An aliquot of 500 mg fine powder of pulp was extracted sequentially with ethanol (80 and 50%), dried in a Speed-Vac, and re-dissolved in 2.5 mL de-ionized water. Glucose and fructose content were measured enzymatically with an automated micro-plate reader (Elx800UV, Biotek Instruments Inc., Winooski, VT, USA) according to the method of Gomez et al. (2007). Tartaric acid content was assessed by using the colorimetric method based on ammonium vanadate reactions (Pereira et al., 2006). Malic acid was determined using an enzyme-coupled spectrophotometric method that measures the change in absorbance at 340 nm from the reduction of NAD+ to NADH (Pereira et al., 2006). The amino acid content was determined after derivatization (Cohen and Michaud, 1993) using a Waters 2695 HPLC system equipped with Waters 474 fluorescence detector (Waters, Milford, MA, USA). Twenty amino acids were identified and quantified as described by Pereira et al. (2006). The results were expressed in μmol.L⁻¹ juice.

An aliquot of 500 mg of berry skin powder was freeze-dried for 72 h and the dried powder (50 mg) was extracted in 1.0 mL methanol containing 0.1% HCl (v/v). Extracts were filtered through a 0.45 μm polypropylene syringe filter (Pall Gelman Corp., Ann Harbor, MI, USA) for HPLC analysis. Each individual anthocyanin was analyzed with HPLC as described in Soubeyrand et al. (2014). Quantification was carried out by peak area integration at 520 nm. The concentration of individual anthocyanins was calculated in milligrams per gram (mg. g⁻¹) of dry skin weight (DW) using malvidin 3-O-glucoside (Extrasynthese, Genay, France) as external standard.

Four hundred twenty seven DEGs were related to the functional category “Stress” and mainly belonged to the “abiotic/heat stress” cluster (Supplementary Table 5; 98 out of the 427 “Stress” DEGs). Most of the transcripts associated to “heat stress” category were predominantly up-regulated (Supplementary Figure 2A) and belong to the HSP family (Heat Shock Protein). Transcripts encoding universal stress proteins (USPs; VIT_17s0000g04260, VIT_04s0079g00610, VIT_08s0032g00590) also accumulated in response to HT.

HT deeply affects protein homeostasis. Indeed, numerous HSP and chaperones genes were up-regulated in HT berries (Supplementary Tables 5, 6). These proteins play an important role in protein-protein interactions (Kotak et al., 2007a; Bokszczanin and Fragkostefanakis, 2013). The abundance of several FK506-binding protein (FKBP) related transcripts was also increased by HT (VIT_19s0015g01100, VIT_07s0031g01150, VIT_08s0007g04340, VIT_13s0064g00580, VIT_01s0011g00930, VIT_00s0260g00070). These proteins belong to the large family of peptidyl prolyl cis–trans isomerases that can function as chaperones. Protein synthesis and degradation were also strongly affected in heated berries as suggested by the 60 DEGs linked to protein synthesis category and the 348 genes belonging to proteolysis (Supplementary Tables 5, 6).

Protein degradation through the ubiquitin-proteasome system (UPS) plays an essential role in diverse cellular pathways, cell-cycle progression, DNA repair, and degradation of damaged proteins as well as in signal transduction. Particularly, UPS components are major players in plant acclimation to abiotic stresses (Stone, 2014; Guerra et al., 2015). In the present study, 167 transcripts potentially linked to the ubiquitin machinery were deregulated after HT (102 up-, 56 down-, 9 both up- and down-regulated transcripts; Supplementary Table 6). Most of these DEGs encode different putative ubiquitin ligases (E3). Orthologs of each of the 3 major E3 classes (namely RING-type (Really Interesting New Gene); HECT-type (Homology to E6-Associated Carboxyl- Terminus), and U-box-type) were up-regulated in heat-stressed berries. While some E3-like transcripts accumulated whatever the developmental stage (VIT_09s0002g00220, VIT_12s0034g01390, VIT_05s0124g00230, VIT_06s0009g03670, VIT_08s0040g02600, VIT_19s0027g00320), most of these were transiently or specifically deregulated at a particular stage. For instance, some E3-related transcripts were only HT up-regulated during the herbaceous stage (VIT_17s0000g09790, VIT_18s0041g01090, VIT_14s0068g02150, VIT_14s0066g02580, VIT_08s0056g01410) whereas others responded to HT during veraison or ripening stages (VIT_19s0015g00660, VIT_01s0011g02950, VIT_01s0026g00300, VIT_07s0005g01360, VIT_08s0007g04790, VIT_18s0001g02280, VIT_00s0160g00270, VIT_18s0001g06220). Furthermore, the CRL group (Cullin based Ring E3 ligases), corresponding to the largest class of ubiquitin ligases (Stone, 2014), and especially the CUL1 based E3s, [also referred to as Skp1-Cullin-F-box (SCF)] were strongly impacted by HT. Indeed, an ortholog of the Arabidopsis adaptor protein S-Phase kinase-associated protein (SKP; VIT_03s0038g02480) and many F-box proteins are up-regulated at the transcriptional level upon HT.

The secondary metabolism produces compounds of critical importance for berry quality and wine bitterness and astringency (Lund and Bohlmann, 2006; Ali et al., 2010; Kuhn et al., 2014; Robinson et al., 2014). Three hundred twenty seven DEGs were related to “Secondary metabolism” and mainly belonged to the subcategories “isoprenoids,” “phenylpropanoid-lignin,” and “flavonoids” (Supplementary Table 7).

