses to temperature and light fluctuation as well as oscillation of circadian clock components.
identified expression profiles suggest that regulatory genes such as a vvrve <dig> circadian clock homologous gene as well as constans-like, wrky and gras family tfs might promote night-activated processes in all analysed experiments.
evidence of circadian variations in the expression of these elements in pre-ripe grapes was shown for the first time in this study.
certain tissue-specific oscillatory expression profiles were related to berry skin and flesh features.
circadian expression profiles in secondary metabolism-related transcripts may impact on the progression of ripening and the final composition of berries, which could result in part from the cyclic expression of chief regulatory genes like vvmyba <dig>  vvmyba <dig>  vvmybf <dig>  vvmyb <dig> or a hy <dig> homolog.
in summary, our results could contribute to understanding the progress of berry ripening over sub-daily time scales in response to circadian environmental changes.
these findings could lead to new considerations in models directed to predict the effect of ripening season environmental conditions on the final berry composition.

 the grapevine fruit is a characteristic berry consisting of an external skin surrounding a fleshy pulp that encloses seeds. grape biochemical composition is crucial for the different uses of grapes such as winemaking, production of juice and liqueur, fresh consumption or elaboration of raisins, and the final composition is mostly achieved during the ripening phase. grape ripening is triggered once the seeds have developed and radically changes fruit features from frugivore-repulsive to -attractive. this shift comprises sugar accumulation in the vacuoles of mesocarp cells accompanied by organic acid metabolisation and titratable acidity reduction. mechanisms to protect seeds from biotic and abiotic stress sources are also activated, mainly in the berry skin. phenolic compounds are accumulated, including phytoalexins, damaging light-absorbing compounds and animal-attractive anthocyanin pigments. changes in terpenoid composition result in attractive aromatic profiles, while other compounds of the same family accumulate to accomplish protective functions. although grapevine ripening is regulated by an intrinsic program that is partially triggered by hormonal signals, the process is also strongly modulated by external factors that influence the final berry composition and its commercial quality. temperature is a major factor altering grape ripening with high temperatures hastening organic acid metabolisation and inhibiting anthocyanin accumulation. irradiation intensity and quality perceived in the berry skin produces changes in secondary metabolism. light promotes flavonols and terpenoids accumulation with some effects being more specifically related with ultraviolet radiation, which is also able to enhance stilbenoids accumulation. moreover, temperature and light conditions cyclically fluctuate under field environments due to the earth's rotation and, in fact, the strength of their daily oscillation has been shown to affect grape ripening. for instance, reduction in temperature fluctuation intensity hastens berry ripening and alters flavonoid partitioning and high temperatures applied during night-times are also able to reduce anthocyanin accumulation. in contrast, light pulses applied during the night-time enhance anthocyanin accumulation. these effects of environmental variation on grape ripening rely, at least in part, on changes at the level of gene expression. thus, it can be hypothesised that daily oscillations in factors such as temperature, light or humidity could influence grape ripening progression and consequently on its final composition by means of conditioning circadian fluctuations on the grape transcriptome. an internal molecular clock is another daily cycling element modulating plant physiology. core clock components are transcription factors that reciprocally regulate their expression resulting in characteristic circadian expression profiles. altogether, they determine daily rhythms of expression in a great proportion of plant transcriptomes. in addition, the circadian clock integrates inputs from fluctuating conditions like light and temperature, which allows for plants to discriminate daily and seasonal changes. in this way, the circadian clock together with environmental factors control several plant physiological and developmental outputs to be triggered at the proper time of day or season such as photosynthesis, starch and nitrogen metabolism, growth, cold acclimation, bud dormancy, flowering time, tuber formation or stomata and organ movements. since grape ripening is modulated by light and temperature inputs, it is possible that part of this modulation could be integrated by circadian clock components as shown in other plant developmental processes. however, the role of circadian clock on fruit ripening control has scarcely been considered before. in this study, nimblegen grapevine whole genome microarrays were used to follow transcriptome variations in ripening grapes throughout a 24 h daily cycle. tempranillo and verdejo cultivars were analysed as genotypes appreciated in spanish viticulture for the production of red and white quality wines, respectively. verdejo is a cultivar with different berry features than tempranillo bearing white skin colour and a characteristic aromatic profile, giving rise to wines distinguishable from other white wines. given that berry flesh and skin ripening transcriptional programs significantly differ, and assuming that this divergence could be more pronounced in cultivars bearing black-skinned berries accordingly with the presumably higher gene expression activity related to the phenylpropanoid pathway in its berry skin, both pericarp tissues were separately analysed in tempranillo to be more precise. this experiment was carried out using potted plants grown under controlled greenhouse conditions that allowed for identifying tissue-shared as well as tissue-specific cyclic expression profiles under monitored variation of environmental parameters. part of this response was confirmed in a different experiment in verdejo berries grown under field environmental conditions despite divergences in genotype, berry colour, berry total soluble solids or sampling day photocycles and thermocycles.  