domain: sdv sde

text:
David Lecourieux
email: david.lecourieux@inra.fr

Christian Kappel

Stéphane Claverol

Philippe Pieri

Regina Feil

John E Lunn

Marc Bonneu

Lijun Wang

Eric Gomès

Serge Delrot

Fatma Lecourieux

Proteomic and metabolomic profiling underlines the stage-and time-dependent effects of high temperature on grape berry metabolism

Keywords: Vitis vinifera, berry, heat stress, proteomic, primary and secondary metabolisms

des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

INTRODUCTION

Grapevine is economically the most important fruit crop in the world, providing dried fruits, table grapes and the basis for wine making (www.oiv.int; www.fao.org). The combination of climate, soil and viticultural practices in a given region constitutes a highly interactive system known as "terroir" [START_REF] Deloire | Grapevine responses to terroir: a global approach[END_REF]. The interaction between "terroir" and the plant's genotype (cultivar) significantly affects vine development and berry composition, and thereafter determines wine quality and character [START_REF] Van Leeuwen | The concept of terroir in viticulture[END_REF]. Grapevine requires suitable temperatures, radiation intensities and duration, and water availability during the growth cycle for optimal fruit production and quality [START_REF] Kuhn | Berry ripening: recently heard through the grapevine[END_REF]. Because of on-going climate change, the environmental parameters in most wine producing regions around the world are expected to change, potentially lowering productivity and altering the traditional characteristics ("typicity") of the wine from a given region [START_REF] Schultz | Climate induced historic and future changes in viticulture[END_REF]. While some of these climatic constraints can be overcome, at least partially, by viticultural practices, temperature remains more difficult to control under field conditions [START_REF] Van Leeuwen | Why climate change will not dramatically decrease viticultural suitability in main wine-producing areas by 2050[END_REF]. Climate change scenarios predict an increase in global mean temperatures between 1.5 and 5°C, during the 21 th century (IPCC, 2014), in addition to an increase in the frequency, intensity, and duration of extreme temperature events [START_REF] Lobell | Prioritizing climate change adaptation needs for food security in 2030[END_REF]. Considering that air temperature is a key climate variable affecting grapevine phenology, berry metabolism and composition [START_REF] Jones | Climate change and global wine quality[END_REF][START_REF] Martínez-Lüscher | Sensitivity of grapevine phenology to water availability, temperature and CO2 concentration[END_REF], global warming represents a serious threat for wine production and/or typicity in many wine-growing regions worldwide [START_REF] Fraga | Modelling climate change impacts on viticultural yield, phenology and stress conditions in Europe[END_REF].

Temperatures above the optimum are sensed as heat stress by living organisms. Heat stress disturbs cellular homeostasis and can lead to severe retardation in growth and development and even death [START_REF] Wahid | Heat tolerance in plants: an overview[END_REF]. In grapevine, warming promotes vegetative growth and disturbs the plant's carbon balance [START_REF] Gouot | Modelling photosynthetic responses to temperature of grapevine (Vitis vinifera cv. Semillon) leaves on vines grown in a hot climate[END_REF], adversely affecting flower set and young berry development [START_REF] Greer | Heat stress affects flowering, berry growth, sugar accumulation and photosynthesis of Vitis vinifera cv. Semillon grapevines grown in a controlled environment[END_REF]. HS also impacts fruit primary and secondary metabolism, desynchronizing sugar and organic acid metabolism and delaying sugar and polyphenol accumulation during ripening [START_REF] Torregrosa | Developmental, molecular and genetic studies on grapevine response to temperature open breeding strategies for adaptation to warming[END_REF][START_REF] Gouot | Modelling photosynthetic responses to temperature of grapevine (Vitis vinifera cv. Semillon) leaves on vines grown in a hot climate[END_REF]). As a consequence, heat waves occurring during key berry development stages can dramatically affect the organoleptic properties of wine even when more favourable weather conditions prevail for the remaining of the season.