Numerous genes affected by HT correspond to aroma and aroma-precursor related gene. Terpenes (predominantly eucalyptol, β-caryophyllene, and α-humulene) are usually present at low levels in Cabernet Sauvignon grapes, accumulating during the preveraison stage whereas benzene derivatives (2-phenylethanol and 2-phenylethanal) appear at late ripening (Kalua and Boss, 2009; Robinson et al., 2014). The strong repression of genes encoding the 1-deoxy-D-xylulose-5-phosphate synthase (VIT_05s0020g02130, VIT_09s0002g02050, VIT_11s0052g01730, VIT_11s0052g01780) suggests that volatile terpenoids biosynthesis may be decreased by HT (Supplementary Table 7). The 1-deoxy-D-xylulose-5-phosphate synthase catalyzes the synthesis of isopentenylpyrophosphate (IPP), which is the precursor of all terpenes. The condensation of IPP and its isomer DMAPP (dimethylallylpyrophosphate) forms geranyl diphosphate (GPP) that is used by terpene synthase (TPS) to produce monoterpenes and derivatives. The Pinot Noir reference genome contains 89 putative TPS, among which half have been functionally characterized (Martin et al., 2010). Among the 55 probe sets giving reliable results and representing transcripts of functional, partial and pseudo TPS on the NimbleGen array (Cramer et al., 2014), 16 transcripts showed differential abundance in HT berries (Supplementary Table 8). Thirteen TPS were transiently repressed in GHT, VHT and or RHT clusters whereas only three TPS were upregulated by HT (VviTPS25, VviTPS26, VviTPS50). From the literature, most of these TPS accumulate at the late stages of ripening in vineyard conditions (Cramer et al., 2014). Finally, the transcript abundance of several terpene-related genes decreased in berries directly exposed to HT, in a similar way to that observed after exposing the whole vine to HT (Rienth et al., 2014). These repressed genes encode geraniol 10-hydroxylase, (-)-germacrene D synthase and linalool synthase (Supplementary Table 8).

Within the terpene family, carotenoids are a complex subgroup of isoprenoid pigments playing diverse roles in plants and providing nutritional value. Carotenoids also lead to C₁₃-norisoprenoids which contribute the characteristic aromas of Vitis vinifera varieties (Mendes-Pinto, 2009). Among the 42 putative grape carotenoid metabolic genes (Young et al., 2012), 21 transcripts showed differential abundance after HT in at least one of the 9 conditions, most of these being down-regulated (Supplementary Tables 7, 9). The abundance of 2 out of 3 phytoene synthase transcripts (VviPSY, VIT_12s0028g00960, VIT_06s0004g00820), encoding the enzyme that catalyzes the first step committed to carotenoid biosynthesis, decreased upon HT. This down-regulation was also observed for downstream genes, namely phytoene dehydrogenase (VIT_04s0023g01790), carotene desaturase (VIT_14s0030g01740), carotenoid isomerase (VIT_08s0032g00800, VIT_12s0035g01080), lycopene cyclase (VIT_06s0080g00810, VIT_11s0016g01880) and carotene hydroxylase (VIT_04s0023g00080). The β-carotene hydroxylase 2 transcripts (VviBCH2, VIT_16s0050g01090) were the only ones strongly accumulating under HT, and especially at the herbaceous stage.

Methoxypyrazines (MPs) are strongly odorant volatile molecules with vegetable-like fragrances that participate to the distinct herbaceous/ bell pepper characters of some wines such as Cabernet Sauvignon (Dunlevy et al., 2009; Darriet et al., 2012; Kuhn et al., 2014). Isobutyl methoxypyrazine (IBMP) is the predominant MP in Cabernet Sauvignon berries, accumulating throughout the pre-veraison stage before declining during the ripening phase. The last step of its biosynthesis was recently deciphered in grapevine, through the unambiguous identification of VviOMT3 (VIT_03s0038g03090), an O-methyltransferase capable of converting the nonvolatile precursor 2-hydroxy-3-isobutylpyrazine (IBHP) into IBMP (Dunlevy et al., 2013; Guillaumie et al., 2013). By contrast to VviOMT4, VviOMT1 and VviOMT2 are active with a broad range of substrates but methylate IBHP in vitro, although with poor affinity (Dunlevy et al., 2010). Interestingly, VviOMT3 transcripts were strongly repressed in green berries exposed to HT (Supplementary Table 7). A similar decrease was also observed in GHT berries for VviOMT4 (VIT_03s0038g03080) and VviOMT1 (VIT_12s0059g01790) transcripts, whereas VviOMT2 (VIT_12s0059g01750) remained unaffected.