circadian oscillatory gene expression profiles in tempranillo berries grown under controlled conditions the question of whether sub-daily changes in environmental conditions produce specific transcriptome shifts that could influence grapevine berry ripening was addressed. alterations putatively related with changes in metabolism taking place at the last steps of berry ripening, in the absence of earlier processes like berry softening that are more related to the veraison stage, were analysed. for this purpose, pre-ripe berries of tempranillo were collected at six different time points throughout a 24 h period under a controlled oscillation of light, temperature and humidity conditions. sampling time points were selected to include times of maximum and minimum temperatures and the end of light and dark periods in order to maximize differences in the environmental conditions. global transcriptome changes were first separately followed in berry skin and flesh. after applying a 5% false discovery rate in limma and 2-fold change cut-offs over normalized expression data on each tissue, a higher number of differentially expressed genes was identified in the berry skin than in the flesh. four main expression profiles were observed in both analysed tissues, which involved oscillatory patterns following inflexion points determined by temperature or light presence variations. they included transcripts up-regulated at 12:45 pm, transcripts down-regulated at 12:45 pm, transcripts up-regulated during the daytime, and transcripts up-regulated during the night-time. night responses were maintained until the early morning in flesh clusters f and f. in both berry tissues, responses to heat were activated at 12:45 pm as indicated by the significant over-representation of ‘hsp-mediated protein folding’ and ‘hsf family transcription factor ’ functional categories in clusters s and f. other functional categories were enriched only in specific flesh or skin clusters. noticeably, among them, is the enrichment of ‘ethylene-mediated signalling pathway’, ‘circadian clock signalling pathway’, ‘biotic stress response’, and ‘mapk cascade’ categories as well as of ap constans-like, wrky and gras families of tfs in skin cluster s gene silencing by mirna was specifically enriched in cluster f thus, daily light and temperature variations are related with the detection of gene expression oscillations in the skin and the flesh of pre-ripe berries, whereas genes showing oscillating expression were more frequent in the skin than in the flesh. in order to search for those transcripts showing similar expression fluctuation in both pericarp tissues, a similar analysis was run considering skin and flesh samples in the same time point as replicates. in that way, deg were identified, however, this analysis could underestimate transcripts similarly oscillating in expression in both tissues but involving differences in absolute expression levels. this result indicates that a great proportion of daily oscillating transcripts were shared between both pericarp tissues. in fact, four expression profiles were identified for pericarp deg that resembled those identified in individual tissues and shared with them enriched functional categories. cluster sf grouping transcripts up-regulated at 12:45 pm in both pericarp tissues was enriched in ‘hsp-mediated protein folding’ and ‘hsf family tf’ and moreover in ‘vacuolar malate transport’ due to the presence of two transcripts putatively coding for tonoplast dicarboxylate transporters. on the other hand, cluster sf grouping transcripts down-regulated at 12:45 pm in both pericarp tissues was enriched in ‘jaz family tf’; while transcripts in cluster sf up-regulated in the pericarp during the night-time, included an over-representation of ‘circadian clock signalling pathway’ as well as in kip santa, gras and constans-like families of tfs. thus, a considerable number of transcripts showed similar circadian expression oscillation in grape skin and flesh, including responses to temperature and light cycles as well as circadian clock-related genes. transcripts differentially oscillating in expression between tempranillo berry flesh and skin once shown that berry skin and flesh shared daily expression changes in tempranillo, tissue specific oscillations in expression in berry flesh or skin were searched by a direct comparison of both tissues. the circadian series on each tempranillo berry tissue were compared in a two-class masigpro time series analysis, identifying deg between tissues. among them, more transcripts were specifically oscillating in expression in the skin than in the flesh confirming results of analyses performed on each tissue. four different skin-specific and two flesh-specific oscillatory expression profiles were identified. moreover, transcripts that were