BACKGROUND
grapes  are a highly valued horticultural crop with production covering approximately  <dig> million ha in  <dig> countries.
the grape berry is extremely rich in secondary metabolites ranging from anthocyanins, carotenoids, norisoprenoids, tannins, terpenes, and other volatile organic compounds.
these metabolites are very important to wine production as they affect wine quality by determining its color, aroma, and flavor  <cit> .
wine-grape growers adopt various vineyard management practices in order to optimize grape and wine quality attributes.
some of these practices involve producing  and/or selecting  berries according to size with the belief that better wines are produced from small berries due to a higher skin to berry weight ratio.
the reasoning being that this higher skin to berry weight ratio results in higher concentrations of key secondary metabolites accumulated in the skin  <cit> .
these practices are becoming increasingly popular and machines are even sold to automatically sort berries based on size.
the grape berry is a non-climacteric fruit with a double-sigmoidal growth curve that can be separated into three major stages  <cit> .
the first stage of development sees a rapid increase in berry size due to high rates of cell division and expansion in the berry pericarp.
coinciding with the rapid growth is the biosynthesis of phenolic compounds, such as tannins and hydroxycinnamates, and organic acids, such as tartaric and malic acid.
in the second stage, the berry experiences a lag phase, where pericarp growth is arrested and the embryo completes its development.
at the end of the second stage, the berry undergoes veraison  and enters the third and final stage.
during this phase, the berry experiences a second period of rapid cell expansion as the pericarp grows to its final size.
many changes in berry metabolism occur: accumulation of sugars, decrease in organic acid concentration, and production of various secondary metabolites.
thus, berry size and composition will differ depending on the stage of development.
hormones play central roles in berry ripening and as environmental mediators .
during the first phase of development, auxins are present at high levels, and then decrease to very low levels as the berry undergoes veraison  <cit> .
just prior to veraison, a small transient peak in ethylene is observed  <cit>  as well as sharp increases in abscisic acid  .
these observations indicate that aba and ethylene are strong candidates as promoters of ripening.
treating berries with these hormones can affect the timing of veraison as well as other parameters, including fruit size and composition .
for example, treating berries with auxin prior to veraison temporarily delays berry growth, and sugar and anthocyanin accumulation  <cit> .
many factors influencing berry size are intrinsic, being related to the individual berry itself, such as seed number  <cit>  and seed weight  <cit> .
recent evidence has shown that pinot noir and cabernet sauvignon seeded berries showed greater cell division and expansion than unseeded ones, resulting in larger berries  <cit> .
gouthu and deluc  <cit>  found a relationship between seed relative abundance and hormone content, where there were distinct differences in auxin and aba levels between berries with different seed to berry weight ratios prior to veraison.
external factors, such as the environment and cultural practices, can also play a remarkable role in determining berry size.
water deficit  <cit>  and some pruning strategies  <cit>  can result in smaller berries with higher phenolic content in the skin.
however, other treatments may lower the concentration of some phenolics along with a reduction in berry size.
this is seen with differences in the microclimate of clusters, where shading results in smaller berries with a lower phenolic content in the skin compared to light exposed clusters  <cit> .
there are contrasting conclusions regarding differences in berry composition when comparing berries strictly on size.
some research indicates no differences between sizes  <cit> , while other research shows smaller sauvignon blanc berries having lower methoxypirazine concentrations  compared to larger berries  <cit> .
similarly, there is no consensus on whether smaller berries make superior wines.
gil et al.
<cit>  demonstrated that smaller grapes produced wines of deeper color and that size is inversely correlated with the concentration of phenolics, such as anthocyanins and stilbenes.
in contrast, others have found that smaller berries do not translate into discernable differences in the resulting wines  <cit> .
changes in gene expression often parallel changes in berry skin metabolism throughout berry development  and in response to stresses, such as water deficit  <cit>  and uv  <cit> .
many transcripts related to berry ripening and quality traits, such as those involved in hormone, phenylpropanoid, terpenoid, fatty acid, and cell wall metabolism, are modulated during development and in response to the environment.
however, despite the strong impact on wine composition and quality, the regulation of transcripts/pathways related to ripening and berry composition in skins of differently sized berries is still unknown.
in this study, our goal was to better understand the relationship between berry size, fruit ripening, and berry metabolism.
we characterized how field grown merlot berries differ in their ripening, metabolism, and transcriptional program in relation to berry size.
we considered two extreme size classes and compared berry physiology, composition, and whole genome gene expression  in the skin throughout development in order to determine the biological processes that discriminate between the two classes of berries.
methods
sample collection and physiological measurements
v. vinifera l.’merlot’ clone  <dig> vines were grown in a vineyard located in oliver .
three biological replicates of fruit samples were harvested from three separate rows.
each replicate had  <dig> to  <dig> berries randomly collected from  <dig> vines per row at  <dig>   <dig>   <dig>   <dig>   <dig>   <dig>   <dig>   <dig>   <dig>   <dig>   <dig>  and 121 days after anthesis  in  <dig>  care was taken to avoid physical damage as berries were trimmed off the cluster at the pedicel and placed into aluminized mylar zip-lock bags to prevent water and turgor loss  <cit> .
the bags were then immediately placed into a cooling box at ambient temperature and transported to the laboratory.
berry diameter and elasticity , a precise quantification of berry firmness  <cit> , were recorded for each berry  <cit>  and berries were individually bagged, labeled, and stored at −80 °c until the following analyses were conducted.
from the population of berries at each sampling point, for each biological replicate seven berries were selected and pooled according to their size.
two pools were created: large berries, consisting of berries included in the top 90–95th percentile and small berries, consisting of berries included in the bottom 10–15th.
these berries were used to calculate the evolution of e and total soluble solids , using a manual refractometer, across the season.
additional pools of seven berries selected from the same percentile at  <dig> ,  <dig> ,  <dig>  and  <dig>  were used for both metabolite  and rna-sequencing  analyses.
three biological replicates for each treatment were considered for both metabolite and rna-seq analysis.
liquid chromatography  and lc-mass spectrophotometric  analysis
berries were peeled while still frozen using a scalpel.
flesh tissue was then used for determining sugar and organic acid concentration, and skin tissue for anthocyanin content.
for the determination of sugar  and organic acid  content, flesh tissues were grounded to a fine powder and centrifuged at  <dig>  g for 10 min.
afterwards, the supernatant was passed through a  <dig>  μm pvdf 13 mm filter  and measured via hplc on an agilent  <dig> hplc system with a refractive index detector .
an aliquot of 20 μl of extract was injected into a nucleogel® ion  <dig> oa column  , maintained at 71 °c.
the mobile phase was  <dig>  mm h2so <dig> with a flow rate of  <dig>  ml/min.
quantification of metabolite concentration  was based on calibration curves of authentic standards.
for the extraction of anthocyanins,  <dig>  g of skins were grounded and extracted in 2 ml 50 %  methanol in water for 3 h with rigorous shaking.
the supernatant was centrifuged, filtered and measured on an agilent  <dig> lc/msd trap xct plus mass spectrometer.
separation of anthocyanin compounds was achieved on an agilent zorbax sb-c <dig> column  held at 57 °c.
the mobile phases used were: water-formic acid , solvent a, and acetonitrile-formic acid , solvent b. flow rate was  <dig>  ml/min.
the solvent gradient program was  <dig>  min, 6 % b; 4 min, 10 % b; 13 min, 25 % b; 20 min, 35 % b; 25 min, 60 % b; 30 min, 95 % b; and 32 min, 6 % b. the anthocyanins mass spectra were analyzed after electrospray ionization  in alternating positive and negative ionization mode with a scan range between  <dig> and 850 m/z.
quantification of single and total anthocyanins was based on malvidin 3-glucoside equivalents .
the percentages of 3′4′-oh, 3′4′5′-oh, and methoxylated anthocyanins were calculated among monoglucoside anthocyanins.
rna extraction, sequencing, and data analysis
extraction of total rna was achieved with the spectrum plant total rna kit  using ~80 mg of grounded skins according to the manufacturer’s protocol.
the integrity of the extracted rna was determined on an agilent  <dig> bioanalyzer  ensuring a rin score > <dig>  prior to library construction.
ribosomal rna depleted library construction was performed using an in-house workflow using customized kits from neb at the canada’s michael smith genome sciences center  followed by sequencing  on an illumina hiseq  <dig> platform  and in-house quality control and filtering of 75-bp paired-end generated reads.
filtered pair-end reads were aligned against the 12× reference genome  <cit>  using burrows-wheeler aligner with default parameters  <cit> .
read summarization was performed with htseq-count  with intersection non-empty settings  <cit>  using the grapevine 12× genome assembly available from ensemblplants .
differential gene expression analysis was carried out using edger   <cit> .
genes were deemed differentially expressed  between pairwise comparisons at a threshold of false discovery rate  <  <dig> .
transcript abundance was calculated as fragments per kilobase of exon per million  mapped reads using edger.
transcripts having fpkm values < <dig>  and assigned counts < <dig> were discarded.
clustering, functional enrichment, and promoter analysis
clustering of de genes using edger’s estimated gene expression  log <dig> fold change  between small/large berries was performed using the k-means method with  <dig> iterations and the speaman’s rank correlation as the similarity metric.
the latest grapevine gene annotations based the 12× v <dig> models were obtained from  <cit> .
a separate functional annotation of transcripts was performed using the mercator pipeline  <cit>  to ascribe potential gene function and mapman bins  <cit>  prior to gene enrichment analysis.
mapman bin categories were considered significantly enriched  as determined by fisher’s exact test adjusted with bonferroni correction for multiple testing correction.
based on the 12× grapevine genome assembly, all grapevine promoter sequences  were retrieved from the cribi grape genome database   <cit> .
target sequences  of  <dig> plant transcription factors  representing  <dig> families recently characterized in arabidopsis thaliana  <cit>  and the ones from place  <cit>  were retrieved.
these sequences were scanned in promoter regions of de genes within clusters and selected gene families.
motif overrepresentation was calculated according to  <cit>  using hypergeometric test and adjusted with false discovery rate  for multiple testing correction when appropriate.
putative cres was considered significantly enriched if the associated fdr <  <dig> .
statistical analysis
a one-way anova was performed using jmp  <dig>  to detect significant differences  in berry components, e, tss, and sugar, organic acid, and anthocyanin levels between small and large berries treatments at each sampling point.
RESULTS
analysis of physiological and compositional parameters between small and large berries
the diameters of all the berries considered in the experiment are reported in fig.
1a.
berry diameter, total soluble solids , and elasticity  evolution in small and large berries during fruit development are reported in fig.
1b, c, and d. despite the differences in absolute values, the evolution of berry diameter was similar between small and large berries throughout the season .
the tss levels of small and large berries increased from 51 daa until harvest .
a faster increase in tss was observed for large berries from 60 to 67 daa.
tss levels of small berries remained relatively low until 67 daa; afterward, a sharp increase was observed.
e measures berry firmness, so a firm berry has higher e values , and as a berry softens e decreases  <cit> .
e of the small and large berries strongly decreased from  <dig> to  <dig> daa, afterward the decrease was gradual .
differences in e levels and in the rate of e decrease between small and large berries were apparent at certain developmental stages.
for example, at  <dig> and  <dig> daa, large berries had higher e than small berries, while between  <dig> and  <dig> daa, small berries had higher e than large ones.
although no significant differences in e levels were observed at  <dig> and  <dig> daa, a steeper drop of e was observed in large than in small berries from  <dig> to  <dig> daa .fig.
<dig> evolution of berry diameter, total soluble solids, and elasticity.
berry diameter of the entire sampled population.
diameter,  total soluble solids, and  elasticity of small and large berries during development.
green and purple indicates the recorded color of each individual berry.
means and standard errors are reported for each berry group at each developmental stage.
* indicates a significant difference  between small and large berries
berries from  <dig>   <dig>   <dig>  and  <dig> daa were selected for further physiological and biochemical analysis .
the skin to berry weight ratio was significantly higher in small than large berries at all four developmental stages ; while large berries had significantly higher seed to berry weight ratios at  <dig> and  <dig> daa .
in addition, significant differences in the number of seeds/berry were observed between sizes, where large berries have more seeds , consistent with previous studies  <cit> .fig.
<dig> berry features.
skin, flesh, and seed weight,  skin to berry weight ratio,  seed/berry weight ratio, and  seeds/berry number in small and large berries at  <dig>   <dig>   <dig>  and  <dig> daa.