In this context, the common viticultural practice of removing leaves around the fruit zone to enhance aroma development and avoid fungal infections [START_REF] Alem | Impact of agronomic practices on grape aroma composition: a review[END_REF], might be questioned since it affects light exposure and temperature of the berries. Because of the direct irradiative effects, temperature within the fruit may differ very significantly from that of the whole plant or surrounding air (Cola et al., 2009). The microclimate (mostly temperature and light conditions) strongly influences the metabolite composition of berries, affecting flavonols, anthocyanins, amino acids and aroma precursors [START_REF] Downey | Cultural practice and environmental impacts on the flavonoid composition of grapes and wine: a review of recent research[END_REF][START_REF] Pereira | Microclimate influence on mineral and metabolic profiles of grape berries[END_REF][START_REF] Asproudi | Grape aroma precursors in cv. Nebbiolo as affected by vine microclimate[END_REF]Young et al. 2016;[START_REF] Reshef | Sunlight Modulates Fruit Metabolic Profile and Shapes the Spatial Pattern of Compound Accumulation within the Grape Cluster[END_REF]. These effects can be explained, at least in part, by the substantial remodelling of the fruit transcriptome observed under these conditions [START_REF] Du Plessis | The Transcriptional Responses and Metabolic Consequences of Acclimation to Elevated Light Exposure in Grapevine Berries[END_REF][START_REF] Lecourieux | Dissecting the Biochemical and Transcriptomic Effects of a Locally Applied Heat Treatment on Developing Cabernet Sauvignon Grape Berries[END_REF].

In a previous report, we showed dramatic biochemical and transcriptomic changes in heat-exposed berries, depending on both the developmental stage and the stress duration [START_REF] Lecourieux | Dissecting the Biochemical and Transcriptomic Effects of a Locally Applied Heat Treatment on Developing Cabernet Sauvignon Grape Berries[END_REF].

Although transcriptomic analyses have offered comprehensive insights into the mechanisms underlying the HS responses in grape [START_REF] Liu | Transcriptomic analysis of grape (Vitis vinifera L.) leaves during and after recovery from heat stress[END_REF][START_REF] Carbonell-Bejerano | Thermotolerance responses in ripening berries of Vitis vinifera L. cv Muscat Hamburg[END_REF]Rienth et al. 2014b;[START_REF] Jiang | Integrating Omics and Alternative Splicing Reveals Insights into Grape Response to High Temperature[END_REF][START_REF] Lecourieux | Dissecting the Biochemical and Transcriptomic Effects of a Locally Applied Heat Treatment on Developing Cabernet Sauvignon Grape Berries[END_REF], it is recognised that changes in transcript abundance do not always correlate with the levels of the encoded proteins [START_REF] Keller | The coupling of transcriptome and proteome adaptation during development and heat stress response of tomato pollen[END_REF]. Furthermore, proteins represent a more functional level of gene expression than transcripts because they have a direct impact on metabolism and other cellular processes. Therefore, state of the art mass spectrometry analysis of the proteome is a powerful tool to study molecular mechanisms and biological traits in plants, including responses to abiotic stresses [START_REF] Kosova | Plant Abiotic Stress Proteomics: The Major Factors Determining Alterations in Cellular Proteome[END_REF]. Using shotgun proteomic tools such as iTRAQ (isobaric Tags for Relative and Absolute Quantitation) and label-free quantification techniques, several proteomic studies have recently investigated the heat responses of grapevine leaves [START_REF] Liu | Differential proteomic analysis of grapevine leaves by iTRAQ reveals responses to heat stress and subsequent recovery[END_REF][START_REF] Jiang | Integrating Omics and Alternative Splicing Reveals Insights into Grape Response to High Temperature[END_REF] or grape cell cultures [START_REF] George | Quantitative proteomic analysis of cabernet sauvignon grape cells exposed to thermal stresses reveals alterations in sugar and phenylpropanoid metabolism. Proteomics: Giribaldi M[END_REF].

To better understand the consequences of high temperature (HT), inherent to leaf removal, on berry development and to improve our knowledge about the molecular mechanisms involved in grapevine fruit response to HT, the present work combines transcriptomic, proteomic and metabolite analysis using Cabernet Sauvignon fruit-bearing cuttings. A label-free proteomic approach was used to investigate changes in the grape berry proteome resulting from direct exposure to elevated temperature. The collected proteomic data were compared with our previous transcriptomic analysis performed on the same berry samples [START_REF] Lecourieux | Dissecting the Biochemical and Transcriptomic Effects of a Locally Applied Heat Treatment on Developing Cabernet Sauvignon Grape Berries[END_REF]. These data showed a strong and negative effect of HT on proteins related to carbohydrate and energy metabolism, which led us to analyse the impact of HT on primary metabolites using tandem mass spectrometry (LC-MS/MS).