Grape berry phenolics derived from the phenylpropanoid pathway participate to sensory properties, color and protection against environmental stress (Teixeira et al., 2013). Our biochemical analysis highlighted the dramatic effects of local warming on the onset of veraison and on the final anthocyanin contents in berries at harvest (Table 1; Figure 2). MapMan analysis (Figure 6) shows the contrasted heat responses of DEGs related to the general phenylpropanoid and to the flavonoid biosynthesis pathways (Figure 6; Supplementary Figure 3; Supplementary Table 10). The transcriptomic responses of GHT samples clearly differed from both VHT and RHT samples. During the herbaceous stage, genes involved in the general phenylpropanoid pathway (phenylalanine ammonia-lyase PAL, cinnamate 4-hydroxylase C4H, 4-coumarate-CoA ligase 4CL) were induced by HT regardless to the stress duration. By contrast, in VHT and RHT samples, the same genes were up-regulated after 1 day, but strongly repressed after 7 and 14 days of treatment. Many transcripts related to the flavonoid biosynthesis pathway were repressed in GHT clusters. Two chalcone synthase transcripts (VviCHS1: VIT_14s0068g00920, VviCHS2: VIT_14s0068g00930) encoding the first committed enzyme in flavonoid biosynthesis (Parage et al., 2012) were repressed after 7 days. A significant repression by HT was also observed for many flavonoid transcripts of the late biosynthetic pathway, such as flavonoid 3′-hydroxylase (VviF3′H, VIT_11s0016g01020, VIT_11s0016g01030, VIT_09s0002g01090), flavonoid 3′5′-hydroxylase (VviF3′5′H), dihydroflavonol 4-reductase (VviDFR, VIT_16s0039g02350, VIT_18s0001g1280) and leucoanthocyanidin dioxygenase (VviLDOX, VIT_08s0105g00380), whereas the flavanone 3-hydroxylase gene (VviF3H, VIT_16s0098g00860) was up-regulated (Figure 6; Supplementary Figure 3; Supplementary Table 10). The HT led to contrasted effects on the same set of genes in VHT and RHT clusters when compared with GHT. VviF3H transcript was significantly less abundant whereas VviCHI (chalcone isomerase, VIT_19s0014g00100) and VviF3′H genes were mostly up-regulated. Moreover, the repressive effect of HT on VviF3′5′H isoforms was not observed in VHT clusters and only transiently detected in RHT berries. In addition, the structural genes VviANR (anthocyanidin reductase, VIT_00s0361g00040), and VviLAR (leucoanthocyanidin reductase, VIT_01s0011g02960, VIT_17s0000g04150) involved in proanthocyanidins (PA) synthesis were significantly repressed under HT, according to the stage and/or duration of the stress.

The anthocyanidin aglycones are further modified through glycosylation, methylation and acylation events, leading to the production of a wide variety of anthocyanin compounds. Glycosylation of flavonoids is catalyzed by enzymes from the large glycosyltranferase (GT) family, represented by 240 genes in the grape genome (Ono et al., 2010). Glycosylation enhances the structural diversity and modifies the functional properties of these secondary metabolites. For instance, the expression of UFGT [UDP-glucose:flavonoid 3-O-glycosyltransferase, renamed VviGT1, VIT_16s0039g02230 (Parage et al., 2012)], catalyzing the 3-O-specific glycosylation of anthocyanidin is critical for the coloration of grape skin (Boss et al., 1996). Twenty-nine putative VviGT were deregulated in berries exposed to HT (Figure 6; Supplementary Figure 3; Supplementary Table 10). Despite the loss of anthocyanins upon HT, VviGT1 expression remained unaffected by HT. By contrast, VviGT5 (VIT_11s0052g01600) and VviGT6 (VIT_04s0023g01290) transcripts were strongly down-regulated in VHT and RHT clusters. VviGT5 and VviGT6 drive the glycosylation of flavonols, which increase their water solubility and their accumulation (Ono et al., 2010).

In grapevine, anthocyanins can also be modified through the action of O-methyltransferases (AOMTs) and acyltransferases (ACTs) before being transported into the vacuole (Fournier-Level et al., 2011; Rinaldo et al., 2015). These modifications modulate berry color by reducing anthocyanin reactivity and increasing their stability and solubility in water. None of the two grape AOMTs (VviAOMT1; VIT_01s0010g03510, VviAOMT2; VIT_01s0010g03490) that were previously described as effective anthocyanin 3′- and 3′,5′-O-methyltransferase (Hugueney et al., 2009; Lücker et al., 2010; Fournier-Level et al., 2011) was significantly deregulated by HT (Supplementary Table 10). However, other putative VviAOMTs transcripts (VIT_11s0016g02610, VIT_07s0031g00350, VIT_03s0063g00140, VIT_12s0028g03110), were transiently up- or down-regulated by HT depending on the ripening stage and stress duration. HT also modified the transcript amounts of various ACTs (Supplementary Table 10). Indeed, 5 putative ACTs were transiently repressed in GHT berries (VIT_12s0134g00590, VIT_12s0134g00630, VIT_12s0134g00600, VIT_12s0134g00650, VIT_12s0134g0660), whereas 3 ACTs (VIT_03s0017g00870, VIT_12s0134g00590, VIT_12s0134g00630) were up-regulated in both VHT and RHT berries. VIT_03s0017g00870 corresponds to Vvi3AT, an enzyme recently associated to the production of the common grape berry acylated anthocyanins (Rinaldo et al., 2015).

Stilbenes and lignins represent branching points in the phenylpropanoid pathway. Stilbenes are a small family of phytoalexins synthesized by plants in response to biotic and abiotic stresses. Under normal growth conditions, berries stilbene content increases from veraison to ripening, with significant differences among V. vinifera varieties (Gatto et al., 2008). Forty-eight stilbene synthases (STSs) genes catalyzing the biosynthesis of the stilbene backbone were found in grapevine genome. This represents an unusual example of functional redundancy (Parage et al., 2012). Most of these (39 genes) were impacted in berries exposed to HT, displaying a quite similar and noticeable expression profile (Figure 6; Supplementary Figure 3; Supplementary Table 10). In GHT clusters, 18 STS transcripts accumulated after 1 and 7 d of treatment before being repressed at 14 d. In VHT and RHT fruits, the STSs were transiently induced at 1 d and then strongly repressed over the 2 weeks of experiment.