*, **, and *** indicate level of significance of p <  <dig> , p <  <dig> , and p <  <dig> , respectively
fig.
<dig> berry composition.
glucose + fructose,  malic acid, and  tartaric acid concentration, expressed as mg/g berry, in small and large berries at  <dig>   <dig>   <dig>  and  <dig> daa.
anthocyanin levels expressed as  mg/g skin,  mg/g berry, and  mg/berry in small and large berries at  <dig>   <dig>   <dig>  and  <dig> daa.
means and standard errors are reported for each berry group at each sampling.
*, **, and *** indicate level of significance of p <  <dig> , p <  <dig> , and p <  <dig> , respectively
glucose and fructose levels increased sharply from  <dig> to  <dig> daa and continue to increase until  <dig> daa .
malic and tartaric acids  were at high levels at  <dig> daa, and progressively decreased from  <dig> daa onwards.
the levels of sugars and organic acids reported here are comparable with the levels found in different cultivars at the same developmental stage  <cit> .
no significant differences in the concentrations of sugars, glucose and fructose , and malic acid  were observed between small and large berries for any of the sampling dates.
tartaric acid concentration was significantly higher in small than in large berries at  <dig> and  <dig> daa .
the total anthocyanin content  was significantly lower in small berries at  <dig> daa .
although the recorded veraison  date in the vineyard was 60 daa, the large increase in sugar levels in large berries prior to 70 daa  could have stimulated an earlier accumulation of anthocyanin in these berries  <cit> .
the accelerated drop in e observed in large berries, as well as the faster increase in sugar and anthocyanin levels, suggest that large berries approach ripening faster than small berries.
although the anthocyanin concentration in skins  was similar between small and large berries at  <dig> and  <dig> daa , small berries had a significantly higher anthocyanin concentration expressed as mg/g of berry .
vice versa, total anthocyanin content, expressed as mg/berry, was higher in large berries .
this suggests that the increase in anthocyanin concentration observed in small berries at  <dig> and  <dig> daa was not due to a higher synthesis of these pigments but to the higher skin to berry weight ratio .
this higher proportion of skin tissue determines the higher concentration of anthocyanin in small berries as observed in  <cit> .
analysis of transcript differences between small and large berries
to determine the influence of berry size on the berry skin transcriptome, illumina mrna sequencing was performed on berry skins at the four selected developmental stages .
using the 12× v <dig> genome as reference  <cit> , an average of  <dig> million high-quality paired-end reads per sample were successfully mapped, which typically corresponds to  <dig>  % of paired-end reads for each library .
an average of  <dig> million paired-end reads per sample was assigned to transcripts.
we detected the presence of  <dig>  unique transcripts expressed in at least one of four developmental stages.
approximately  <dig> ,  <dig> ,  <dig> , and  <dig>  transcripts were expressed in berry skins at  <dig>   <dig>   <dig>  and  <dig> daa, respectively.
a principal component analysis  was undertaken to analyze the level of similarity of the transcriptomes analyzed.
the first three principal components explained a cumulative variance of  <dig>  %; with the first, second, and third principal component explaining  <dig> ,  <dig> ,  <dig>  % of the variance, respectively .
inspection of the pca plots revealed a clear separation of berry skin transcriptome based on the developmental stage and not on the biological variation within a developmental stage.
furthermore, a separation of the transcriptomes driven by the berry size was more evident at  <dig>   <dig>  and  <dig> daa, while it was undetected at  <dig> daa.
a total of  <dig> unique genes  were identified to be differentially  expressed between small and large berries in at least one developmental stage .
a total of  <dig> ,  <dig> ,  <dig>  and  <dig>  were differentially expressed  between small and large berries at  <dig>   <dig>   <dig>  and  <dig> daa, respectively .
gouthu et al.
<cit>  demonstrated that differences in the ripening program  was reflected at the transcript level with large differential expression of berry transcripts when comparing between berry classes differing in softness and coloration at mid-veraison , regardless of tissue.
these differences were drastically reduced to near zero at maturity.
our study revealed that differences in the skin transcriptomes between small and large berries are larger at earlier stages of development  than at later stages of development .
this difference, together with the differences in fruit physiology and composition observed at the corresponding stages further suggests an asynchrony between small and large berries at early stages of development .
the reduction of de  suggests a process of resynchronization of the transcriptome between small and large berries during development.
interestingly, there was a second increase of the number of de transcripts at  <dig> daa.
this might reflect the activation of an over-ripening/senescence program in the skins of small berries  <cit> .
cramer et al.
<cit>  investigated the evolution of the skin transcriptome at late developmental stages, including over-ripening stages.
approximately 15 % of the top  <dig> de genes were the same when results were compared between the de genes between small and large berries at  <dig> daa  in this study and between  <dig> and  <dig> °brix in cramer et al.
<cit> , suggesting that small berries are ahead in ripening at these later stages.fig.
<dig> analysis of the berry skin transcriptome of small and large berries.
principal component analysis  of the berry skin transcriptome of small  and large  berries at  <dig> ,  <dig> ,  <dig> , and  <dig>  daa.
the venn diagram represents the common and unique genes differentially expressed between small and large berries at  <dig>   <dig>   <dig>  and  <dig> daa.
differentially expressed genes in each intersection of the venn diagram are described in additional file 1: table s <dig>   the box plot and smoothed line plot represents the response of small versus large berries and dynamic change of gene expression during berry development, respectively.
differentially expressed genes were clustered using the k-means algorithm.
the log <dig> fold changes between small and large and log <dig>  values at  <dig>   <dig>   <dig>  and  <dig> daa were used.
outlier log <dig> fold change values are represented as grey circles
differentially expressed genes were clustered into  <dig> clusters based on their log <dig> fold change differences in small with respect to large berries at each development stage using the k-means clustering .
functional annotation of de genes according to high-level mapman ontology categories  <cit>  showed that the largest proportion was involved in rna regulation and protein metabolism , while transport, cell wall, signaling, and primary and secondary metabolism categories contributed between  <dig> and 8 % of the total de genes .
a total of  <dig>  transcripts annotated as potential grapevine transcriptional regulators  <cit>  were de between small and large berries within each sampling date , indicating a difference in transcript regulatory programs between small and large berries.
validating the observation that a large number of tf were de between small and large berries, many cis-regulatory elements  related to binding sites of various tf families were also enriched among the de genes , which may play an important role in the regulation of de genes.
the frequent occurrences of multiple enriched cres within promoter regions of de transcripts will be discussed further in the context of metabolic pathway regulation .table  <dig> enrichment of mapman functional categories  of k-means assigned clusters containing differentially expressed transcripts comparing skins of large and small berries.
the table contains detailed information of the cluster size, bin code and associated description, and the bonferroni-adjusted p-value .
only high-level bins are presented in this table
because of the observed differences between small and large berries at the physiological level and of the potential role of some of the de genes on determining fruit and wine quality, we focused our discussion on pathways related to hormone metabolism and signaling, cell wall modifications, and flavonoid, stilbenoid, and fatty acid biosynthesis.
modulation of hormone metabolism and signaling in small and large berries
regulation of fruit development and ripening involves major metabolic changes regulated by complex interactions among hormones and not by a single hormone in isolation  <cit> .
in grapes, aba promotes berry ripening and auxins delay this process, while results on ethylene are mixed  <cit> .
one hundred fifty-seven genes annotated to function in hormone metabolism  were de between small and large berries .
a total of  <dig>   <dig>  and  <dig> transcripts related to aba, auxin, and ethylene metabolism and signaling, respectively, were the most represented among hormone related genes, followed by  <dig>   <dig>   <dig>  and  <dig> transcripts related to jasmonic/salicylic acid, cytokinin, brassinosteroid, and gibberellin metabolism and signaling, respectively.
due to their major role in the ripening process  <cit> , emphasis will be given to the transcripts involved in the biosynthetic pathways of aba, auxin, and ethylene .fig.
<dig> evolution during development and fold change between small and large berries of abscisic acid , auxin, and ethylene genes differentially expressed at  <dig>   <dig>   <dig>  and  <dig> daa.
evolution, based on the mean log <dig>  of small and large berries, and  log <dig> fold  changes.
the relative log <dig>  values registered in small and large berries on average during berry development in a are depicted by green  and blue .
grey color indicates the absence  of detectable transcripts at the corresponding stage.
blue and red colors in b indicate downregulated and upregulated transcripts, respectively, in small berries in relation to large berries.
boxes with bold margins indicate significant differences  between berry size treatments at a given developmental stage.
the cluster column in b indicates the cluster number the associated transcript belongs to nced, 9-cis-epoxycarotenoid dioxygenase; aba <dig>  xanthoxin dehydrogenase; taa/tar, tryptophan aminotransferase of arabidopsis1/tryptophan aminotransferase related ; yuc, yucca; gh, iaa-amido synthetase; aco, 1-aminocyclopropane-1-carboxylic acid oxidase
transcripts encoding structural enzymes of the aba biosynthetic pathway were modulated during fruit development and were de between small and large berries .
transcripts encoding 9-cis epoxycarotenoid dioxygenase  regulate aba biosynthesis and the alteration of nced expression have direct implications in aba concentration and ripening-related traits in fruits  <cit> .
the expression patterns of three de nced transcripts in the skin were distinct .
for example, nced <dig>  transcripts peak at  <dig> daa and gradually decrease towards  <dig> daa, nced <dig>  transcripts peak at  <dig> daa and again at  <dig> daa, while nced <dig>  transcripts progressively increase towards  <dig> daa.
these observations were consistent with previous studies showing that nced transcripts are under complex regulation in the berry  and that expression of nceds is required to maintain in situ berry aba biosynthesis.
several studies have highlighted that nced <dig> is the enzymatic isoform which is correlated with berry aba accumulation  <cit> .
therefore, the upregulation of this gene in small berries at  <dig> daa may contribute to higher aba levels at this stage; however, later during ripening when aba levels are still high  <cit>  no significant difference in nced <dig> transcripts was observed in the skins of small and large berries.
instead, nced <dig> transcripts were highly downregulated at  <dig> daa in skins of small with respect to large berries.
this might have contributed to differences in overall aba accumulation and lower aba accumulation in small berries at early stages of ripening would be consistent with the delay in sugar and anthocyanin accumulation observed at  <dig> and  <dig> daa, respectively .
in addition, the downregulation of ripening-associated nced <dig> gene in small compared to large berries from  <dig> daa onwards might also reflect the lower aba biosynthetic capacity of small compared to large berries at  <dig> and  <dig> daa.
nevertheless, nced <dig> was observed to be upregulated in small compared to large berries at  <dig> daa.
collectively, different sized berries exhibit a different regulation of nced transcripts, and significantly lower nced transcripts levels were found in small berries than in large ones during berry ripening, especially at  <dig> daa, with potential effects on the ripening process.
the genes encoding tryptophan aminotransferase of arabidopsis1/tryptophan aminotransferase related  and yucca  are critical for regulating auxin levels and ripening in young berries  <cit> .
the expression of transcripts encoding taa1/tar <dig> –  <dig> and yuc <dig> throughout berry development  have also been reported in shiraz berries  <cit> .
these genes were modulated during fruit development and were also de between small and large berries in this study .
three taa/tar transcripts  and another  peaked in the skins of berries harvested at  <dig> and  <dig> daa, respectively.
one yuc transcript  was specifically present at  <dig> daa, another yuc transcript  peaked around veraison , and other two yucs  were mostly present during late ripening.
these observations were consistent with previous studies showing taa/tar and yuc transcript evolution and auxin accumulation during berry development and ripening  <cit> .
at  <dig> daa, when auxins levels are high, transcripts encoding tar <dig>  and a yuc  were highly upregulated in small with respect to large berries.
similarly, tar <dig>  transcripts were also upregulated at  <dig> daa.
however, tar <dig>  transcripts, which have been shown to relate with the accumulation of auxin at early stages  <cit> , were downregulated in small with respect to large berries at  <dig> daa.
several studies have indicated that auxin conjugation through the action of iaa-amido synthetases , which conjugates aspartic acid to auxin, is critical for ripening initiation and the regulation of auxins in the berry during development  <cit> .
interestingly, gh3- <dig> transcripts  which display a ripening-associated developmental accumulation were significantly upregulated in the skins of small compared to large berries.
therefore, significant differences in ripening physiology of the two berry classes  and significant modulation of auxin metabolic pathways points to the possible involvement of auxin and its conjugates in determining asynchrony between berries of different sizes.