up-regulated during the night-time in both tissues but showing inductive profile maintained until the early morning only in the flesh were grouped in cluster svsf finally, transcripts showing an increased expression throughout the 24 h sampling cycle specifically in the skin were grouped in cluster svsf the most abundant expression profiles were those including transcripts up-regulated specifically in the berry skin either during the daytime or the night-time. cluster svsf was significantly enriched in ‘wounding’ functional category. in addition, this cluster included transcripts encoding phenylpropanoid biosynthetic enzymes like a ferulate 5-hydroxylase and an anthocyanidin glycosylase; terpenoid biosynthetic enzymes including a vinorine synthase and two β-amyrin synthase; and myb tfs. cluster svsf was enriched in ‘protein kinase’, ‘wrky family tf’ and ‘ap family tf’ functional categories. cluster svsf included transcripts whose expression was down-regulated specifically in the skin at time points in the middle of dark and light periods. notably this cluster is enriched for ‘r proteins from plant-pathogen interaction’ and ‘auxin transport’ categories, the last one resulting from the presence of five transcripts encoding putative auxin transporters. heavy metal responsive transcripts and c2c2-dof family tfs were also enriched within this cluster. interestingly, the group of transcripts up-regulated during the daytime only in the flesh was enriched in ‘secondary metabolism’ classified transcripts, mainly related to the presence of flavonoid biosynthetic transcripts including one phenylalanine ammonia-lyase, three chalcone synthase, flavonoid hydroxylases and two flavonol synthases. other phenylpropanoid biosynthetic genes and two monoterpenoid biosynthetic transcripts coding for 10-geraniol hydroxylase were also found in cluster svsf together with vvmyba and vvmyba tfs; the former being a key regulator promoting anthocyanin biosynthesis in grapevine. some of these transcripts underwent a net increase in expression in the flesh during the studied 24 h period. ‘stilbenoid biosynthesis’ was prominently over-represented in both clusters that involved profiles with expression peaking in the flesh at the early morning. this was due to the presence of a set of stilbenoid biosynthetic transcripts among transcripts more specifically up-regulated in the flesh and another set of transcripts within the group of transcripts that peaked earlier in the skin at the end of the night-time. in this manner, out of stilbenoid biosynthetic genes annotated in the microarray were differentially involved in these two expression profiles and all of them were located in two gene clusters at chromosomes and, which suggest a functional diversification of grapevine stilbene synthase promoters before the extensive duplication of these genes. cluster svsf was also enriched in ‘nitrogen metabolism’ and ‘auxin signalling’ resulting both from the presence of seven nitrilase encoding transcripts. an iaa31-like and an auxin-responsive d encoding transcript were also included within this profile. the enrichment of ‘oxidative stress response’ in cluster svsf was determined by the presence of six laccases, one lactoylglutathione lyase and one glutathione s-transferase. concerning cluster svsf it was enriched in ‘wrky family tf’ and ‘ammonium transport’ and included other phenylpropanoid biosynthetic transcripts in addition to stss and the vvmyb tf. finally, cluster svsf was enriched in transcripts belonging to the ‘oxidative stress response’ category. therefore, circadian gene expression patterns in the flesh and the skin of pre-ripe berries include both tissue-shared and tissue-specific oscillatory genes that in several cases might be related to specific tissue functions. circadian oscillatory gene expression profiles in verdejo berries grown under field conditions a parallel circadian experiment was set up on field grown cultivar verdejo plants to test whether comparable daily transcriptome changes would take place in different cultivars and under vineyard conditions. environmental conditions were slightly different during the sampling day at the verdejo vineyard. temperature showed a wider oscillation, dark periods were slightly longer and relative humidity was lower. therefore, although differential expression detected between tempranillo and verdejo could not be undoubtedly explained, at least those shared expression profiles could be considered as consistent cycling responses. as an important proportion of circadian regulated genes showed the same oscillatory expression profile either in tempranillo berry skin or flesh, the analysis of verdejo whole pericarp from pre-ripe berries was considered suitable to be compared. a total of genes significantly oscillating in expression were identified in verdejo berry pericarps. almost one third of verdejo deg was also significant in any of the comparisons carried out in tempranillo indicating that an important part of this response was shared between experiments regardless of genotypic, environmental and developmental differences. when verdejo deg were clustered, only three main expression profile groups were detected because profiles of transcripts up-regulated around noontime could not be distinguished from those up-regulated during the daytime contrasting with the tempranillo experiment. such discrepancy could result from differential oscillation in environmental conditions as berries experienced the maximum temperature near noontime in the tempranillo experiment and close to the end of the daytime in the verdejo one. moreover, putative