Lignification can also be induced as a response to various biotic and abiotic stresses, as shown in Citrus fruit after postharvest HT (Yun et al., 2013). Conversely, the cell wall lignin content was reduced in the skin of grape mature berries experiencing a water stress (Vannozzi et al., 2012; Fernandes et al., 2015). In the present study, numerous transcripts (39) potentially involved in the lignin biosynthetic pathway differentially accumulated in HT berries (Figure 6; Supplementary Figure 3; Supplementary Table 10). Two cinnamoyl-CoA reductase (VviCCR) gene isoforms were affected in an opposite way by HT, VIT_14s0066g01150 being induced, and VIT_09s0070g00240 being repressed. In GHT and VHT clusters, the transcript levels of 16 cinnamyl alcohol dehydrogenase (VviCAD) genes were mainly repressed whereas a few ones accumulated in RHT berries. The content of 3 ferulate 5-hydroxylase transcripts (VviF5H) was reduced in both VHT and RHT berries. Finally, HT led to contrasted effects on caffeic acid O-methyltransferase (VviCOMT) and peroxidase transcripts (catalyzing the polymerization of monolignols into lignins).

While many structural genes were transcriptionally affected in HT berries, the positive regulatory genes identified so far in grapevine were either weakly (VviMYBPA1, VviMYB14, VviMYB15, VviMYBF1) or not deregulated (VviMYBA1, VviMYBA2, VviMYBA3, VviMYC1, VviMYCA1, VviMYBPA2) in HT berries (Supplementary Table 11). Accordingly, HT did not affect the amount of VviGT1 transcript (VIT_16s0039g02230) that encodes the anthocyanidin glycosyltransferase catalyzing the limiting step for anthocyanin accumulation and are a direct target of VviMybA1 transcription factor (TF; VIT_02s0033g00410) (Cutanda-Perez et al., 2009). For comparison, no effect of HT was observed on VviMYBA1 transcripts in berries from fruiting cuttings (Carbonell-Bejerano et al., 2013), whereas a VviMYBA1 repression was reported in two others studies (Yamane et al., 2006; Rienth et al., 2014). It is also noteworthy that both VviMYBC2-L3 (VIT_14s0006g01620) and VviMYB4b (VIT_04s0023g0371) transcripts were transiently enhanced in VHT and RHT berries, respectively. These two TFs were recently described as negative regulators of the phenylpropanoid pathway in grape (Cavallini et al., 2015). While HT did not significantly impact the expression of genes known to act in the transport of flavonoids (reviewed by Zhao (2015), either directly (VviAM1, VviAM3, VviABCC1, VviMATE1, VviMATE2) or indirectly (VviGST1, VviGST4), the present work pointed out a significant deregulation of many putative ABC or MATE transporters and Glutathion S-transferase family members (Supplementary Tables 1, 11). It cannot be excluded that these proteins could be involved in the control of flavonoid transport in HT grape berries, as well.

In addition to synthesis, stabilization and vacuolar sequestration, the anthocyanin content can also be modulated through degradation that may involve enzymes such as laccases, polyphenol oxidases, class III peroxidases, and β-glucosidases (Oren-Shamir, 2009). Our experiments showed a strong HT effect on the expression of laccase genes, potentially impacting the polymerization rate of various phenolic compounds. Out of the 93 laccase-annotated genes in the grapevine genome, 33 transcripts were deregulated by HT (Supplementary Table 12). Interestingly, the laccase TT10 (Transparent Testa 10, VIT_18s0075g00600) showed a maintained up-regulation by HT whatever the stage of development. In Arabidopsis, TT10 was proposed to participate in the oxidative polymerization of phenolic compounds (Pourcel et al., 2005). In a similar way, numerous genes encoding putative polyphenol oxidases (4), peroxidases (25) and β-glucosidases (16) were impacted by HT (Supplementary Table 12). In the present study, HT induced several peroxidase genes including class III type (VIT_07s0130g00220, VIT_18s0001g06850) whereas others were repressed (VIT_18s0001g06840, VIT_18s0001g06890). Recently, Movahed et al. (2016) showed that the expression of 3 of the 5 peroxidase genes (VIT_14s0066g01850, VIT_06s0004g07770, VIT_07s0191g00050, VIT_11s0016g05320 and VIT_18s0072g00160) that were most strongly expressed in grape berry pericarp during ripening was influenced by temperature elevation. In agreement with these results, the present data also pinpoints the alteration of 4 of these genes (VIT_14s0066g01850, VIT_06s0004g07770, VIT_11s0016g05320 and VIT_18s0072g00160) in response to HT but with different kinetics and intensities.

Essential for cell growth and viability, HSPs function as molecular chaperones in maintaining protein quality and folding, and are required for the acquisition of the thermotolerance (Bokszczanin and Fragkostefanakis, 2013). A massive up-regulation of HSP genes is a highly conserved response in heat-exposed plants (Finka et al., 2011). This response is a result of the complex signaling cascade, whose final steps consist in the activation of heat shock TFs (HSFs) and their binding to the HSP promoters (Mittler et al., 2012). HSF activity is regulated at the transcriptional, post-transcriptional and post-translational levels (Mittler et al., 2012) and 21 Arabidopsis HSF transcripts are up-regulated in response to various environmental stress conditions (Miller and Mittler, 2006). Data mining of the 12X version of the grape genome allowed the identification of 19 putative VviHSFs (Pillet et al., 2012; Scharf et al., 2012) among which 6 were up-regulated in HT berries (Supplementary Table 14). Among these, VIT_04s0008g01110 corresponds to VviHSFA2 that was recently reported as involved in the regulation of heat responses in stressed berries (Pillet et al., 2012). HSFA2 is a key player for basal and acquired thermotolerance, extending the effect of heat acclimation in both tomato and Arabidopsis (Charng et al., 2007). AtHSFA2 which is the most highly HT-induced HSF (Busch et al., 2005), serves as a regulatory amplifier of a subset of genes (Schramm et al., 2008) and is required for the induction and maintenance of HT memory-related genes (Lämke et al., 2016). The transcriptional regulation of the heat-induced responses may also be due to VviMBF1c (Multiprotein bridging factor 1c; VIT_11s0016g04080) that showed enhanced expression pattern in HT berries (Supplementary Table 14), in good agreement with two recent studies (Carbonell-Bejerano et al., 2013; Rienth et al., 2014). In Arabidopsis, AtMBF1c acts as a thermotolerance mediator through the heat-dependent regulation of 36 different transcripts (Suzuki et al., 2011). The existence of a similar MBF1c regulon was proposed in ripening berries of microvines submitted to HT (Rienth et al., 2014). In the present study, the relationship between VviMBF1c and putative members of this regulon was not so tight, probably due to a much longer heat exposure (14 days vs. few hours).