the biosynthesis of ethylene is determined, in part, by the action of 1-aminocyclopropane 1-carboxylate  oxidase , a key enzyme involved in the production of ethylene using acc as precursor  <cit> .
several aco-encoding members were also de between small and large berries , many of which exhibit highest expression at  <dig> daa followed by a gradual decrease until  <dig> daa .
although grapevine is a non-climacteric fruit, previous findings have shown an increase of aco activity and ethylene production at stages before veraison  <cit> .
two aco transcripts  displayed strong upregulation at  <dig> daa in small compared to large berries.
this might provide evidence that in small berries at pre-veraison stages a higher production of ethylene may in fact be delaying the ripening process and not enhancing ripening, as exemplified by the delayed sugar accumulation at  <dig> daa and the lower anthocyanin levels at  <dig> daa in small berries .
indeed, recent studies have demonstrated application of ethylene at pre-veraison stages delays ripening through the activation of auxin biosynthesis pathways, significantly postponing sugar and anthocyanin accumulation, as well as the berry growth  <cit> .
ethylene may also play a role later in ripening, especially in the skin, as the abundance of many transcripts involved in ethylene biosynthesis and signaling peaked from  <dig> °brix onwards, stages when desirable flavor and aroma compounds accumulate  <cit> .
in this study, the significant upregulation of two aco transcripts  from  <dig> daa onwards, indicates that ethylene might contribute to the induction of pathways important for fruit flavor in the skin of small berries compared to large ones  <cit> .
among hormone related transcription factors, the grapevine bzip tf, vvabf <dig>  have been shown to modulate aba-dependent berry ripening processes through the induction of cell wall hydrolytic enzymes and complex modulation of multiple hormonal pathways including aba, auxin, and ethylene  <cit> .
expression of vvabf <dig> transcript  was highest at  <dig> daa corroborating its ripening-associated expression and its role in berry ripening .
interestingly, vvabf <dig> was first upregulated in small berries at  <dig> daa and subsequently downregulated at  <dig> daa compared to large berries.
this modulation might mediate the altered hormonal metabolism profiles involving nced, taa/yuc, and aco transcripts between small and large berries at  <dig> and  <dig> daa.
also, the regulation of cell wall degradation and expansion via abf <dig> may explain the slower softening rate experienced in small compared to large berries observed at the onset of fruit ripening.
in summary, the differential regulation of several nced, taa/yuc, and aco transcripts between the berry size classes suggest that hormonal pathways are differential regulated and might therefore contribute to variations in aba, auxin, and ethylene levels in the skin of small and large berries with potential effects on skin metabolism and composition.
however, additional studies with intensive sampling strategies and measurement of these hormones in the two berry classes will be needed to gain a deeper understanding on the relative contribution of each hormone and its influence on berry size and fruit composition traits.
modulation of cell wall metabolism in small and large berries
in fleshy fruits, the remodeling of cell wall architecture during fruit development affects fruit softening  <cit>  and involves concerted changes in cell wall-related gene expression and multiple enzyme activities  <cit> .
influence of berry skins in determining post-veraison berry growth  <cit>  and the role of cell wall degradation and modification enzymes in mediating this process  <cit>  have been previously reported.
the altered transcriptional profiles of cell wall genes in skins between small and large berries highlights the potential role of cell wall modification genes in modulating berry growth and softening.
in this study, more than  <dig> annotated cell wall  transcripts exhibited de between small and large berries during fruit development .
transcripts related to cellulose synthesis , cell wall modification , degradation , and pectin esterification  were significantly modulated among the berry size classes in these clusters.
a decrease in berry e has been shown to be the earliest ripening-associated event observed at the onset of ripening  <cit> .
emphasis will be given on the cell wall genes differentially expressed at  <dig> daa , given that this is the developmental stage that immediately follows the large decrease in berry e and the stage when small and large berries displayed differences in e.fig.
<dig> evolution during development, fold change values between small and large berries, and selected cis-regulatory element profile of cell wall transcripts differentially expressed at  <dig>   <dig>   <dig>  and  <dig> daa.
the heat map represents the evolution , based on the mean log <dig>  of small and large berries, and log <dig> fold  changes  of cell wall degradation  and modification  genes.
the relative log <dig>  values registered in small and large berries on average during berry development in a and c are depicted by green  and blue .
grey color indicates the absence  of detectable transcripts at the corresponding stage.
blue and red boxes in b and d indicate downregulated and upregulated transcripts, respectively, in small berries in relation to large berries.
boxes with bold margins indicate significant differences  between small and large berries at a given developmental stage.
the cluster column in b and d indicates the cluster number the associated transcript belongs to.
em,  <dig> -beta-mannan endohydrolase; pl pectate lyase; pg polygalacturonase; xet/xth xyloglucan endotransglucosylase/hydrolase.
the heat map illustrates the presence of ap2/erf and bhlh/nac cis regulatory elements in cell wall genes.
purple and white colors depict the presence and absence of the respective cre in the promoter regions of the gene
we observed that  <dig> cell wall degradation genes , consisting of six xyloglucan endo-transglycosylase/trans-hydrolase, two  <dig> -beta-mannan endohydrolase, five pectate lyases, and three polygalacturonases, were downregulated in skins of small compared to large berries at  <dig> daa .
this observation may reflect lower cell wall degradation activity in small than in large berries, which supports the slower rate of softening from  <dig> to  <dig> daa.
pectin degrading enzymes, such as polygalacturonases and pectate lyases, are two principal enzymes involved in tomato fruit softening through active cell wall pectin de-polymerization  <cit> .
studies in tomato  <cit> , apple  <cit> , and strawberries  <cit>  have demonstrated that silencing of polygalacturonase gene expression does not always affect fruit softening.
pose et al.
<cit>  found large differences in strawberry fruit firmness at harvest in strawberry polygalacturonase 1-suppressed lines, but the changes in firmness in tomato during softening were very small  <cit>  and in apple most transgenic polygalacturonase suppression lines had no effect on fruit firmness  <cit> .
nevertheless, silencing of pectate lyases transcripts result in firmer fruits both during softening and at harvest  <cit> .
in plants, the transcription factor apetala2/ethylene responsive factor  belongs to a multigenic family involved in the control of metabolism, stress response, and plant development through the binding of dre and gcc-related motifs in promoters regions of regulatory targets .
several studies in fruits have demonstrated that ap2/erf tfs contribute in regulating ripening-related processes, especially fruit softening, by targeting cell wall degradation genes .
in this study, significant enrichment of ethylene related cres, such as drecrtcoreat, cbfhv, and ltrecoreatcor <dig> , in cluster  <dig> genes also corresponds to the presence of ap2/erf cres in promoter regions of seven  predicted cell wall degrading transcripts.
in agreement, we observed eight de ap2/erf tfs which were significantly downregulated in small compared to large berries at  <dig> daa; four of which were ripening-associated .
this suggest that ap2/erf tfs may be critical in regulating cell wall degrading transcripts in grapevines, and differences in ap2/erf tf regulation between small compared to large berries prior to ripening, may have affected the progression of softening reaching this stage .
expansins act as primary cell wall loosening agents and incorporators of new cell wall material during the first growth phase and as mediators of cell wall disassembly by facilitating the contact between cell wall material and degradation enzymes during stage iii of growth in grapevine berries  <cit> .
the grapevine genome contains  <dig> expansin genes classified into four distinct subfamilies   <cit> , of which two grapevine expansins were demonstrated to promote cell expansion  <cit> .
many cell wall expansin transcripts  were highly expressed at  <dig> daa and six of these transcripts were significantly downregulated in skins of small with respect to large berries.
moreover, other six transcripts were downregulated at  <dig> daa when their level of expression was on average low .
the downregulation of expansin transcripts observed in the skins of small compared to large berries at  <dig> and  <dig> daa indicates that cell wall loosening in the skin may be generally reduced, potentially limiting skin and mesocarp expansion and resulting in smaller berries.
indeed, fruits of tomatoes overexpressing expansin displayed enhanced softening and were significantly larger compared to controls at various stages of fruit development  <cit> .
the regulation of some expansin transcripts such as expa <dig>  and expb <dig>  by vvceb <dig> have also been proposed  <cit> .
analysis of vvceb <dig>  a fruit ripening-specific bhlh transcription factor, gene expression from  <dig> different varieties exhibiting differences in berry size revealed a strong correlation between fruit size and vvceb <dig> transcript accumulation  <cit> .
in this study, vvceb <dig> transcript  reached a maximum at  <dig> daa and slowly decreased until  <dig> daa .
vvceb <dig> was downregulated in skins of small compared to large berries at  <dig> and  <dig> daa, but were only significant at  <dig> daa.
the down regulation of vvceb <dig> in small berries at  <dig> and  <dig> daa paralleled the down regulation of many expansin transcripts.
promoter analysis performed on de expansins for bhlh-related cre revealed that the catgtg element, a typical dna binding domain for bhlh tfs  <cit> , was enriched in the promoter region of  <dig> out of   <dig> de expansin genes .
this suggests that upstream control of expansins differentially expressed in small and large berries may in part be mediated by vvceb <dig> during ripening.
nevertheless, previous studies have also shown that nac tfs can bind specifically to catgtg elements  <cit> , which might provide evidence for the regulation of expansin transcripts by nac tfs.
we observed that the majority of nacs, such as a ripening-associated nac <dig>  which is highly expressed at  <dig> and  <dig> daa, were downregulated in small compared to large berries at  <dig> daa in parallel with downregulation of several expansin genes .
a recent study has also shown that polymorphisms in vvnac <dig> were associated with berry size variation among  <dig> grapevine varieties  <cit> .
together, this transcriptional network involving bhlh and nac tfs may be critical for regulating berry growth and size determination in grapevine.
modulation of flavonoid and stilbenoid pathways in small and large berries
flavonoids and stilbenes are plant secondary metabolites commonly found across the plant kingdom.
these specialized metabolites fulfill diverse physiological roles in stress response, as antioxidants, and during reproduction  <cit> ; in grapevine, they strongly affect grape and wine quality  <cit> .
we observed that differences in berry size were related to large differences in the expression of flavonoid genes .
the de flavonoid genes were predominantly assigned to clusters  <dig>  and  <dig> .fig.
<dig> modulation in berry skin transcripts involved in the phenylpropanoid and flavonoid pathway in small and large berries at  <dig>   <dig>   <dig>  and  <dig> daa.
blue and red boxes indicate downregulated and upregulated transcripts, respectively, in small berries in relation to large berries.
boxes with bold margins indicate significant differences  between berry size treatments at a given developmental stage.
transcription factors  involved in the regulation of the phenylpropanoid and/or the flavonoid pathway transcripts are depicted in dotted lines.
pal phenylalanine lyase; c4h cinnamate-4-hydroxylase; 4cl 4-coumarate:coenzyme a ligase; chi chalcone isomerase; chs chalcone synthase; f3h flavanone 3-hydroxylase; f3′h flavonoid 3′-hydroxylase; f3′5′h flavonoid 3′5′-hydroxylase; fls flavonol synthase; dfr dihydroflavonol 4-reductase; lar leucoanthocyanin reductase; ldox leucoanthocyanin dioxygenase; ufgt udpglucose:flavonoid 3-o-glucosyltransferase; and aomt anthocyanin o-methyltransferase.
all other information is available at additional file 1: table s5
three chalcone synthases , and three chalcone isomerases  -coding genes were differentially modulated in small and large berries.
one chs  and one chi  were downregulated in small relative to large berries at  <dig> daa when the pathway precursors are used for the production of flavan-3-ol and proanthocyanidins.
later during development, one chs  was upregulated at  <dig> daa, while two  out of three chss and all the chis mentioned above were downregulated in small in comparison to large berries at  <dig> daa.
of the total 16 flavonoid 3′5′-hydroxylases  found in the grapevine genome, we observed seven f3′5′h transcripts  that were significantly downregulated in small berries at  <dig> and  <dig> daa .
similarly, two flavonoid 3′-hydroxylases  and one flavanone 3-hydroxylase  -coding genes, were also significantly downregulated in small berries at  <dig> daa .
f3′5′h and f3′h enzymes have been shown to be critical in determining the accumulation of di- and tri-hydroxylated flavonoids and the ratio between blue and red anthocyanin in grapes .
the simultaneous downregulation of both f3′h and f3′5′h transcripts in small compared to large berries, particularly at  <dig> daa, did not affect the relative abundance of the different anthocyanin forms .