responses to night conditions were maintained until the early morning in verdejo pericarp as observed for tempranillo berry flesh. the number of transcripts up-regulated during the daytime in verdejo was almost half to that of transcripts up-regulated during the night-time. one expression profile included all daytime up-regulated transcripts. similarly to tempranillo transcripts up-regulated at 12:45 pm, cluster v was enriched in ‘hsp-mediated protein folding’; however it also showed enrichment in ‘erf subfamily tf’ and ‘plant photosystem i supercomplex’ among other categories. noticeably, cluster v included a hy5-like bzip tf. concerning night up-regulated transcripts in verdejo pericarp, the absence of light in the first time point of this experiment made it possible to discriminate between transcripts putatively up-regulated by low temperatures or by darkness. functional categories over-represented among transcripts up-regulated in tempranillo berries during the night-time were also enriched in these two verdejo clusters such as ‘gras family tf’ in cluster v and ‘constans-like family tf’ in cluster v additionally, cluster v was enriched in ‘biotic stress response’ and ‘abc multidrug transport’. two putative 1-deoxy-d-xylulose 5-phosphate synthase encoding transcripts were present within cluster v, while the first one was also induced during the night-time in tempranillo berry skin together with another putative dxs. verdejo cluster v was enriched in ‘xyloglucan modification’ because it contained eight xyloglucan endotransglucosylase encoding transcripts. ‘cytokinin-mediated signalling pathway’ was also over-represented in cluster v which included three transcripts coding for authentic-response regulator type-a and one for histidine kinase ahk2-like cytokinin receptor, while ‘response regulator family tf’ was enriched in both v and v clusters of night-time up-regulated transcripts also due to the presence of several arr genes. noticeably, vvgai, encoding a repressor of gibberellin responses, was also grouped within the v cluster. although differential expression profiles between tempranillo and verdejo could not be clearly attributable to differences in genotype or growth conditions, time series in both cultivars were explicitly compared. considering that tempranillo berry skin and flesh shared circadian oscillatory gene expression and that a considerable portion of the ripening transcriptional program is common to both pericarp tissues, tempranillo skin and flesh samples on each time point were considered as replicates to be compared to verdejo pericarp samples. in this manner, deg were identified. more transcripts specifically oscillating in expression were identified in verdejo than in tempranillo. however, they were enriched in similar functional categories as those regulated in tempranillo. they involved ‘hsp-mediated protein folding’ and ‘response regulator family tf’ among transcripts up-regulated only in verdejo during the daytime or during the night-time, respectively. apart from these similarities, differences in secondary metabolism were observed. three caffeoyl-coa o-methyltransferase and one secoisolariciresinol dehydrogenase phenylpropanoid biosynthetic transcripts were up-regulated during the daytime only in tempranillo. another secoisolariciresinol dehydrogenase and a flavonoid hydroxylase were induced during the night-time specifically in tempranillo. in contrast, terpenoid biosynthetic transcripts specifically oscillated in expression in verdejo including a linalool synthase, a myrcene synthase, two β-amyrin synthase and three lupeol synthase that were up-regulated during the daytime; while an isoprene synthase, a β-ocimene synthase and a germacrene synthase were up-regulated during the night-time. interestingly, vvmybf, a light inducible tf, was up-regulated during the daytime only in the verdejo experiment. these results indicate that circadian expression changes in grapes are mostly consistent among different genotypes. divergences putatively related with genotypic or environmental differences were also found. circadian clock homologous genes oscillating in expression in grapevine berries given that expression profiles enriched in circadian clock signalling-related functional categories were identified in all analysed datasets, homologs to core clock components within the grapevine 12× v genome gene predictions were searched for, in order to analyse their expression profiles throughout the studied 24 h cycles. vvlhy was the only identified grapevine ortholog to arabidopsis thaliana cca and lhy partially redundant morning core clock myb genes. this indicates that the lhy duplication observed in other angiosperms took place after the separation of clades giving rise to the vitaceae as has previously been reported. vvlhy did not show any significant oscillation in expression in the experiments contrasting to the results obtained for another homolog to morning myb tfs, vvrve. this grapevine gene was previously annotated as cir1/rve, despite it is the closest homolog to arabidopsis reveille circadian clock gene. transcription of vvrve was up-regulated during the night-time and peaked in the morning in tempranillo and verdejo pericarps. in addition, vvppr7_ was up-regulated during the daytime in tempranillo pericarp, while vvelf3_ expression peaked in the morning in verdejo berries. several grapevine homologs to night core clock genes could be identified in the grapevine genome including vvtoc, although they did not significantly change in expression. therefore, core circadian clock gene homologs are present in the grapevine