Several transcripts encoding putative USPs also accumulated in response to HT (Supplementary Table 5). The USP family plays an important role in stress resistance in bacteria (Kvint et al., 2003). Based on expression profiles, a similar role in abiotic stress tolerance was suggested for various plant USPs (Kerk et al., 2003; Maqbool et al., 2009; Isokpehi et al., 2011). Recently, the Arabidopsis AtUSP was shown to exhibit a redox-dependant chaperone function and to enhance plant tolerance to heat shock and oxidative stress (Young Jun et al., 2015). The role of USPs in grapevine remains to be elucidated.

The maintenance of protein homeostasis, which includes the control of synthesis, intracellular sorting, folding, the function and degradation of proteins, is fundamental to ensure growth and development of plants under normal and stressful environmental conditions. In Arabidopsis, FKBP62 (ROF1) and FKBP65 (ROF2) are involved in acquired thermotolerance through the interaction with HSP90.1 and HSFA2 (Meiri and Breiman, 2009; Meiri et al., 2010). ROF1 contributes to the transcription activity of HSFA2 but ROF2, in the presence of ROF1, abolishes this activity. This suggests that ROF2 acts as a HT modulator through a negative feedback regulation of HSFA2 (Meiri et al., 2010). While expression of the grapevine VviROF1 ortholog (VIT_00s0769g00010) was not affected in our experiments (Supplementary Table 6), VviROF2 transcripts (VIT_00s0260g00070) accumulated upon HT at green and ripening stages (G7D, R1D and R7D). Half of the DEGs linked to the functional category “protein synthesis” encode ribosomal proteins known to prevent inhibition of protein synthesis under HT (Muñoz and Castellano, 2012). A massive heat-dependent deregulation of ribosomal protein genes was also described in wheat (Qin et al., 2008). Interestingly, one of the constantly and strongly up-regulated genes encodes a chloroplast ribosomal protein (RPS1; VIT_13s0101g00050), a heat responsive protein involved in retrograde activation of heat responses in Arabidopsis (Yu et al., 2012). Particularly, RPS1 was proposed as a critical factor in the activation of HSFA2 and of its target genes. Many genes related to protein targeting (34 DEGs) were also affected in HT berries and mainly up-regulated during the green stage (Supplementary Table 6).

HT-induced protein degradation is a complex process involving a multitude of proteolytic pathways, with many DEGs encoding cysteine protease, serine protease, arginine protease, subtilase or metalloprotease activities. For instance, 24 transcripts encoding AAA/FtsH metalloproteases were deregulated in heated berries (Supplementary Table 6). This protease family is essential for the protein quality control of mitochondrial and chloroplastic membranes, and to prevent damages caused by stress conditions (Janska et al., 2013). For example, VviFtsH6 transcripts (VIT_14s0108g00590) strongly accumulated in berries when the HT was applied during the herbaceous stage. In Arabidopsis, the FtsH6 protease is involved in the degradation of the light-harvesting complex II (LHC II) during high light acclimatization and dark-induced senescence (Zelisko et al., 2005). The involvement of FtsH proteases in the quality control of the photosystem II was also described under moderate HT (Yoshioka et al., 2006) and a similar role may be attributed to FtsH6 in green photosynthetic berries exposed to HT. Another metalloprotease encoding gene (VIT_03s0088g00320) was highly up-regulated by HT at all stages. This transcript encodes an Arabidopsis EGY3 ortholog (At1g17870) belonging to the EGY family (ethylene-dependent gravitropism-deficient and yellow-green-like 3). AtEGY3 is an AtHSFA2 target gene (Nishizawa et al., 2006). The involvement of this putative membrane and plastidial metalloprotease in heat responses was recently pinpointed (Laranjeira and coworkers, unpublished, PhD thesis; http://hdl.handle.net/1822/18268). Both FtsH6 and EGY3 genes were also recently identified as heat responsive genes in Brassica napus siliques (Yu et al., 2014). The deregulation of numerous E3 ligase genes in HT berries may reflect an important role in triggering adaptive responses. Indeed, recent findings highlighted the role of various E3 ligases in mediating abiotic stress tolerance in Arabidopsis and in different crop species (Stone, 2014; Guerra et al., 2015). Most stress-related E3 ligases identified so far facilitate responses to environmental stimuli by modulating the abundance of key downstream stress-responsive TFs, thus affecting stress-related changes in gene expression. A non-proteolytic function of ubiquitin modification has also been reported in abiotic stress tolerance (Stone, 2014). For example, the rice E3 ligase OsHCI1 (Heat and Cold induced 1) monoubiquitinates some nuclear proteins in plants exposed to HT, which results in their translocation to the cytoplasm, and promotes HT tolerance (Lim et al., 2013). In apple, the E3 ligases MdCOP1s negatively regulate the peel anthocyanins content of fruits by modulating the degradation of the MdMYB1 protein (Li et al., 2012). A similar role can be envisaged in heat exposed berries to control the stability of flavonoid associated TFs. Many E2 conjugating enzyme genes are stress-inducible (Stone, 2014; Guerra et al., 2015). Two E2 transcripts were up-regulated when HT was applied on ripening berries. The first one (VIT_06s0004g08200) is an E2 enzyme ortholog of the Arabidopsis ubiquitin-conjugating enzyme 28 (UBC28, At1g64230). The second one (VIT_18s0001g10100) encodes a SUMO Conjugating Enzyme (SCE1a) binding small ubiquitin-like modifiers (SUMOs) to a wide range of cellular proteins. SUMOylation of AtHSFA2 represses its transcriptional activity and SUMO overexpression decreases sHSPs accumulation after HT (Cohen-Peer et al., 2010).