flavonol synthase  is the key enzyme for the production of flavonols in grapevines  <cit> .
of the five fls transcripts encoded in the genome, the two transcriptionally active fls  exhibited a ripening-associated accumulation in the skin ; similar to that observed in previous studies in developing grape berries  <cit> .
transcripts encoding grapevine fls <dig>  were significantly upregulated in small berries at  <dig>   <dig>  and  <dig> daa .
similarly, the fls <dig>  gene was significantly upregulated in small with respect to large berries at  <dig> daa.
previous studies have shown that fls transcript expression provides an excellent indicator for cluster light exposure and marker for flavonol synthase activity given strong positive relationship  of solar radiation intensity with fls transcripts and flavonol content in berry skins  <cit> .
in the current study, berries of different sizes were harvested from the same clusters controlling for differences in microclimate among berries.
thus differences in fls expression between berries of different sizes must have arose from endogenous mechanisms.
consistently, previous studies reported that wine produced from small berries contain more flavonols compared to wines obtained from medium and large berries  <cit> .
the expression level of key flavan-3-ol/proanthocyanidin genes codifying for leucoanthocyanidin reductase  and anthocyanidin reductase  were highly expressed at  <dig> daa and decreased in the expression at  <dig> daa, consistently with the early flavan-3-ols and proanthocyanidin accumulation during berry development  <cit> .
downregulation of anr transcript at  <dig> daa and upregulation of lar transcripts at  <dig> daa were observed in skins of small compared to large berries indicating a differential regulation of flavan-3-ol biosynthesis during stages that are critical for the accumulation of these compounds and their polymeric forms  in the skin .
glycosylation of cyanidin and delphinidin via udp glucose:flavonoid 3-o-glucosyltransferase   <cit>  is critical in anthocyanin synthesis.
the expression of grapevine ufgt  was significantly downregulated at  <dig> daa .
myba <dig> and myba <dig> tfs redundantly regulates ufgt expression and modulates anthocyanin accumulation .
the myba transcripts were specifically activated at  <dig> daa and decreases approaching  <dig> daa .
myba <dig>  was significantly downregulated in small compared to large berries at  <dig> and  <dig> daa, while the others mybas were all downregulated at  <dig> daa.
the observed downregulation of grapevine myba <dig> in small compared to large berries at  <dig> daa is consistent with the lag of anthocyanin accumulation in smaller berries suggesting a delayed onset of ripening.
although no concurrent downregulation of ufgt with myba <dig> was observed in small compared to large berries at  <dig> daa, significant reduction of anthocyanin in small berries at  <dig> daa might be strongly related to altered regulation of anthocyanin modification and transport pathways regulated by myba tfs  <cit> .
concurrent downregulation of myba <dig>  myba <dig>  and myba <dig> with many flavonoid pathways transcripts  targeted by mybas  <cit>  did not significantly reduce the concentration of anthocyanin in the skin at  <dig> daa.
anthocyanin accumulation ceased from  <dig> daa in both small and large berries.
this indicates that the precursors for anthocyanin production  are channeled to the production of other phenolic compounds, such as stilbenes, at late stages of ripening  <cit> .
a marked upregulation of genes encoding enzymes involved in the general phenylpropanoid and stilbenoid pathway, namely phenylalanine ammonia lyase , cinnamic acid 4-hydroxylase , 4-coumarate:coa ligase , and stilbene synthase , was observed at  <dig> and  <dig> daa .
of particular interests are transcripts encoding sts enzymes.
in plants producing stilbenes, sts catalyzes the production of cis- or trans-resveratrol using 4-coumaroyl-coa and three molecules of malonyl-coa.
interestingly, trends in gene expression of the majority of transcripts encoding sts and pal were very similar .
several studies have reported that multiple stss are responsible for the production of stilbene/resveratrol accumulation in the skin during berry development and in response to abiotic and biotic stress .
a large proportion of pal and sts transcripts were significantly upregulated in small berries especially at  <dig> and  <dig> daa .
similar to that of anthocyanin, stilbenes largely accumulate at the onset of ripening .
however, no de expression was observed at  <dig> daa where their expression tends to peak.
concerted downregulation of chs and upregulation of sts at  <dig> daa in small compared to large berries might relieve the competition for coumaroyl-coa and malonyl-coa substrates by chs; thereby favoring the flux to stilbene production, since anthocyanin levels have plateaued in both berries from  <dig> daa onwards .
although the higher skin to berry weight ratio often determines a higher concentration of the anthocyanin and stilbenes accumulated in the skin  <cit> , concerted upregulation of the stilbene branch in small berries may enhance stilbene levels in wines made from small berries compared to normal and large sized berries harvested at the same time  <cit> .
this induction of pal and sts genes in small compared to large berries at  <dig> daa might also be a consequence of altered hormone levels.
hormones such as ethylene and jasmonic acids are not only involved in the induction of key stilbene genes  and levels in grapevines  but also, in the case of ethylene, might take part to the over ripening process  <cit> .
two grapevine myb tfs, myb <dig>  and myb <dig>  directly regulate the biosynthesis of stilbene in grapevines  <cit> .
in this study, myb <dig> had consistent expression trends, in both small and large berries, peaking at  <dig> daa, while myb <dig> transcript evolution was not consistent between small and large berries.
in large berries, myb <dig> transcripts progressively increase until  <dig> daa before decreasing at  <dig> daa; while, in small berries, myb <dig> expression peaked at  <dig> daa before decreasing slightly at  <dig> daa and increasing again later during ripening .
nonetheless, grapevine myb <dig> and myb <dig> were significantly upregulated in small berries at  <dig> and  <dig> daa, respectively .
this result indicates their role in the regulation of sts expression and stilbene production in the skin.
modulation of fatty acid degradation pathways in small and large berries
aromatic alcohols and aldehydes derived from the fatty acid metabolism pathways , such as hexanol/hexenol and heptanal/hexanal, also contribute to late-ripening associated flavor/aromas in fruits and finished wines  <cit> .
several successive steps involving lipoxygenase , hydroperoxide lyase , alcohol dehydrogenases , and alcohol acyl transferases  enzymes regulate the production of these compounds in plant tissues  <cit> .
many transcripts encoding enzymes of the aforementioned steps were modulated during fruit development and differed in the abundance between small and large berries .
lipoxygenase enzymes are involved in the formation of hydroperoxides from linolenic acid, which can lead to the accumulation of key wine aromas in the grape.
two lox genes differentially expressed between small and large berries were highly expressed at  <dig> and  <dig> daa before decreasing towards  <dig> daa.
one lox transcript  was significantly downregulated at  <dig> daa and another lox transcript  was significantly upregulated at  <dig> daa in small compared to large berries.
hydroperoxides are then converted to c <dig> related aldehydes, which often confer grassy-related aromas via hpl.
one hpl transcript , mostly accumulated at  <dig> and  <dig> daa, was downregulated in small compared to large berries.
subsequently, adh converts c <dig> related aldehydes to c <dig> related alcohols, which can be further converted to produce volatile aromas via aats.
interestingly, all seven adh transcripts encoded in the grapevine genome were de.
four adh transcripts were highly expressed at  <dig> daa and sharply decrease from  <dig> daa and onwards; one adh transcript peaked at  <dig> daa while another two were highly expressed at  <dig> daa .
the four early developmental related adh transcripts , as well as two  of the three late-ripening adh genes, displayed significant upregulation in small compared to large berries at  <dig> daa .
on the contrary, at  <dig> daa, one  of the two adhs that are expressed at high levels at this stage was downregulated in small berries.
the accumulation profiles of the different aldehydes, alcohols, and esters in cabernet sauvignon grapes during berry development and ripening has been reported and their formation is thought to involve a synergy between the biosynthesis and catabolism of various steps  <cit> .
although the molecular mechanisms are not fully understood, high levels of volatile esters in young berries  and high levels of alcohol compounds accumulated in later stages of berry development  are reported  <cit> .
therefore, the strong upregulation of four early ripening adh and one lox transcripts in small compared to large berries might favor a higher production of c <dig> aldehydes and subsequent turnover/flux to alcohols/esters  during early development  in small compared to large berries.fig.
<dig> berry skin transcript and selected cis-regulatory element profile of small and large berries of the fatty acid degradation/c <dig> volatile biosynthesis pathway at  <dig>   <dig>   <dig>  and  <dig> daa.
simplified pathway schematic.
the heat map represents the transcript evolution , based on the mean log <dig>  in small and large berries, and log <dig> fold  changes .
the relative log <dig>  values from the four time points in b are depicted by green  and blue .
grey color indicates the absence  of detectable transcripts at the corresponding stage.
blue and red boxes in c indicate downregulated and upregulated transcripts, respectively, in small berries in relation to large berries.
boxes with bold margins indicate significant differences  between small and large berries at a given developmental stage.
the cluster column in c indicates the cluster the associated transcript belongs to.
the heat map illustrates the distribution of mads box cres in promoter regions of aroma-related transcripts differentially expressed between small and large berries.
purple and white colors depict the presence and absence of each cres, respectively, in the promoter regions of the relevant transcripts.
lox lipoxygenase; hpl hydroperoxide lyase; adh alcohol dehydrogenase; aat alcohol acyl transferases
in tomato, a mads-box transcription factor  is a negative regulator of fruit ripening and acts as a modulator of aroma production in fruits via direct regulation of adh, lox, and hpl promoters  <cit> .
we identified nine mads-box tfs differentially expressed between small and large berries: four genes were allocated to clusters  <dig>  two to clusters  <dig>  and one to cluster  <dig>   <dig>  and  <dig> individually.
of note are the predicted homologs of tomato rin , allocated into cluster  <dig>  that share high expression similarity with four adh transcripts from  <dig> to  <dig> daa in small and large berries .
analysis of the promoter regions of the total de fatty acid degradation pathway genes  for various mads-box binding site  revealed that one motif  was located within all tested promoter regions except promoter region of an adh transcript .
these observations indicate that berry size may be associated with a different accumulation of aromatics in the fruit, likely through the coordinate regulation of mads-box tfs and structural genes of the fatty acid degradation pathway.
CONCLUSIONS
significant differences in the physiology, biochemistry, and transcripts are found in different sized berries during fruit development.
as small and large berries approach the onset of ripening, clear differences in the rate of development were apparent.
around the onset of ripening, the steeper drop in elasticity, more rapid accumulation of sugars, lower tartaric acid, and greater anthocyanin levels in large compared to small berries suggest that fruit size is associated to changes in the ripening physiology of the berry, where large berries approach ripening faster.
these differences correspond to congruent changes in the hormonal pathways related to aba, auxin, and ethylene via genes encoding nced for aba, taa1/tar and yuc for auxin, and aco for ethylene.
genes encoding pathways contributing to fruit texture, flavor, and aroma were also differentially modulated accordingly to the berry size.
the modulation of cell wall degradation and modification genes  may contribute to the differences in elasticity decreases and berry growth/size.
upregulation of fatty acid degradation genes, especially adh, during early development might favor production of desirable aromatics in small berries.
in the late ripening stages, concurrent upregulation of phenylpropanoid and stilbenoid pathway genes with a parallel downregulation of the flavonoid pathway in skins of small compared to large berries indicates that smaller berries may have higher concentrations of aromatics and stilbenes, major antioxidants produced in the berry.
further investigation on endogenous and exogenous factors that regulate the fruit metabolism in berries of different sizes is necessary to identify the key factors – e.g.
hormone signals, advantage of position within a cluster that favors mineral or water uptake, or a better microclimate – that determine the berry size itself as well as the transcriptome response.
finally, a deeper investigation of the fruit composition in relation to berry size may lead to the adoption of screening strategies based on size for tailoring fruit and wine quality.
additional file
additional file 1: table s <dig>  summary of rna sequencing analysis metrics.
rna sequencing were carried in skins of small and large berries at four berry developmental stages namely  <dig> ,  <dig> ,  <dig> , and  <dig>  days after anthesis .
table s <dig>  transcript abundance of the de genes, reported as log <dig>  values, for each individual biological replicate  in each treatment  at  <dig>   <dig>   <dig>  and 121 days after anthesis.
table s <dig>  summary of differentially expressed genes between small and large berries at four berry developmental stages.
all differentially expressed genes  and detailed description of the 12×v <dig> gene id, log <dig> fold change values , differential expression calls across four developmental stages, average log <dig>  values  of small and large  berries together and individually, k-means assigned cluster , functional annotations based on grimplet et al.