genome and only some of them seem to oscillate in expression in berry tissues. acoverage of the arabidopsis protein by the grapevine predicted protein sequence. bclusters of significant transcripts in figures  and and additional files and discussion berry transcriptome plasticity in the adaptation to daily environmental changes plants as sessile organisms have evolved physiological and developmental adaptations to changes in the surrounding environment. grape transcriptome changes promoted by the soil composition and weather conditions of different years and locations have been described as examples of plasticity displayed by the berry ripening transcriptome. herein changes are shown in the berry ripening transcriptome taking place within the same plants as a response to sub-daily fluctuations in the surrounding environment. after applying consistent statistical analyses and cut-offs, a total of and transcripts showing circadian oscillation in expression were respectively identified in tempranillo and verdejo experiments. although these daily oscillations could involve a smaller proportion of circadian cycling genes than in other species or tissues, they are larger than those transcriptome changes observed between consecutive berry ripening stages near maturity, consistently described to be low under diverse experimental conditions and genotypes. thus, sub-daily variation of gene expression in the berry could be more decisive for the final berry composition than other developmental cues at late ripening stages. most detected cycling profiles were apparently directed to adapt berry cells to changing environmental conditions such as temperature and light. they included the over-expression of hsps, erf tfs and bag5-like bcl- associated athanogene positively correlating with high temperatures as well as the down-regulation of abc transporters expression negatively correlating with them as described for thermotolerance responses in muscat hamburg berries. the identification of a higher number of hsps specifically induced during the daytime in verdejo berries, which experienced more extreme temperatures than the tempranillo ones, also suggests the existence of plasticity in berry thermotolerance responses. nonetheless, some of these as well as other circadian transcriptional responses observed herein may not result in physiological responses as they might not always involve changes at the protein level. on the other hand, transcriptional changes related to biotic stress responses were activated during the night-time and more greatly in samples including berry skin. induction of pathogen r-gene-mediated resistance before dawn, the time when the pathogen normally disperses the spores, has been described as a mechanism controlled by core clock genes in arabidopsis to anticipate infection. an analogous mechanism in the grape berry, an organ particularly susceptible to pathogen attacks when approaching ripeness, could have been evolutionarily selected to ensure seeds dispersal and species survival. tfs of the wrky family were also induced around dawn in all analysed datasets suggesting that they could participate in the regulation of this defence response. stss were simultaneously induced only in tempranillo skin and flesh. higher expression of sts transcripts has also been correlated with low temperatures in corvina berries grown under different environmental conditions. circadian regulation of sts genes could be partially controlled by vvmyb which was also induced at dawn only in tempranillo berry skin, since this tf is able to activate sts promoters in grapevine. in contrast, activation of defence pathways against virus at warmest times of day could be characteristic of grape berries in view that rna silencing argonaute homologues were induced around noontime in all experiments. in fact, plant rna silencing-mediated defence pathways are generally less active under low temperatures and more active at high temperatures concomitantly with plant virus activity. constans-like genes participate in the integration of light and circadian clock signalling, whereas transcripts within this family consistently peaked around dawn both in tempranillo and verdejo berries. relative to circadian clock signalling, cca and lhy are considered the core morning loop genes in the arabidopsis molecular clock. vvlhy was the only ortholog to these genes found in the grapevine reference genome and did not oscillated in expression in our experiments. in contrast, in all analysed grape samples, vvrve showed a cyclic expression profile paralleling these of lhy/cca genes in arabidopsis leaves. this gene is the ortholog of rve another myb cca subfamily circadian clock-related gene that, like lhy and cca is able to bind to the ‘evening element’ in the promoter of evening-phased circadian rhythmic genes. taking into account these coincidences, it might be interesting to test whether vvrve could act as a core clock morning gene in grapevine pre-ripe fruits. absence of significant regulation in other central clock homologs in the grape analyses may suggest that a simplified version of the clock would operate in grapevine late ripening fruits as shown in other non-leaf arabidopsis organs like roots. the biology of grapevine fruits is extremely different to that of plant tissues and species where the circadian clock signalling has been explained. therefore, further research is required to confirm how distinct can this pathway be in particular grapevine tissues. on the other hand, hormone signalling was enriched within several expression profiles peaking in the morning in tempranillo and