Low temperatures are beneficial to aroma production in the cool climate white grape cultivars (Duchêne and Schneider, 2005). Conversely, the aromatic potential of berries exposed to HT may be reduced (Belancic et al., 1997; Falcão et al., 2007), possibly due to heat-induced transcriptional changes (Rienth et al., 2014). Cabernet Sauvignon berries contain various aromatic molecules such as terpenes and C₁₃-norisoprenoids (Robinson et al., 2014) and are typified by specific volatile thiols and methoxypyrazines (Bouchilloux et al., 1998). Our transcriptomic data indicate that direct exposure of Cabernet Sauvignon berries to HT may decrease their aromatic potential through deregulation of numerous aroma and aroma precursor-related genes (Supplementary Tables 7, 9). Indeed, volatile terpenoids biosynthesis may be decreased by HT, as suggested by the heat repression of many key enzymes of the biosynthetic pathway (1-deoxy-D-xylulose-5-phosphate synthase, terpene synthase, geraniol 10-hydroxylase, (-)-germacrene D synthase and linalool synthase). Comparable results were obtained after exposure of the whole vine to HT (Rienth et al., 2014). Our data also revealed that most of 21 DEGs linked to carotenoid metabolism were down-regulated after HT, with the exception of VviBCH2. In Arabidopsis, overexpression of BCH2 improved tolerance to high light and HT by catalyzing the conversion of β-carotene to zeaxanthin and therefore preventing membranes from oxidative damage (Davison et al., 2002). While a constant decline in carotenoid abundance is generally observed after veraison in various cultivars including Cabernet Sauvignon (Deluc et al., 2009), our results suggest that HT may contribute to the decrease of carotenoid (except zeaxanthin) concentration before veraison, and/or accelerate its decrease after veraison. However, the possible consequences in term of aroma potential are not clear since only one type of CCD transcripts (VviCCD4a, VIT_02s0087g00910) was decreased by HT. Another gene (VviCCD4b, VIT_02s0087g00930) was transiently up-regulated by HT at veraison. The CCD enzymes convert their carotenoid substrates to C₁₃-norisoprenoids, which encompass desirable flavor and aroma compounds in grapes and wine. In normal growth conditions, up-regulation of VviCCD4a and VviCCD4b was observed during ripening in Sauvignon Blanc and Pinotage (Young et al., 2012; Lashbrooke et al., 2013), whereas VviCCD4a expression pattern during berry development was dependent on the growth temperature regime applied to Cabernet Sauvignon fruit cuttings (Guillaumie et al., 2011). Carotenoid concentration in grape berries is influenced by microclimate through an effect of light exposure on the cluster (Kwasniewski et al., 2010; Young et al., 2015) and possibly through an impact of the temperature as suggested by our results. Finally, the present work highlights the HT repressive effects on 3 out of 4 VviOMTs (Supplementary Table 7), including VviOMT3, responsible for the synthesis of the predominant methoxypyrazine IBMP (Guillaumie et al., 2013). Natural (climate, soil) and viticultural factors impact on IBMP concentration in grapes and wines, and grapes ripened under HT produce wines with reduced IBMP contents (Darriet et al., 2012). Considering the strong correlation between VviOMT3 expression and berry MP content, our data implies that HT lead to a strong reduction in IBMP synthesis during the herbaceous stage, resulting from the repression of the key gene VviOMT3, thus drastically reducing IBMP content in ripe berries.

Grape berry phenolic compounds contribute to organoleptic properties, color and protection against environmental cues. Our data show that a local warming of developing berries strongly affects the phenylpropanoid metabolic pathway. The effects depend on the stress duration and on the developmental stage (Figure 6; Supplementary Figures 2B, 3). Interestingly, the repression of VviPAL genes that correlated well with the accumulation of its substrate PHE and the deregulation of genes involved in anthocyanin stabilization (VviAOMTs and VviACTs) may contribute to the significant decrease of anthocyanin contents in HT berries at harvest (Table 1). Therefore, the delayed onset of veraison observed for GHT clusters could be considered at the transcriptional level. Additionally, together with the repression of VviFLS1 (flavonol synthase, VIT_18s0001g03470; also named FLS4, Fujita et al., 2006; Czemmel et al., 2009), the down-regulation of VviUFGT genes (VviGT5 and VviGT6) may contribute to the decrease of the flavonol content in heated berries. This result agrees with a recent work reporting the repression of VviGT5 in detached berries subjected to elevated temperatures (Loyola et al., 2016). Since previous work reported little or no effect on skin flavonol amounts (Spayd et al., 2002), the consequences of a locally applied HT on flavonol content still have to be determined. The transcriptional regulation of many flavonoid structural genes under HT suggests a control through the associated TFs. Since no or weak effect was observed on the expression of the positive TFs identified so far (Supplementary Table 11), several hypothesis can be proposed among which an up-regulation of negative regulators such as VviMYBC2-L3 and VviMYB4b (Supplementary Table 11; Cavallini et al., 2015), and a post-transcriptional control of the flavonoid associated genes as observed for the light-controlled stability of MYB1 protein in apple (Li et al., 2012).