<cit>  , and mapman pipeline.
table s <dig>  summary of place- and pbm-curated cis-regulatory elements  analysis of k-means assigned clusters and selected group of genes.
all information on the number of promoters with the specified cre , the number of genes within each group, number of promoters in the genome containing the specified cre , p-value and fdr of enriched cre , motif name and description, and a short description on selected cre instances in groups.
table s <dig>  summary of selected hormone, cell wall, flavonoid/phenylpropanoid, and aroma/flavor metabolic pathways transcripts differentially expressed genes between small and large berries at four berry developmental stages which have been divided into separate classes and sub-categories.
we thank mission hill family estate for providing the grapes used in this study.
we thank dr. patricia bowen  for providing laboratory space during the sample collection.
we thank canada’s michael smith genome sciences centre for the sequencing services.
funding
this study was funded by the university of british columbia, genome bc, and natural sciences and engineering research council of canada.
availability of data and materials
all raw sequence reads have been deposited in ncbi sequence read archive .
the bioproject and sra accessions are prjna <dig> and srp <dig>  respectively.
authors’ contributions
dcjw performed the physiological measurements, liquid chromatography  and lc-mass spectrophotometric analysis, rna extractions, transcriptome data analysis, cis-regulatory sequence analysis, interpreted the results, and drafted the manuscript.
rlg performed the transcriptome and cis-regulatory sequence data analysis.
nd performed part of the physiological measurements.
gag contributed to the experimental design, expert advice throughout the study, interpreted the results, and drafted part of the manuscript.
sdc conceived the study, coordinated the field experiments, performed the physiological measurements, supervised the entire study, interpreted the results, and drafted part of the manuscript.
all authors read and approved the final manuscript.
competing interests
the authors declare that they have no competing interests.

 grapes are a highly valued horticultural crop with production covering approximately million ha in countries. the grape berry is extremely rich in secondary metabolites ranging from anthocyanins, carotenoids, norisoprenoids, tannins, terpenes, and other volatile organic compounds. these metabolites are very important to wine production as they affect wine quality by determining its color, aroma, and flavor. wine-grape growers adopt various vineyard management practices in order to optimize grape and wine quality attributes. some of these practices involve producing and/or selecting berries according to size with the belief that better wines are produced from small berries due to a higher skin to berry weight ratio. the reasoning being that this higher skin to berry weight ratio results in higher concentrations of key secondary metabolites accumulated in the skin. these practices are becoming increasingly popular and machines are even sold to automatically sort berries based on size. the grape berry is a non-climacteric fruit with a double-sigmoidal growth curve that can be separated into three major stages. the first stage of development sees a rapid increase in berry size due to high rates of cell division and expansion in the berry pericarp. coinciding with the rapid growth is the biosynthesis of phenolic compounds, such as tannins and hydroxycinnamates, and organic acids, such as tartaric and malic acid. in the second stage, the berry experiences a lag phase, where pericarp growth is arrested and the embryo completes its development. at the end of the second stage, the berry undergoes veraison and enters the third and final stage. during this phase, the berry experiences a second period of rapid cell expansion as the pericarp grows to its final size. many changes in berry metabolism occur: accumulation of sugars, decrease in organic acid concentration, and production of various secondary metabolites. thus, berry size and composition will differ depending on the stage of development. hormones play central roles in berry ripening and as environmental mediators. during the first phase of development, auxins are present at high levels, and then decrease to very low levels as the berry undergoes veraison. just prior to veraison, a small transient peak in ethylene is observed as well as sharp increases in abscisic acid. these observations indicate that aba and ethylene are strong candidates as promoters of ripening. treating berries with these hormones can affect the timing of veraison as well as other parameters, including fruit size and composition. for example, treating berries with auxin prior to veraison temporarily delays berry growth, and sugar and anthocyanin accumulation. many factors influencing berry size are intrinsic, being related to the individual berry itself, such as seed number and seed weight. recent evidence has shown that pinot noir and cabernet sauvignon seeded berries showed greater cell division and expansion than unseeded ones, resulting in larger berries. gouthu and deluc found a relationship between seed relative abundance and hormone content, where there were distinct differences in auxin and aba levels between berries with different seed to berry weight ratios prior to veraison. external factors, such as the environment and cultural practices, can also play a remarkable role in determining berry size. water deficit and some pruning strategies can result in smaller berries with higher phenolic content in the skin. however, other treatments may lower the concentration of some phenolics along with a reduction in berry size. this is seen with differences in the microclimate of clusters, where shading results in smaller berries with a lower phenolic content in the skin compared to light exposed clusters. there are contrasting conclusions regarding differences in berry composition when comparing berries strictly on size. some research indicates no differences between sizes, while other research shows smaller sauvignon blanc berries having lower methoxypirazine concentrations compared to larger berries. similarly, there is no consensus on whether smaller berries make superior wines. gil et al. demonstrated that smaller grapes produced wines of deeper color and that size is inversely correlated with the concentration of phenolics, such as anthocyanins and stilbenes. in contrast, others have found that smaller berries do not translate into discernable differences in the resulting wines. changes in gene expression often parallel changes in berry skin metabolism throughout berry development and in response to stresses, such as water deficit and uv. many transcripts related to berry ripening and quality traits, such as those involved in hormone, phenylpropanoid, terpenoid, fatty acid, and cell wall metabolism, are modulated during development and in response to the environment. however, despite the strong impact on wine composition and quality, the regulation of transcripts/pathways related to ripening and berry composition in skins of differently sized berries is still unknown. in this study, our goal was to better understand the relationship between berry size, fruit ripening, and berry metabolism. we characterized how field grown merlot berries differ in their ripening, metabolism, and transcriptional program in relation to berry size. we considered two extreme size classes and compared berry physiology, composition, and whole genome gene expression in the skin throughout development in order to determine the biological processes that discriminate between the two classes of berries. methods sample collection and physiological measurements v. vinifera l.’merlot’ clone vines were grown in a vineyard located in oliver. three biological replicates of fruit samples were harvested from three separate rows. each replicate had to berries randomly collected from vines per row at and 121 days after anthesis in care was taken to avoid physical damage as berries were trimmed off the cluster at the pedicel and placed into aluminized mylar zip-lock bags to prevent water and turgor loss. the bags were then immediately placed into a cooling box at ambient temperature and transported to the laboratory. berry diameter and elasticity, a precise quantification of berry firmness, were recorded for each berry and berries were individually bagged, labeled, and stored at −80 °c until the following analyses were conducted. from the population of berries at each sampling point, for each biological replicate seven berries were selected and pooled according to their size. two pools were created: large berries, consisting of berries included in the top 90–95th percentile and small berries, consisting of berries included in the bottom 10–15th. these berries were used to calculate the evolution of e and total soluble solids, using a manual refractometer, across the season. additional pools of seven berries selected from the same percentile at,, and were used for both metabolite and rna-sequencing analyses. three biological replicates for each treatment were considered for both metabolite and rna-seq analysis. liquid chromatography and lc-mass spectrophotometric analysis berries were peeled while still frozen using a scalpel. flesh tissue was then used for determining sugar and organic acid concentration, and skin tissue for anthocyanin content. for the determination of sugar and organic acid content, flesh tissues were grounded to a fine powder and centrifuged at  g for 10 min. afterwards, the supernatant was passed through a  μm pvdf 13 mm filter and measured via hplc on an agilent hplc system with a refractive index detector. an aliquot of 20 μl of extract was injected into a nucleogel® ion oa column, maintained at 71 °c. the mobile phase was  mm h2so with a flow rate of  ml/min. quantification of metabolite concentration was based on calibration curves of authentic standards. for the extraction of anthocyanins,  g of skins were grounded and extracted in 2 ml 50 % methanol in water for 3 h with rigorous shaking. the supernatant was centrifuged, filtered and measured on an agilent lc/msd trap xct plus mass spectrometer. separation of anthocyanin compounds was achieved on an agilent zorbax sb-c column held at 57 °c. the mobile phases used were: water-formic acid, solvent a, and acetonitrile-formic acid, solvent b. flow rate was  ml/min. the solvent gradient program was  min, 6 % b; 4 min, 10 % b; 13 min, 25 % b; 20 min, 35 % b; 25 min, 60 % b; 30 min, 95 % b; and 32 min, 6 % b. the anthocyanins mass spectra were analyzed after electrospray ionization in alternating positive and negative ionization mode with a scan range between and 850 m/z. quantification of single and total anthocyanins was based on malvidin 3-glucoside equivalents. the percentages of 3′4′-oh, 3′4′5′-oh, and methoxylated anthocyanins were calculated among monoglucoside anthocyanins. rna extraction, sequencing, and data analysis extraction of total rna was achieved with the spectrum plant total rna kit using ~80 mg of grounded skins according to the manufacturer’s protocol. the integrity of the extracted rna was determined on an agilent bioanalyzer ensuring a rin score > prior to library construction. ribosomal rna depleted library construction was performed using an in-house workflow using customized kits from neb at the canada’s michael smith genome sciences center followed by sequencing on an illumina hiseq platform and in-house quality control and filtering of 75-bp paired-end generated reads. filtered pair-end reads were aligned against the 12× reference genome using burrows-wheeler aligner with default parameters. read summarization was performed with htseq-count with intersection non-empty settings using the grapevine 12× genome assembly available from ensemblplants. differential gene expression analysis was carried out using edger. genes were deemed differentially expressed between pairwise comparisons at a threshold of false discovery rate  < . transcript abundance was calculated as fragments per kilobase of exon per million mapped reads using edger. transcripts having fpkm values < and assigned counts < were discarded. clustering, functional enrichment, and promoter analysis clustering of de genes using edger’s estimated gene expression log fold change between small/large berries was performed using the k-means method with iterations and the speaman’s rank correlation as the similarity metric. the latest grapevine gene annotations based the 12× v models were obtained from. a separate functional annotation of transcripts was performed using the mercator pipeline to ascribe potential gene function and mapman bins prior to gene enrichment analysis. mapman bin categories were considered significantly enriched as determined by fisher’s exact test adjusted with bonferroni correction for multiple testing correction. based on the 12× grapevine genome assembly, all grapevine promoter sequences were retrieved from the cribi grape genome database. target sequences of plant transcription factors representing families recently characterized in arabidopsis thaliana and the ones from place were retrieved. these sequences were scanned in promoter regions of de genes within clusters and selected gene families. motif overrepresentation was calculated according to using hypergeometric test and adjusted with false discovery rate for multiple testing correction when appropriate. putative cres was considered significantly enriched if the associated fdr < . statistical analysis a one-way anova was performed using jmp to detect significant differences in berry components, e, tss, and sugar, organic acid, and anthocyanin levels between small and large berries treatments at each sampling point.  