verdejo berries. hormonal responses coordinated by light- and circadian clock-mediated signalling were shown important for daily promotion of growth in arabidopsis. thus, it might be worth testing whether similar mechanisms could modulate grape growth and ripening processes that are also under the control of phytohormones. for instance, relative to gibberellins signalling, rhythmic expression of della genes peaking around dawn under circadian clock control has been described in arabidopsis seedlings and similar expression profile of vvgai was observed in verdejo pericarp. berry composition features affected by daily changing conditions final composition of mature grape berries is highly related with activity of secondary metabolism. the differential daily cycling responses between berry tissues and cultivars included greater variations in the expression of phenylpropanoid biosynthetic transcripts in the tempranillo experiment despite these results could be underestimated in our approach. vvmyba and vvmyba tfs were co-induced during the daytime only in tempranillo berry flesh together with a large group of flavonoid biosynthetic transcripts. the hybridization signal for vvmyba and vvmyba microarray probe sets was constitutively higher in tempranillo berry skin and in verdejo pericarp when compared to tempranillo flesh samples. this could indicate that cross hybridization between both tf probe sets and transcripts takes place in the microarray because, as known, vvmyba should be expressed in verdejo berries but not vvmyba whose expression is not expected in white cultivars. indeed, vvgt, a direct target of vvmyba tf and considered to encode the anthocyanidin-glycosyltransferase catalysing the limiting step for anthocyanin accumulation in berry skin, was constitutively more highly expressed in tempranillo berry skin than in flesh and very much less in verdejo pericarp. further research would be required to show whether vvmyba or vvmyba positively regulate flavonoid biosynthesis in grape mesocarp cells in a circadian cycle-dependent manner. the activation of terpenoid biosynthetic genes during the daytime, including monoterpenoid synthases, concurrently to that of light signalling components took place only in verdejo white and aromatic berries. these light signalling genes included a homolog of hy a key gene involved in light perception transduction in arabidopsis, and vvmybf which is induced by sunlight and promotes flavonol biosynthesis in grapes. absolute expression of vvmybf and this hy homolog during the daytime was higher in verdejo than in tempranillo samples. photosynthetic genes activated during the daytime only in verdejo could also be indicative of greater light responses in these berries as some of them are induced by hy and by the circadian clock during the daytime in arabidopsis. considering these results together with the fact that scent terpenoids accumulation in grapes is enhanced by light, it would be interesting to test whether hy and vvmybf tfs regulate light induced terpenoid biosynthesis in grapes. additionally, the extension of circadian gene expression analysis to other black and white cultivars under different cycles of controlled temperature and irradiation conditions would help to explain if some of these specific responses are related to the experimental conditions, the berry skin colour or to other cultivar-specific features. concerning primary metabolism-related ripening processes, breakdown of malic acid in grapes is hastened by high temperatures. only a few genes related to this process cycled in expression despite great temperature oscillations took place during the sampling time courses. interestingly, two dicarboxylate vacuolar transporters, with a likely function in organic acids import into the vacuole, were induced at 12:45 pm in tempranillo berries opposing high temperature effects on malic acid metabolisation. in general, results suggest that circadian changes of expression in grapes at late ripening stages affect more deeply secondary than primary metabolism pathways. part of these expression changes may have important consequences on the final berry composition including berry features depending either on the plant genotype or on the circadian fluctuations in the surrounding environment.  our study demonstrates the existence of daily oscillatory changes in the grapevine berry transcriptome at late ripening stages. these expression profiles involved responses to temperature and light fluctuation as well as oscillation of circadian clock components. identified expression profiles suggest that regulatory genes such as a vvrve circadian clock homologous gene as well as constans-like, wrky and gras family tfs might promote night-activated processes in all analysed experiments. evidence of circadian variations in the expression of these elements in pre-ripe grapes was shown for the first time in this study. certain tissue-specific oscillatory expression profiles were related to berry skin and flesh features. circadian expression profiles in secondary metabolism-related transcripts may impact on the progression of ripening and the final composition of berries, which could result in part from the cyclic expression of chief regulatory genes like vvmyba vvmyba vvmybf vvmyb or a hy homolog. in summary, our results could contribute to understanding the progress of berry ripening over sub-daily time scales in response to circadian environmental changes. these findings could lead to new considerations in models directed to predict the effect of ripening season environmental conditions on the final berry composition. 