Finally, we focused on proteins accumulating in berries exposed to HT that are potentially involved in thermotolerance. Altogether, this work helps to better understand the consequences of heat stress on developing grape berries and provides a list of potential heat-tolerance molecular markers that could be useful for breeders and viticulturists.

RESULTS AND DISCUSSION

Quantitative analysis of heat stress responsive proteins in grape berries

Experiments were performed on fruit-bearing cuttings of V. vinifera Cabernet Sauvignon as described in [START_REF] Lecourieux | Dissecting the Biochemical and Transcriptomic Effects of a Locally Applied Heat Treatment on Developing Cabernet Sauvignon Grape Berries[END_REF]. In brief, berries at three different stages of development were exposed to elevated temperatures for 12 h (7:00 am to 7:00 pm) during each day of treatment and harvested for analysis after 1 and 7 days of treatment. The average pulp temperature of the heat-treated berries was about 8°C higher than in control berries. A label-free proteomic approach was performed to identify proteins with differential abundance between control and heat-treated berry samples.

Using the parameters and filters described in the Material and Methods, the conducted label-free experiment led to the identification of 3972 protein groups (from here on referred to as proteins) in the combined dataset from the six conditions (three stages x two treatment durations), of which 2279 showed two or more unique peptides (Table 1). Protein abundance changes between control and heatexposed fruits were considered to be significant when a minimum fold change of 1.5 (log 2 FC > 0.58 or < -0.58) was reached, with a significance threshold of p < 0.05. Only proteins identified in all three biological replicates were considered for quantification. Among the quantified proteins with a minimum of two unique peptides, 592 DAPs (differentially abundant proteins) displayed significant differences in abundance between control and HT berries in at least one of the six conditions. The entire dataset is available via ProteomeXchange (identifier PXD014693) and in Supplementary Table S1. Further information on the 592 DAPs is provided in Supplementary Table S2. Among these 592 DAPs, 501 proteins were identified in only one condition (139 up-regulated and 362 down-regulated) while the 91 remaining proteins were retrieved in more than one condition (16 up-regulated, 61 downregulated, and 14 proteins that were up-regulated in some conditions but down-regulated in others)

(Table 1 andTable S2). More DAPs were identified at the ripening stage (304 DAPs) than at the green (146 DAPs) or veraison stages (222 DAPs). Whereas the numbers of up-and down-regulated proteins were similar at veraison (115 up, 107 down), more down-regulated proteins were found when the HT was applied at the green stage (24 up, 122 down) or ripening (49 up, 255 down) stages (Figure 1A). The DAPs identified after short (1 day) treatments were mostly different from those observed after long (7 days) treatments. Indeed, only 1 (up), 9 (4 up, 5 down) and 18 (2 up, 16 down) common DAPs between 1D or 7D of HT were observed at the green, veraison and middle ripening stages, respectively (Figure 1B and Tables S2,S3, S4 & S5). Similarly, at all three stages, only one common down-regulated DAP was identified after 1 day of HT (VIT_12s0057g01020, fasciclin-like protein), with none being detected after 7 days of treatment (Table S2). The number of common DAPs per stage increased when looking for proteins with modified abundance after 1 and/or 7 days treatment. In this case, 7 DAPs (3 up, 4 down) were found in common for the three stages, and up to 31 DAPs were obtained when comparing heat effects between green and veraison stages (Figure 1C and

Supplementary Table 2).

To determine the functional categories affected by HT, the identified DAPs were functionally classified using MapMan BIN codes [START_REF] Usadel | Extension of the visualization tool MapMan to allow statistical analysis of arrays, display of corresponding genes, and comparison with known responses[END_REF]) and a V. vinifera mapping file (Lecourieux et al., 2017). The predominant proteins changed in berries in response to HT were assigned to "protein metabolism" (114/592 DAPs, 19.3%), "stress" (54/592 DAPs, 9.1%), "secondary metabolism" (31/592 DAPs, 5.2%) and "cell wall" (25/592 DAPs, 4.2%) (Figure 2). Except for the categories "stress" (36 up, 18 down) and "RNA" (10 up, 9 down), all the others categories contained more downregulated proteins that up-regulated ones, with particularly asymmetrical changes in some categories, such as "lipid metabolism" (0 up, 16 down) and "transport" (2 up, 17 down). No particular effect of HT according to the developmental stage was observed on these functional categories, whereas categories linked to carbohydrate and energy metabolism (glycolysis, tricarboxylic acid (TCA) cycle, fermentation, gluconeogenesis, minor and major carbohydrate (CHO) metabolism) or related to "photosynthesis", "cell wall" and "redox" were mostly impacted during veraison and/or ripening stages (Supplementary Table 2).