A relationship between increased anthocyanin catabolism and elevated temperatures was proposed for grape berries (Mori et al., 2007). Although it is poorly known, anthocyanin degradation may involve enzymes such as laccases, polyphenol oxidases, class III peroxidases, and β-glucosidases (Oren-Shamir, 2009). In plants, the role of laccases remains largely unknown but their involvement in different steps of phenylpropanoid metabolism has been suggested. Fang et al. (2015) identified a vacuolar located ADE/LAC protein (anthocyanin degradation enzyme/laccase) responsible for the epicatechin-mediated anthocyanin degradation in litchi fruit during pericarp browning. Likewise, a gene encoding a putative rice laccase plays an important role in responses to abiotic stress (Cho et al., 2014). Our work strengthens the idea that the decrease in berry flavonoid content induced by HT involves an enzymatic-mediated degradation of these molecules in the vacuolar compartment. Indeed, laccases, polyphenol oxidase, class III peroxidase, and β-glucosidase genes were deregulated in local HT conditions (Supplementary Table 12). Particularly, the role of vacuolar class III peroxidase in anthocyanin degradation has been highlighted in different plant species including grape (Calderon et al., 1992; Vaknin et al., 2005; Zipor and Oren-Shamir, 2013; Zipor et al., 2015). For instance, a transcriptional up-regulation of VviPrx31 (VIT_14s0066g01850) was observed in berries exposed to HT (Movahed et al., 2016; and Supplementary Table 1). In Petunia, its overexpression reduced anthocyanin contents in petals exposed to heat.

Finally, genes involved in stilbenes and lignins biosynthesis were also specifically altered by HT during berry development. These alterations may modify berry texture and tolerance to the environment.

Many genes encoding proteins related to transport processes were enriched in GHT conditions (Supplementary Table 15). These genes were mainly repressed in GHT berries, in agreement with previous reports indicating that HT exposure led to a decrease in micronutrient transport. Genes involved in the calcium homeostasis were also downregulated by applying HT to green berries, and particularly CNGCs genes. CNGCs are nonspecific cation channels that are regulated by cyclic nucleotides such as cAMP or cyclic GMP (Ward et al., 2009) and that contribute to Ca²⁺ signaling in the context of developmental processes, biotic and abiotic stress responses (Jha et al., 2016). Calcium may act through facilitating developmental and stress response signaling, stabilizing membranes, influencing water relations and modifying cell wall properties through cross-linking of de-esterified pectins (Hocking et al., 2016). Beside a sum of DEGs potentially linked to calcium homeostasis, the HT also impacted numerous genes related to signaling (protein kinase, transcription factors, Ca²⁺-binding proteins) (Supplementary Table 1). Potassium transporter gene expression was also altered after GHT exposure. As potassium is required for cell expansion, alteration in the expression of its transporters may affect berry growth. Additionally, as K⁺ transporters and channels are known targets of ABA (that play an important role in berry ripening) (Davies et al., 2006), the alteration of their expression during the herbaceous stage may contribute to delay fruit ripening. MATE efflux transporters and ABC transporters were specifically affected by HT. For instance, while members of subfamily B were up-regulated at GHT, MATE, and ABCC and G were down-regulated. Interestingly, ABCG, the most affected subfamily, is involved in fruit maturation and exhibits a protective role (detoxification, vacuolar transport of ABA and glucosyl ester, anion transport) (Andolfo et al., 2015). It is the largest plant ABC transporter subfamily divided in two groups: WBCs and PDRs. The first group is involved in the extrusion of cuticular lipids and the second, in resistance to pathogens, antimicrobial terpenoids and auxinic herbicides, and in transport of signaling molecules or in secretion of volatile compounds (Kretzschmar et al., 2011; Andolfo et al., 2015). The repression of most of these transporters could potentially contribute to decrease anthocyanin accumulation.

The down-regulation of sugar transporter genes by HT fits well with earlier reports showing that HT results in the delay or arrest of the ripening process and of the accumulation of sugars (Greer and Weston, 2010; Greer and Weedon, 2013). However, in our experimental conditions, the postponing of the ripening process appears only when HT is directly applied on green clusters (GHT). At later stages, most of these sugar transporters were either down-regulated or not affected by HT (Supplementary Table 15). Altogether, these modifications in the amount of transporter transcripts may affect the berry physiology by modifying the pH and the nutrient content. As a consequence, amounts of certain primary and secondary metabolites were also affected in heated berries, resulting in the delayed onset of veraison.

Genes involved in cell wall composition were strongly altered in green berries during HT, highlighting the importance of cell wall adjustment (Supplementary Table 15). Heating effects on cell wall metabolism were also observed in VHT and RHT clusters. The fine-tuning of the cellulose-hemicellulose networks appears to be crucial for tolerance to heat (Tenhaken, 2014; Le Gall et al., 2015). The present transcriptomic data suggest differential cell wall synthesis and remodeling in heat-exposed berries, potentially affecting the overall fruit growth even if the exact consequences remain to be determined. In vineyard conditions, Dal Santo et al. (2013) found a correlation between the season climate and some differentially expressed genes encoding enzymes involved in cell wall structural modifications (especially CESs, PAEs, and XETs). XETs contribute to cell wall expansion and loosening, through their action on xyloglucan frame. After whole grape exposure to heat, an increased level of XETs transcripts was measured in warmed green berries. It was postulated that enhanced expression could be related to the adaptation of berry volume to temperature and the need for more flexible cell walls (Rienth et al., 2014). Our data reinforce these observations with 19 up-regulated XETs in GHT clusters. Cell wall loosening through increase level of expansin might help to maintain cellular functions during HT (Le Gall et al., 2015).