additionally, a molecular circadian clock regulates daily cyclic expression in a large proportion of the plant transcriptome modulating multiple developmental processes in diverse plant organs and developmental phases. indeed, homologs of core clock genes were identified in the grapevine genome and, among them, vvreveille, showed a consistent circadian expression rhythm in every grape berry tissue analysed. light signalling components and terpenoid biosynthetic transcripts were specifically induced during the daytime in verdejo, a cultivar bearing white-skinned and aromatic berries, whereas transcripts involved in phenylpropanoid biosynthesis were more prominently regulated in tempranillo, a cultivar bearing black-skinned berries. thus, in this study, gene expression throughout 24 h periods in pre-ripe berries of tempranillo and verdejo grapevine cultivars was followed to determine whether different ripening transcriptional programs are activated during certain times of day in different grape tissues and genotypes. circadian cycling of fruit transcriptomes has not been characterized in detail despite their putative relevance in the final composition of the fruit. microarray analyses identified oscillatory transcriptional profiles following circadian variations in the photocycle and the thermocycle. the transcriptome of ripening fruits varies in response to daily environmental changes, which might partially be under the control of circadian clock components.
 temperature and solar radiation influence vitis vinifera l. berry ripening. both environmental conditions fluctuate cyclically on a daily period basis and the strength of this fluctuation affects grape ripening too. additionally, a molecular circadian clock regulates daily cyclic expression in a large proportion of the plant transcriptome modulating multiple developmental processes in diverse plant organs and developmental phases. circadian cycling of fruit transcriptomes has not been characterized in detail despite their putative relevance in the final composition of the fruit. thus, in this study, gene expression throughout 24 h periods in pre-ripe berries of tempranillo and verdejo grapevine cultivars was followed to determine whether different ripening transcriptional programs are activated during certain times of day in different grape tissues and genotypes.  microarray analyses identified oscillatory transcriptional profiles following circadian variations in the photocycle and the thermocycle. a higher number of expression oscillating transcripts were detected in samples carrying exocarp tissue including biotic stress-responsive transcripts activated around dawn. thermotolerance-like responses and regulation of circadian clock-related genes were observed in all studied samples. indeed, homologs of core clock genes were identified in the grapevine genome and, among them, vvreveille, showed a consistent circadian expression rhythm in every grape berry tissue analysed. light signalling components and terpenoid biosynthetic transcripts were specifically induced during the daytime in verdejo, a cultivar bearing white-skinned and aromatic berries, whereas transcripts involved in phenylpropanoid biosynthesis were more prominently regulated in tempranillo, a cultivar bearing black-skinned berries.  the transcriptome of ripening fruits varies in response to daily environmental changes, which might partially be under the control of circadian clock components. certain cultivar and berry tissue features could rely on specific circadian oscillatory expression profiles. these findings may help to a better understanding of the progress of berry ripening in short term time scales. circadianfruit ripeninggene expressiongrapevinelightmicroarrayphenylpropanoidtemperatureterpenevitis vinifera 