Poor correlation between the transcriptome and proteome in heat-stressed berries

Global analyses based on gene expression and protein profiles provide a powerful tool for understanding how organisms respond to environment changes. To integrate proteomic data with transcriptomic results, the present protein abundance data were compared to each corresponding differentially expressed gene (DEG) identified in the same grape berry samples exposed to HT [START_REF] Lecourieux | Dissecting the Biochemical and Transcriptomic Effects of a Locally Applied Heat Treatment on Developing Cabernet Sauvignon Grape Berries[END_REF]. Whereas 592 DAPs were retrieved in at least one of the six HT conditions (Table 1, Supplementary Table S1), 6848 DEGs were identified using the same berry samples [START_REF] Lecourieux | Dissecting the Biochemical and Transcriptomic Effects of a Locally Applied Heat Treatment on Developing Cabernet Sauvignon Grape Berries[END_REF]. A linear correlation between differentially expressed transcripts and the corresponding proteins was established for 64 out of 592 DAPs (Figure 4, Supplementary Table S11). Two types of correlation profiles were found. The first one corresponds to proteins and transcripts that exhibit profiles with the same tendency (up-or down-regulation in response to HT) and the second one includes proteins and transcripts that are inversely correlated for a same gene. The best relationship between mRNA and protein levels (39 DAPs) was observed when HT was applied for one day at veraison, and for proteins belonging to functional BINs associated to "heat stress" (25 members), "hormone" (4), and "secondary metabolism" (5) (Figure 4, Supplementary Table S11).

Our results underline the poor correlation between the proteome and the transcriptome in HT berries, with only 11% (64/592 DAPs) of the differentially abundant proteins that were also impacted at the mRNA expression level. This is not unexpected since HT leads to denaturation and aggregation of proteins, and because translation, especially under elevated temperature, is governed by a variety of regulatory mechanisms independent of transcription rate (Bokszczanin and Fragkostefanakis 2013).

These include the impacts of miRNAs on mRNA stability and translatability, mRNA half-life, global and transcript specific translational rates, as well as protein turnover. Any lag between the synthesis of a gene transcript and its translation into a protein could also give rise to an apparent discrepancy between transcript and protein abundance at a given sampling time [START_REF] Keller | The coupling of transcriptome and proteome adaptation during development and heat stress response of tomato pollen[END_REF]. Last but not least, the poor correlation between transcript and protein levels in heat-treated berry samples may also be due to alternative splicing (AS) events. Increasing evidence has shown that AS is a critical post-transcriptional event and plays an important role in plant stress responses [START_REF] Baurle | Plant Heat Adaptation: priming in response to heat stress. F1000Research[END_REF].

Particularly, many genes are under transcriptional regulation by the circadian clock, and several circadian clock genes show AS in response to even small changes in temperature [START_REF] James | How does temperature affect splicing events? Isoform switching of splicing factors regulates splicing of LATE ELONGATED HYPOCOTYL (LHY)[END_REF].

In grapevine, AS changes were observed for nearly 70% of the genes in leaves exposed to temperatures above 35°C. In the same study, only 64 out of the 808 identified DAPs were retrieved as differentially expressed at the transcript level [START_REF] Jiang | Integrating Omics and Alternative Splicing Reveals Insights into Grape Response to High Temperature[END_REF], that fits well with the weak transcript/protein correlation observed in our work. [START_REF] Jiang | Integrating Omics and Alternative Splicing Reveals Insights into Grape Response to High Temperature[END_REF] suggested that AS changes under high temperature could provide molecular plasticity allowing the plants to adapt to stress conditions. Using Arabidopsis (Arabidopsis thaliana) plants exposed or not to a non-lethal HT, referred to as priming, [START_REF] Ling | Thermopriming triggers splicing memory in Arabidopsis[END_REF] have recently demonstrated that AS functions as a novel component of heat-shock memory.