analysis of physiological and compositional parameters between small and large berries the diameters of all the berries considered in the experiment are reported in fig. 1a. berry diameter, total soluble solids, and elasticity evolution in small and large berries during fruit development are reported in fig. 1b, c, and d. despite the differences in absolute values, the evolution of berry diameter was similar between small and large berries throughout the season. the tss levels of small and large berries increased from 51 daa until harvest. a faster increase in tss was observed for large berries from 60 to 67 daa. tss levels of small berries remained relatively low until 67 daa; afterward, a sharp increase was observed. e measures berry firmness, so a firm berry has higher e values, and as a berry softens e decreases. e of the small and large berries strongly decreased from to daa, afterward the decrease was gradual. differences in e levels and in the rate of e decrease between small and large berries were apparent at certain developmental stages. for example, at and daa, large berries had higher e than small berries, while between and daa, small berries had higher e than large ones. although no significant differences in e levels were observed at and daa, a steeper drop of e was observed in large than in small berries from to daa.fig. evolution of berry diameter, total soluble solids, and elasticity. berry diameter of the entire sampled population. diameter, total soluble solids, and elasticity of small and large berries during development. green and purple indicates the recorded color of each individual berry. means and standard errors are reported for each berry group at each developmental stage. * indicates a significant difference between small and large berries berries from and daa were selected for further physiological and biochemical analysis. the skin to berry weight ratio was significantly higher in small than large berries at all four developmental stages; while large berries had significantly higher seed to berry weight ratios at and daa. in addition, significant differences in the number of seeds/berry were observed between sizes, where large berries have more seeds, consistent with previous studies.fig. berry features. skin, flesh, and seed weight, skin to berry weight ratio, seed/berry weight ratio, and seeds/berry number in small and large berries at and daa. *, **, and *** indicate level of significance of p < , p < , and p < , respectively fig. berry composition. glucose + fructose, malic acid, and tartaric acid concentration, expressed as mg/g berry, in small and large berries at and daa. anthocyanin levels expressed as mg/g skin, mg/g berry, and mg/berry in small and large berries at and daa. means and standard errors are reported for each berry group at each sampling. *, **, and *** indicate level of significance of p < , p < , and p < , respectively glucose and fructose levels increased sharply from to daa and continue to increase until daa. malic and tartaric acids were at high levels at daa, and progressively decreased from daa onwards. the levels of sugars and organic acids reported here are comparable with the levels found in different cultivars at the same developmental stage. no significant differences in the concentrations of sugars, glucose and fructose, and malic acid were observed between small and large berries for any of the sampling dates. tartaric acid concentration was significantly higher in small than in large berries at and daa. the total anthocyanin content was significantly lower in small berries at daa. although the recorded veraison date in the vineyard was 60 daa, the large increase in sugar levels in large berries prior to 70 daa could have stimulated an earlier accumulation of anthocyanin in these berries. the accelerated drop in e observed in large berries, as well as the faster increase in sugar and anthocyanin levels, suggest that large berries approach ripening faster than small berries. although the anthocyanin concentration in skins was similar between small and large berries at and daa, small berries had a significantly higher anthocyanin concentration expressed as mg/g of berry. vice versa, total anthocyanin content, expressed as mg/berry, was higher in large berries. this suggests that the increase in anthocyanin concentration observed in small berries at and daa was not due to a higher synthesis of these pigments but to the higher skin to berry weight ratio. this higher proportion of skin tissue determines the higher concentration of anthocyanin in small berries as observed in. analysis of transcript differences between small and large berries to determine the influence of berry size on the berry skin transcriptome, illumina mrna sequencing was performed on berry skins at the four selected developmental stages. using the 12× v genome as reference, an average of million high-quality paired-end reads per sample were successfully mapped, which typically corresponds to  % of paired-end reads for each library. an average of million paired-end reads per sample was assigned to transcripts. we detected the presence of unique transcripts expressed in at least one of four developmental stages. approximately,,, and transcripts were expressed in berry skins at and daa, respectively. a principal component analysis was undertaken to analyze the level of similarity of the transcriptomes analyzed. the first three principal components explained a cumulative variance of  %; with the first, second, and third principal component explaining,,  % of the variance, respectively. inspection of the pca plots revealed a clear separation of berry skin transcriptome based on the developmental stage and not on the biological variation within a developmental stage. furthermore, a separation of the transcriptomes driven by the berry size was more evident at and daa, while it was undetected at daa. a total of unique genes were identified to be differentially expressed between small and large berries in at least one developmental stage. a total of,, and were differentially expressed between small and large berries at and daa, respectively. gouthu et al. demonstrated that differences in the ripening program was reflected at the transcript level with large differential expression of berry transcripts when comparing between berry classes differing in softness and coloration at mid-veraison, regardless of tissue. these differences were drastically reduced to near zero at maturity. our study revealed that differences in the skin transcriptomes between small and large berries are larger at earlier stages of development than at later stages of development. this difference, together with the differences in fruit physiology and composition observed at the corresponding stages further suggests an asynchrony between small and large berries at early stages of development. the reduction of de suggests a process of resynchronization of the transcriptome between small and large berries during development. interestingly, there was a second increase of the number of de transcripts at daa. this might reflect the activation of an over-ripening/senescence program in the skins of small berries. cramer et al. investigated the evolution of the skin transcriptome at late developmental stages, including over-ripening stages. approximately 15 % of the top de genes were the same when results were compared between the de genes between small and large berries at daa in this study and between and °brix in cramer et al., suggesting that small berries are ahead in ripening at these later stages.fig. analysis of the berry skin transcriptome of small and large berries. principal component analysis of the berry skin transcriptome of small and large berries at,,, and daa. the venn diagram represents the common and unique genes differentially expressed between small and large berries at and daa. differentially expressed genes in each intersection of the venn diagram are described in additional file 1: table s the box plot and smoothed line plot represents the response of small versus large berries and dynamic change of gene expression during berry development, respectively. differentially expressed genes were clustered using the k-means algorithm. the log fold changes between small and large and log values at and daa were used. outlier log fold change values are represented as grey circles differentially expressed genes were clustered into clusters based on their log fold change differences in small with respect to large berries at each development stage using the k-means clustering. functional annotation of de genes according to high-level mapman ontology categories showed that the largest proportion was involved in rna regulation and protein metabolism, while transport, cell wall, signaling, and primary and secondary metabolism categories contributed between and 8 % of the total de genes. a total of transcripts annotated as potential grapevine transcriptional regulators were de between small and large berries within each sampling date, indicating a difference in transcript regulatory programs between small and large berries. validating the observation that a large number of tf were de between small and large berries, many cis-regulatory elements related to binding sites of various tf families were also enriched among the de genes, which may play an important role in the regulation of de genes. the frequent occurrences of multiple enriched cres within promoter regions of de transcripts will be discussed further in the context of metabolic pathway regulation.table enrichment of mapman functional categories of k-means assigned clusters containing differentially expressed transcripts comparing skins of large and small berries. the table contains detailed information of the cluster size, bin code and associated description, and the bonferroni-adjusted p-value. only high-level bins are presented in this table because of the observed differences between small and large berries at the physiological level and of the potential role of some of the de genes on determining fruit and wine quality, we focused our discussion on pathways related to hormone metabolism and signaling, cell wall modifications, and flavonoid, stilbenoid, and fatty acid biosynthesis. modulation of hormone metabolism and signaling in small and large berries regulation of fruit development and ripening involves major metabolic changes regulated by complex interactions among hormones and not by a single hormone in isolation. in grapes, aba promotes berry ripening and auxins delay this process, while results on ethylene are mixed. one hundred fifty-seven genes annotated to function in hormone metabolism were de between small and large berries. a total of and transcripts related to aba, auxin, and ethylene metabolism and signaling, respectively, were the most represented among hormone related genes, followed by and transcripts related to jasmonic/salicylic acid, cytokinin, brassinosteroid, and gibberellin metabolism and signaling, respectively. due to their major role in the ripening process, emphasis will be given to the transcripts involved in the biosynthetic pathways of aba, auxin, and ethylene.fig. evolution during development and fold change between small and large berries of abscisic acid, auxin, and ethylene genes differentially expressed at and daa. evolution, based on the mean log of small and large berries, and log fold changes. the relative log values registered in small and large berries on average during berry development in a are depicted by green  and blue. grey color indicates the absence of detectable transcripts at the corresponding stage. blue and red colors in b indicate downregulated and upregulated transcripts, respectively, in small berries in relation to large berries. boxes with bold margins indicate significant differences between berry size treatments at a given developmental stage. the cluster column in b indicates the cluster number the associated transcript belongs to nced, 9-cis-epoxycarotenoid dioxygenase; aba xanthoxin dehydrogenase; taa/tar, tryptophan aminotransferase of arabidopsis1/tryptophan aminotransferase related; yuc, yucca; gh, iaa-amido synthetase; aco, 1-aminocyclopropane-1-carboxylic acid oxidase transcripts encoding structural enzymes of the aba biosynthetic pathway were modulated during fruit development and were de between small and large berries. transcripts encoding 9-cis epoxycarotenoid dioxygenase regulate aba biosynthesis and the alteration of nced expression have direct implications in aba concentration and ripening-related traits in fruits. the expression patterns of three de nced transcripts in the skin were distinct. for example, nced transcripts peak at daa and gradually decrease towards daa, nced transcripts peak at daa and again at daa, while nced transcripts progressively increase towards daa. these observations were consistent with previous studies showing that nced transcripts are under complex regulation in the berry and that expression of nceds is required to maintain in situ berry aba biosynthesis. several studies have highlighted that nced is the enzymatic isoform which is correlated with berry aba accumulation. therefore, the upregulation of this gene in small berries at daa may contribute to higher aba levels at this stage; however, later during ripening when aba levels are still high no significant difference in nced transcripts was observed in the skins of small and large berries. instead, nced transcripts were highly downregulated at daa in skins of small with respect to large berries. this might have contributed to differences in overall aba accumulation and lower aba accumulation in small berries at early stages of ripening would be consistent with the delay in sugar and anthocyanin accumulation observed at and daa, respectively. in addition, the downregulation of ripening-associated nced gene in small compared to large berries from daa onwards might also reflect the lower aba biosynthetic capacity of small compared to large berries at and daa. nevertheless, nced was observed to be upregulated in small compared to large berries at daa. collectively, different sized berries exhibit a different regulation of nced transcripts, and significantly lower nced transcripts levels were found in small berries than in large ones during berry ripening, especially at  daa, with potential effects on the ripening process. the genes encoding tryptophan aminotransferase of arabidopsis1/tryptophan aminotransferase related and yucca are critical for regulating auxin levels and ripening in young berries. the expression of transcripts encoding taa1/tar – and yuc throughout berry development have also been reported in shiraz berries. these genes were modulated during fruit development and were also de between small and large berries in this study. three taa/tar transcripts and another peaked in the skins of berries harvested at and daa, respectively. one yuc transcript was specifically present at daa, another yuc transcript peaked around veraison, and other two yucs were mostly present during late ripening. these observations were consistent with previous studies showing taa/tar and yuc transcript evolution and auxin accumulation during berry development and ripening. at daa, when auxins levels are high, transcripts encoding tar and a yuc were highly upregulated in small with respect to large berries. similarly, tar transcripts were also upregulated at daa. however, tar transcripts, which have been shown to relate with the accumulation of auxin at early stages, were downregulated in small with respect to large berries at daa. several studies have indicated that auxin conjugation through the action of iaa-amido synthetases, which conjugates aspartic acid to auxin, is critical for ripening initiation and the regulation of auxins in the berry during development. interestingly, gh3- transcripts which display a ripening-associated developmental accumulation were significantly upregulated in the skins of small compared to large berries. therefore, significant differences in ripening physiology of the two berry classes and significant modulation of auxin metabolic pathways points to the possible involvement of auxin and its conjugates in determining asynchrony between berries of different sizes. the biosynthesis of ethylene is determined, in part, by the action of 1-aminocyclopropane 1-carboxylate oxidase, a key enzyme involved in the production of ethylene using acc as precursor. several aco-encoding members were also de between small and large berries, many of which exhibit highest expression at daa followed by a gradual decrease until daa. although grapevine is a non-climacteric fruit, previous findings have shown an increase of aco activity and ethylene production at stages before veraison. two aco transcripts displayed strong upregulation at daa in small compared to large berries. this might provide evidence that in small berries at pre-veraison stages a higher production of ethylene may in fact be delaying the ripening process and not enhancing ripening, as exemplified by the delayed sugar accumulation at daa and the lower anthocyanin levels at daa in small berries. indeed, recent studies have demonstrated application of ethylene at pre-veraison stages delays ripening through the activation of auxin biosynthesis pathways, significantly postponing sugar and anthocyanin accumulation, as well as the berry growth. ethylene may also play a role later in ripening, especially in the skin, as the abundance of many transcripts involved in ethylene biosynthesis and signaling peaked from °brix onwards, stages when desirable flavor and aroma compounds accumulate. in this study, the significant upregulation of two aco transcripts from daa onwards, indicates that ethylene might contribute to the induction of pathways important for fruit flavor in the skin of small berries compared to large ones. among hormone related transcription factors, the grapevine bzip tf, vvabf have been shown to modulate aba-dependent berry ripening processes through the induction of cell wall hydrolytic enzymes and complex modulation of multiple hormonal pathways including aba, auxin, and ethylene. expression of vvabf transcript was highest at daa corroborating its ripening-associated expression and its role in berry ripening. interestingly, vvabf was first upregulated in small berries at daa and subsequently downregulated at daa compared to large berries. this modulation might mediate the altered hormonal metabolism profiles involving nced, taa/yuc, and aco transcripts between small and large berries at