Fruit development and responses to environmental cues are controlled by plant hormones (Davies and Böttcher, 2009; Kuhn et al., 2014) and particularly by ABA. The present work revealed that berry heating significantly affects the expression of genes involved in ABA metabolism and signaling (Supplementary Table 9). The deregulation of ABA biosynthesis and signaling by applying HT during the herbaceous stage may contribute to the postponing of veraison and/or help to face HT. Interestingly, NCED expression and particularly VviNCED2 and VviNCED4 was impacted in GHT but also in VHT and RHT clusters. The down regulation of these 2 genes contrasts with data from Carbonell-Bejerano et al. (2013) reporting an HT-induction of these two genes after veraison and an increased ABA level in full ripe heated berries. The differences between both studies might be due to differences in the genotype and/or in the experimental conditions (temperature regime, microenvironment versus whole plant exposure, stress kinetic). Genes involved in ABA signaling were also affected. For instance, VviABI3 (VIT_07s0005g05400) strongly accumulated in GHT berries (Supplementary Table 9). ABI3 is a B3-domain TF that is a part of the core ABA signaling network and the corresponding gene is up-regulated in Cabernet Sauvignon berries treated with ABA (Rattanakon et al., 2016). In normal growth conditions, VviABI3 transcripts accumulated during the lag phase prior veraison in Cabernet Sauvignon (Deluc et al., 2007). Thus, heating seems to accelerate the expression of VviABI3 during the herbaceous stage. In Arabidopsis, ABI3 is exclusively expressed in seeds, controlling gene expression programs that are essential to achieve seed maturation (Baud et al., 2016). Whereas the role of ABI3 in nonseed tissues remains unknown, a functional connection was demonstrated between ABI3 and HSFs in sunflowers embryos and during seed development in Arabidopsis (Rojas et al., 1999; Kotak et al., 2007b). Rattanakon et al. (2016) identified others TFs showing modified expression level after ABA treatment of grape organs, these TFs belonging to the AP2/ERF, NAC, bZIP/ABRE, and MYC/MYB families. In berries exposed to heat, more than 20 transcripts from this set of TFs were deregulated what can be potentially due to ABA action (Supplementary Table 15). The strongest deregulated TF of this list corresponds to the bZIP/ABRE Abscisic Acid Insensitive protein 5 (VviABI5; VIT_06s0080g00340, VIT_08s0007g03420) that significantly accumulated in GHT clusters whatever the treatment duration (Supplementary Table 15). ABI5 is depicted as a key regulator in the ABA signaling pathway, and a recent work highlighted its contribution in the ABA-dependent stimulation of plant thermotolerance (Lee et al., 2015). After interaction with the plant-specific RNA-binding protein FCA, ABI5 enhanced antioxidant activity under HT conditions. Particularly, ABI5 promotes the expression of genes encoding antioxidants, including 1-CYSTEINE PEROXIREDOXIN 1 (PER1). In agreement with these data, the heat-induced expression profiles of VviABI5 and VviPER1 (VIT_05s0020g00600) were closely related in berries, suggesting a conserved mechanism in the fruit (Supplementary Table 1). Peroxiredoxins, which are thiol-based peroxidases, enhance plant tolerance to oxidative and heat stresses. More broadly, in this work, 70 DEGs were identified in heated berries as ROS (reactive oxygen species) scavenging/detoxifying enzymes and various antioxidants (Supplementary Table 1). This kind of adaptative response, described in different plants exposed to HT (de Pinto et al., 2015), may help the berries to manage potential damages due to oxidative burst. Indeed, ROS mainly attributable to NADPH oxidase activity accumulate under stress conditions including HT (Miller et al., 2009). Accordingly, two VviRBOH (respiratory burst oxidase homolog; VIT_14s0060g02320, VIT_01s0150g00440) genes were up-regulated in heat-exposed berries (Supplementary Table 1).

Besides ABA, our transcriptomic data (Supplementary Table 1) also suggest that HT affects the metabolism of auxin, ethylene and jasmonic acid. These hormones are involved in the control of grape berry development (Davies and Böttcher, 2009) and in the activation of key genes responsible for HT response (Bokszczanin and Fragkostefanakis, 2013; Sharma and Laxmi, 2015). For instance, indole-3-acetic acid (IAA) inhibits berry growth, sugar and anthocyanin accumulation (Kuhn et al., 2014) and prevents ripening. Indeed, the decrease in IAA content and the increase of its conjugated form are needed to induce ripening (Davies et al., 1997; Böttcher et al., 2010). Interestingly, our work showed that application of a HT to green berries resulted in the reduction of IAA-amido synthetase (VviGH3, VIT_07s0005g00090) transcript abundance and in an increase in the expression of two transcripts encoding IAA-amino acid hydrolases (VIT_11s0016g02700, VIT_08s0007g02740), thus suggesting that heating is disrupting IAA conjugating process in GHT clusters and therefore postponing the onset of ripening. Further work is needed to address the respective role of each hormone and their interactions in the context of grape berry grown under HT.