and daa. also, the regulation of cell wall degradation and expansion via abf may explain the slower softening rate experienced in small compared to large berries observed at the onset of fruit ripening. in summary, the differential regulation of several nced, taa/yuc, and aco transcripts between the berry size classes suggest that hormonal pathways are differential regulated and might therefore contribute to variations in aba, auxin, and ethylene levels in the skin of small and large berries with potential effects on skin metabolism and composition. however, additional studies with intensive sampling strategies and measurement of these hormones in the two berry classes will be needed to gain a deeper understanding on the relative contribution of each hormone and its influence on berry size and fruit composition traits. modulation of cell wall metabolism in small and large berries in fleshy fruits, the remodeling of cell wall architecture during fruit development affects fruit softening and involves concerted changes in cell wall-related gene expression and multiple enzyme activities. influence of berry skins in determining post-veraison berry growth and the role of cell wall degradation and modification enzymes in mediating this process have been previously reported. the altered transcriptional profiles of cell wall genes in skins between small and large berries highlights the potential role of cell wall modification genes in modulating berry growth and softening. in this study, more than annotated cell wall transcripts exhibited de between small and large berries during fruit development. transcripts related to cellulose synthesis, cell wall modification, degradation, and pectin esterification were significantly modulated among the berry size classes in these clusters. a decrease in berry e has been shown to be the earliest ripening-associated event observed at the onset of ripening. emphasis will be given on the cell wall genes differentially expressed at daa, given that this is the developmental stage that immediately follows the large decrease in berry e and the stage when small and large berries displayed differences in e.fig. evolution during development, fold change values between small and large berries, and selected cis-regulatory element profile of cell wall transcripts differentially expressed at and daa. the heat map represents the evolution, based on the mean log of small and large berries, and log fold  changes of cell wall degradation and modification genes. the relative log values registered in small and large berries on average during berry development in a and c are depicted by green  and blue. grey color indicates the absence of detectable transcripts at the corresponding stage. blue and red boxes in b and d indicate downregulated and upregulated transcripts, respectively, in small berries in relation to large berries. boxes with bold margins indicate significant differences between small and large berries at a given developmental stage. the cluster column in b and d indicates the cluster number the associated transcript belongs to. em, -beta-mannan endohydrolase; pl pectate lyase; pg polygalacturonase; xet/xth xyloglucan endotransglucosylase/hydrolase. the heat map illustrates the presence of ap2/erf and bhlh/nac cis regulatory elements in cell wall genes. purple and white colors depict the presence and absence of the respective cre in the promoter regions of the gene we observed that cell wall degradation genes, consisting of six xyloglucan endo-transglycosylase/trans-hydrolase, two -beta-mannan endohydrolase, five pectate lyases, and three polygalacturonases, were downregulated in skins of small compared to large berries at daa. this observation may reflect lower cell wall degradation activity in small than in large berries, which supports the slower rate of softening from to daa. pectin degrading enzymes, such as polygalacturonases and pectate lyases, are two principal enzymes involved in tomato fruit softening through active cell wall pectin de-polymerization. studies in tomato, apple, and strawberries have demonstrated that silencing of polygalacturonase gene expression does not always affect fruit softening. pose et al. found large differences in strawberry fruit firmness at harvest in strawberry polygalacturonase 1-suppressed lines, but the changes in firmness in tomato during softening were very small and in apple most transgenic polygalacturonase suppression lines had no effect on fruit firmness. nevertheless, silencing of pectate lyases transcripts result in firmer fruits both during softening and at harvest. in plants, the transcription factor apetala2/ethylene responsive factor belongs to a multigenic family involved in the control of metabolism, stress response, and plant development through the binding of dre and gcc-related motifs in promoters regions of regulatory targets. several studies in fruits have demonstrated that ap2/erf tfs contribute in regulating ripening-related processes, especially fruit softening, by targeting cell wall degradation genes. in this study, significant enrichment of ethylene related cres, such as drecrtcoreat, cbfhv, and ltrecoreatcor, in cluster genes also corresponds to the presence of ap2/erf cres in promoter regions of seven predicted cell wall degrading transcripts. in agreement, we observed eight de ap2/erf tfs which were significantly downregulated in small compared to large berries at daa; four of which were ripening-associated. this suggest that ap2/erf tfs may be critical in regulating cell wall degrading transcripts in grapevines, and differences in ap2/erf tf regulation between small compared to large berries prior to ripening, may have affected the progression of softening reaching this stage. expansins act as primary cell wall loosening agents and incorporators of new cell wall material during the first growth phase and as mediators of cell wall disassembly by facilitating the contact between cell wall material and degradation enzymes during stage iii of growth in grapevine berries. the grapevine genome contains expansin genes classified into four distinct subfamilies, of which two grapevine expansins were demonstrated to promote cell expansion. many cell wall expansin transcripts were highly expressed at daa and six of these transcripts were significantly downregulated in skins of small with respect to large berries. moreover, other six transcripts were downregulated at daa when their level of expression was on average low. the downregulation of expansin transcripts observed in the skins of small compared to large berries at and daa indicates that cell wall loosening in the skin may be generally reduced, potentially limiting skin and mesocarp expansion and resulting in smaller berries. indeed, fruits of tomatoes overexpressing expansin displayed enhanced softening and were significantly larger compared to controls at various stages of fruit development. the regulation of some expansin transcripts such as expa and expb by vvceb have also been proposed. analysis of vvceb a fruit ripening-specific bhlh transcription factor, gene expression from different varieties exhibiting differences in berry size revealed a strong correlation between fruit size and vvceb transcript accumulation. in this study, vvceb transcript reached a maximum at daa and slowly decreased until daa. vvceb was downregulated in skins of small compared to large berries at and daa, but were only significant at daa. the down regulation of vvceb in small berries at and daa paralleled the down regulation of many expansin transcripts. promoter analysis performed on de expansins for bhlh-related cre revealed that the catgtg element, a typical dna binding domain for bhlh tfs, was enriched in the promoter region of out of  de expansin genes. this suggests that upstream control of expansins differentially expressed in small and large berries may in part be mediated by vvceb during ripening. nevertheless, previous studies have also shown that nac tfs can bind specifically to catgtg elements, which might provide evidence for the regulation of expansin transcripts by nac tfs. we observed that the majority of nacs, such as a ripening-associated nac which is highly expressed at and daa, were downregulated in small compared to large berries at daa in parallel with downregulation of several expansin genes. a recent study has also shown that polymorphisms in vvnac were associated with berry size variation among grapevine varieties. together, this transcriptional network involving bhlh and nac tfs may be critical for regulating berry growth and size determination in grapevine. modulation of flavonoid and stilbenoid pathways in small and large berries flavonoids and stilbenes are plant secondary metabolites commonly found across the plant kingdom. these specialized metabolites fulfill diverse physiological roles in stress response, as antioxidants, and during reproduction; in grapevine, they strongly affect grape and wine quality. we observed that differences in berry size were related to large differences in the expression of flavonoid genes. the de flavonoid genes were predominantly assigned to clusters and.fig. modulation in berry skin transcripts involved in the phenylpropanoid and flavonoid pathway in small and large berries at and daa. blue and red boxes indicate downregulated and upregulated transcripts, respectively, in small berries in relation to large berries. boxes with bold margins indicate significant differences between berry size treatments at a given developmental stage. transcription factors involved in the regulation of the phenylpropanoid and/or the flavonoid pathway transcripts are depicted in dotted lines. pal phenylalanine lyase; c4h cinnamate-4-hydroxylase; 4cl 4-coumarate:coenzyme a ligase; chi chalcone isomerase; chs chalcone synthase; f3h flavanone 3-hydroxylase; f3′h flavonoid 3′-hydroxylase; f3′5′h flavonoid 3′5′-hydroxylase; fls flavonol synthase; dfr dihydroflavonol 4-reductase; lar leucoanthocyanin reductase; ldox leucoanthocyanin dioxygenase; ufgt udpglucose:flavonoid 3-o-glucosyltransferase; and aomt anthocyanin o-methyltransferase. all other information is available at additional file 1: table s5 three chalcone synthases, and three chalcone isomerases -coding genes were differentially modulated in small and large berries. one chs and one chi were downregulated in small relative to large berries at daa when the pathway precursors are used for the production of flavan-3-ol and proanthocyanidins. later during development, one chs was upregulated at daa, while two out of three chss and all the chis mentioned above were downregulated in small in comparison to large berries at daa. of the total 16 flavonoid 3′5′-hydroxylases  found in the grapevine genome, we observed seven f3′5′h transcripts that were significantly downregulated in small berries at and daa. similarly, two flavonoid 3′-hydroxylases and one flavanone 3-hydroxylase  -coding genes, were also significantly downregulated in small berries at daa. f3′5′h and f3′h enzymes have been shown to be critical in determining the accumulation of di- and tri-hydroxylated flavonoids and the ratio between blue and red anthocyanin in grapes. the simultaneous downregulation of both f3′h and f3′5′h transcripts in small compared to large berries, particularly at daa, did not affect the relative abundance of the different anthocyanin forms. flavonol synthase is the key enzyme for the production of flavonols in grapevines. of the five fls transcripts encoded in the genome, the two transcriptionally active fls exhibited a ripening-associated accumulation in the skin; similar to that observed in previous studies in developing grape berries. transcripts encoding grapevine fls were significantly upregulated in small berries at and daa. similarly, the fls gene was significantly upregulated in small with respect to large berries at daa. previous studies have shown that fls transcript expression provides an excellent indicator for cluster light exposure and marker for flavonol synthase activity given strong positive relationship of solar radiation intensity with fls transcripts and flavonol content in berry skins. in the current study, berries of different sizes were harvested from the same clusters controlling for differences in microclimate among berries. thus differences in fls expression between berries of different sizes must have arose from endogenous mechanisms. consistently, previous studies reported that wine produced from small berries contain more flavonols compared to wines obtained from medium and large berries. the expression level of key flavan-3-ol/proanthocyanidin genes codifying for leucoanthocyanidin reductase and anthocyanidin reductase were highly expressed at daa and decreased in the expression at daa, consistently with the early flavan-3-ols and proanthocyanidin accumulation during berry development. downregulation of anr transcript at daa and upregulation of lar transcripts at daa were observed in skins of small compared to large berries indicating a differential regulation of flavan-3-ol biosynthesis during stages that are critical for the accumulation of these compounds and their polymeric forms in the skin. glycosylation of cyanidin and delphinidin via udp glucose:flavonoid 3-o-glucosyltransferase is critical in anthocyanin synthesis. the expression of grapevine ufgt was significantly downregulated at daa. myba and myba tfs redundantly regulates ufgt expression and modulates anthocyanin accumulation. the myba transcripts were specifically activated at daa and decreases approaching daa. myba was significantly downregulated in small compared to large berries at and daa, while the others mybas were all downregulated at daa. the observed downregulation of grapevine myba in small compared to large berries at daa is consistent with the lag of anthocyanin accumulation in smaller berries suggesting a delayed onset of ripening. although no concurrent downregulation of ufgt with myba was observed in small compared to large berries at daa, significant reduction of anthocyanin in small berries at daa might be strongly related to altered regulation of anthocyanin modification and transport pathways regulated by myba tfs. concurrent downregulation of myba myba and myba with many flavonoid pathways transcripts targeted by mybas did not significantly reduce the concentration of anthocyanin in the skin at daa. anthocyanin accumulation ceased from daa in both small and large berries. this indicates that the precursors for anthocyanin production are channeled to the production of other phenolic compounds, such as stilbenes, at late stages of ripening. a marked upregulation of genes encoding enzymes involved in the general phenylpropanoid and stilbenoid pathway, namely phenylalanine ammonia lyase, cinnamic acid 4-hydroxylase, 4-coumarate:coa ligase, and stilbene synthase, was observed at and daa. of particular interests are transcripts encoding sts enzymes. in plants producing stilbenes, sts catalyzes the production of cis- or trans-resveratrol using 4-coumaroyl-coa and three molecules of malonyl-coa. interestingly, trends in gene expression of the majority of transcripts encoding sts and pal were very similar. several studies have reported that multiple stss are responsible for the production of stilbene/resveratrol accumulation in the skin during berry development and in response to abiotic and biotic stress. a large proportion of pal and sts transcripts were significantly upregulated in small berries especially at and daa. similar to that of anthocyanin, stilbenes largely accumulate at the onset of ripening. however, no de expression was observed at daa where their expression tends to peak. concerted downregulation of chs and upregulation of sts at daa in small compared to large berries might relieve the competition for coumaroyl-coa and malonyl-coa substrates by chs; thereby favoring the flux to stilbene production, since anthocyanin levels have plateaued in both berries from daa onwards. although the higher skin to berry weight ratio often determines a higher concentration of the anthocyanin and stilbenes accumulated in the skin, concerted upregulation of the stilbene branch in small berries may enhance stilbene levels in wines made from small berries compared to normal and large sized berries harvested at the same time. this induction of pal and sts genes in small compared to large berries at daa might also be a consequence of altered hormone levels. hormones such as ethylene and jasmonic acids are not only involved in the induction of key stilbene genes and levels in grapevines but also, in the case of ethylene, might take part to the over ripening process. two grapevine myb tfs, myb and myb directly regulate the biosynthesis of stilbene in grapevines. in this study, myb had consistent expression trends, in both small and large berries, peaking at daa, while myb transcript evolution was not consistent between small and large berries. in large berries, myb transcripts progressively increase until daa before decreasing at daa; while, in small berries, myb expression peaked at daa before decreasing slightly at daa and increasing again later during ripening. nonetheless, grapevine myb and myb were significantly upregulated in small berries at and daa, respectively. this result indicates their role in the regulation of sts expression and stilbene production in the skin. modulation of fatty acid degradation pathways in small and large berries aromatic alcohols and aldehydes derived from the fatty acid metabolism pathways, such as hexanol/hexenol and heptanal/hexanal, also contribute to late-ripening associated flavor/aromas in fruits and finished wines. several successive steps involving lipoxygenase, hydroperoxide lyase, alcohol dehydrogenases, and alcohol acyl transferases enzymes regulate the production of these compounds in plant tissues. many transcripts encoding enzymes of the aforementioned steps were modulated during fruit development and differed in the abundance between small and large berries. lipoxygenase enzymes are involved in the formation of hydroperoxides from linolenic acid, which can lead to the accumulation of key wine aromas in the grape. two lox genes differentially expressed between small and large berries were highly expressed at and daa before decreasing towards daa. one lox transcript was significantly downregulated at daa and another lox transcript was significantly upregulated at daa in small compared to large berries. hydroperoxides are then converted to c related aldehydes, which often confer grassy-related aromas via hpl. one hpl transcript, mostly accumulated at and daa, was downregulated in small compared to large berries. subsequently, adh converts c related aldehydes to c related alcohols, which can be further converted to produce volatile aromas via aats. interestingly, all seven adh transcripts encoded in the grapevine genome were de. four adh transcripts were highly expressed at daa and sharply decrease from daa and onwards; one adh transcript peaked at daa while another two were highly expressed at daa. the four early developmental related adh transcripts, as well as two of the three late-ripening adh genes, displayed significant upregulation in small compared to large berries at daa. on the contrary, at daa, one of the two adhs that are expressed at high levels at this stage was downregulated in small berries. the accumulation profiles of the different aldehydes, alcohols, and esters in cabernet sauvignon grapes during berry development and ripening has been reported and their formation is thought to involve a synergy between the biosynthesis and catabolism of various steps. although the molecular mechanisms are not fully understood, high levels of volatile esters in young berries and high levels of alcohol compounds accumulated in later stages of berry development are reported. therefore, the strong upregulation of four early ripening adh and one lox transcripts in small compared to large berries might favor a higher production of c aldehydes and subsequent turnover/flux to alcohols/esters during early development in small compared to large berries.fig. berry skin transcript and selected cis-regulatory element profile of small and large berries of the fatty acid degradation/c volatile biosynthesis pathway at and daa. simplified pathway schematic. the heat map represents the transcript evolution, based on the mean log in small and large berries, and log fold changes. the relative log values from the four time points in b are depicted by green  and blue. grey color indicates the absence of detectable transcripts at the corresponding stage. blue and red boxes in c indicate downregulated and upregulated transcripts, respectively, in small berries in relation to large berries. boxes with bold margins indicate significant differences between small and large berries at a given developmental stage. the cluster column in c indicates the cluster the associated transcript belongs to.  the heat map illustrates the distribution of mads box cres in promoter regions of aroma-related transcripts differentially expressed between small and large berries. purple and white colors depict the presence and absence of each cres, respectively, in the promoter regions of the relevant transcripts. lox lipoxygenase; hpl hydroperoxide lyase; adh alcohol dehydrogenase; aat alcohol acyl transferases in tomato, a mads-box transcription factor is a negative regulator of fruit ripening and acts as a modulator of aroma production in fruits via direct regulation of adh, lox, and hpl promoters. we identified nine mads-box tfs differentially expressed between small and large berries: four genes were allocated to clusters two to clusters and one to cluster and individually. of note are the predicted homologs of tomato rin, allocated into cluster that share high expression similarity with four adh transcripts from to daa in small and large berries. analysis of the promoter regions of the total de fatty acid degradation pathway genes for various mads-box binding site revealed that one motif was located within all tested promoter regions except promoter region of an adh transcript. these observations indicate that berry size may be associated with a different accumulation of aromatics in the fruit, likely through the coordinate regulation of mads-box tfs and structural genes of the fatty acid degradation pathway.  significant differences in the physiology, biochemistry, and transcripts are found in different sized berries during fruit development. as small and large berries approach the onset of ripening, clear differences in the rate of development were apparent. around the onset of ripening, the steeper drop in elasticity, more rapid accumulation of sugars, lower tartaric acid, and greater anthocyanin levels in large compared to small berries suggest that fruit size is associated to changes in the ripening physiology of the berry, where large berries approach ripening faster. these differences correspond to congruent changes in the hormonal pathways related to aba, auxin, and ethylene via genes encoding nced for aba, taa1/tar and yuc for auxin, and aco for ethylene. genes encoding pathways contributing to fruit texture, flavor, and aroma were also differentially modulated accordingly to the berry size. the modulation of cell wall degradation and modification genes may contribute to the differences in elasticity decreases and berry growth/size. upregulation of fatty acid degradation genes, especially adh, during early development might favor production of desirable aromatics in small berries. in the late ripening stages, concurrent upregulation of phenylpropanoid and stilbenoid pathway genes with a parallel downregulation of the flavonoid pathway in skins of small compared to large berries indicates that smaller berries may have higher concentrations of aromatics and stilbenes, major antioxidants produced in the berry. further investigation on endogenous and exogenous factors that regulate the fruit metabolism in berries of different sizes is necessary to identify the key factors – e.g. hormone signals, advantage of position within a cluster that favors mineral or water uptake, or a better microclimate – that determine the berry size itself as well as the transcriptome response. finally, a deeper investigation of the fruit composition in relation to berry size may lead to the adoption of screening strategies based on size for tailoring fruit and wine quality. additional file additional file 1: table s summary of rna sequencing analysis metrics. rna sequencing were carried in skins of small and large berries at four berry developmental stages namely,,, and days after anthesis. table s transcript abundance of the de genes, reported as log values, for each individual biological replicate in each treatment at and 121 days after anthesis. table s summary of differentially expressed genes between small and large berries at four berry developmental stages. all differentially expressed genes and detailed description of the 12×v gene id, log fold change values, differential expression calls across four developmental stages, average log values of small and large  berries together and individually, k-means assigned cluster, functional annotations based on grimplet et al., and mapman pipeline. table s summary of place- and pbm-curated cis-regulatory elements analysis of k-means assigned clusters and selected group of genes. all information on the number of promoters with the specified cre, the number of genes within each group, number of promoters in the genome containing the specified cre, p-value and fdr of enriched cre, motif name and description, and a short description on selected cre instances in groups. table s summary of selected hormone, cell wall, flavonoid/phenylpropanoid, and aroma/flavor metabolic pathways transcripts differentially expressed genes between small and large berries at four berry developmental stages which have been divided into separate classes and sub-categories. we thank mission hill family estate for providing the grapes used in this study. we thank dr. patricia bowen for providing laboratory space during the sample collection. we thank canada’s michael smith genome sciences centre for the sequencing services. funding this study was funded by the university of british columbia, genome bc, and natural sciences and engineering research council of canada. availability of data and materials all raw sequence reads have been deposited in ncbi sequence read archive. the bioproject and sra accessions are prjna and srp respectively. authors’ contributions dcjw performed the physiological measurements, liquid chromatography and lc-mass spectrophotometric analysis, rna extractions, transcriptome data analysis, cis-regulatory sequence analysis, interpreted the results, and drafted the manuscript. rlg performed the transcriptome and cis-regulatory sequence data analysis. nd performed part of the physiological measurements. gag contributed to the experimental design, expert advice throughout the study, interpreted the results, and drafted part of the manuscript. sdc conceived the study, coordinated the field experiments, performed the physiological measurements, supervised the entire study, interpreted the results, and drafted part of the manuscript. all authors read and approved the final manuscript. competing interests the authors declare that they have no competing interests.
fatty acid degradation and stilbenoid pathway genes were upregulated at daa while cell wall degrading and modification genes were downregulated at daa in small compared to large berries. abscisic acid, auxin, and ethylene hormone pathway genes were differentially modulated between berry sizes. the comparison of berry physiology between small and large berries throughout development revealed significant differences in firmness, the rate of softening, and sugar accumulation at specific developmental stages. these results suggest that, in addition to direct effects brought about by differences in size, key aspects involved in the regulation of ripening likely contribute to different quality profiles between small and large berries. small berries had significantly higher skin to berry weight ratio, lower number of seeds per berry, and higher anthocyanin concentration compared to large berries. in the late ripening stage, concerted upregulation of the general phenylpropanoid and stilbenoid pathway genes and downregulation of flavonoid pathway genes were observed in skins of small compared to large berries. aromacell wallflavonoidgrapevinehormonepromoterqualityrna-seqsecondary metabolismtranscriptomicshttp://dx.doi.org/ /501100000233genome british columbia10r1883810r18838http://dx.doi.org/ /501100000038natural sciences and engineering research council of canada10r23082issue-copyright-statement© the author 2016 in wine grape production, management practices have been adopted to optimize grape and wine quality attributes by producing, or screening for, berries of smaller size. fruit size and composition are influenced by numerous factors that include both internal and external factors. rna-sequencing analyses of berry skins at and daa revealed a total of differentially expressed genes between small and large berries. combined physiological, biochemical, and transcriptome analyses were performed to improve our current understanding of metabolic and transcriptional pathways related to berry ripening and composition in berries of different sizes. the study demonstrates that physiological and compositional differences between berries of different sizes parallel transcriptome changes that involve fruit texture, flavor, and aroma pathways. cis-regulatory element analysis of differentially expressed hormone, fruit texture, flavor, and aroma genes revealed an enrichment of specific regulatory motifs related to bzip, bhlh, ap2/erf, nac, myb, and mads-box transcription factors.
 in wine grape production, management practices have been adopted to optimize grape and wine quality attributes by producing, or screening for, berries of smaller size. fruit size and composition are influenced by numerous factors that include both internal and external factors. combined physiological, biochemical, and transcriptome analyses were performed to improve our current understanding of metabolic and transcriptional pathways related to berry ripening and composition in berries of different sizes.  the comparison of berry physiology between small and large berries throughout development revealed significant differences in firmness, the rate of softening, and sugar accumulation at specific developmental stages. small berries had significantly higher skin to berry weight ratio, lower number of seeds per berry, and higher anthocyanin concentration compared to large berries. rna-sequencing analyses of berry skins at and daa revealed a total of differentially expressed genes between small and large berries. abscisic acid, auxin, and ethylene hormone pathway genes were differentially modulated between berry sizes. fatty acid degradation and stilbenoid pathway genes were upregulated at daa while cell wall degrading and modification genes were downregulated at daa in small compared to large berries. in the late ripening stage, concerted upregulation of the general phenylpropanoid and stilbenoid pathway genes and downregulation of flavonoid pathway genes were observed in skins of small compared to large berries. cis-regulatory element analysis of differentially expressed hormone, fruit texture, flavor, and aroma genes revealed an enrichment of specific regulatory motifs related to bzip, bhlh, ap2/erf, nac, myb, and mads-box transcription factors.  the study demonstrates that physiological and compositional differences between berries of different sizes parallel transcriptome changes that involve fruit texture, flavor, and aroma pathways. these results suggest that, in addition to direct effects brought about by differences in size, key aspects involved in the regulation of ripening likely contribute to different quality profiles between small and large berries. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. keywords aromacell wallflavonoidgrapevinehormonepromoterqualityrna-seqsecondary metabolismtranscriptomicshttp://dx.doi.org/ /501100000233genome british columbia10r1883810r18838http://dx.doi.org/ /501100000038natural sciences and engineering research council of canada10r23082issue-copyright-statement© the author 2016 