Despite the generally poor correlation between individual transcripts and proteins levels in berries exposed to HT, there was a much better correspondence at the functional category level, with transcripts and proteins assigned to the "stress", "protein metabolism" and "secondary metabolism" MapMan BINs showing consistent responses as a group (Supplementary Figure S1). We also noticed that, at the transcript level, most of the functional categories were differentially affected according to the developmental stage and the stress duration whereas it was less obvious at the protein level. The best relationship between transcripts and proteins levels was observed with genes related to the category "stress", and especially to the "heat" sub-category. Among the 30 HSPs whose protein abundance increased in response to HT, 25 were also up-regulated at the transcript level (Supplementary Table S12). Most proteins are inherently thermo-labile, with some becoming denatured at the temperatures that grapevines experience during a heat wave. In addition, high temperature is often associated with oxidative stress leading to production of reactive oxygen species that irreversibly damage proteins. Maintaining proteins in stable and functional conformations and preventing their aggregation are essential for survival of cells under high temperature. HSPs function as molecular chaperones in maintaining protein quality and folding, and are required for the acquisition of plant thermotolerance (Bokszczanin and Fragkostefanakis 2013). It is well established that transcription factors from the HSF family regulate the heat stress response in plants, through a massive up-regulation of HSP genes [START_REF] Scharf | The plant heat stress transcription factor (Hsf) family: structure, function and evolution[END_REF]. However, as transcription factors are low abundance proteins they largely escaped proteomic detection, and no HSF was retrieved in the present study despite the observed transcriptional up-regulation of six VviHSF genes in heat-exposed berries [START_REF] Lecourieux | Dissecting the Biochemical and Transcriptomic Effects of a Locally Applied Heat Treatment on Developing Cabernet Sauvignon Grape Berries[END_REF].

In good agreement with our transcriptomic data, the protein abundance of the functional category "Protein metabolism" was strongly impacted with nearly 20% of the identified DAPs that belong to this category being affected, especially those belonging to the sub-categories "protein synthesis" (34 DAPs) and "protein degradation" (55 DAPs) (Supplementary Table S6). However, the HT triggered a strong down-accumulation effect on the "protein metabolism" category (92 out of the 114 DAPs), in contrast with what was observed on transcripts (333 up-and 307 down-regulated genes). A number of studies demonstrated that different abiotic stresses, including heat, result in a general inhibition of protein translation [START_REF] Yanguez | Grapevine plasticity in response to an altered microclimate: Sauvignon Blanc modulates specific metabolites in response to increased berry exposure[END_REF]. Accordingly, we observed a significant decrease in abundance of many proteins related to protein synthesis including various ribosome structural proteins and several translation initiation factors (Supplementary Table S6). Whereas inhibiting translation under stress helps to reduce energy consumption, certain specific mRNAs are selectively translated to produce relevant proteins involved in the proper establishment of the stress adaptation process. By studying the translatome of Arabidopsis seedling exposed to HT, [START_REF] Yanguez | Grapevine plasticity in response to an altered microclimate: Sauvignon Blanc modulates specific metabolites in response to increased berry exposure[END_REF] observed that regulation of gene expression at the translational level is superimposed on regulation at the transcriptional level, and often reinforces the response of individual genes/proteins to heat stress.

Protein translation is accomplished by the combined actions of the ribosomes and ancillary proteins such as initiation, elongation, and termination factors. [START_REF] Yanguez | Grapevine plasticity in response to an altered microclimate: Sauvignon Blanc modulates specific metabolites in response to increased berry exposure[END_REF] showed that translation could be regulated at the initiation phase in Arabidopsis seedlings exposed to HT, even if the mechanisms involved in this regulation remain unclear. To date, there is some evidence for the role of different plant translation initiation factors (eIFs) in various abiotic stresses responses [START_REF] Dutt | Translation initiation in plants: roles and implications beyond protein synthesis[END_REF]. Our proteomic data highlighted the increase in abundance of four eIFs upon HS, namely eIF1A (VIT_15s0046g01500), two isoforms of eIF3 (VIT_14s0060g01630, VIT_00s0880g00020) and eIF5 (VIT_14s0006g01990), whereas others eIF isoforms were decreased (Supplementary Table S6). eIF5 (VIT_14s0006g01990) showed the highest accumulation score (Log 2 FC 5.22) within this functional category. The importance of eIF5 proteins in thermotolerance was highlighted in pea [START_REF] Suragani | The translation initiation factor, PeIF5B, from Pisum sativum displays chaperone activity[END_REF] and Arabidopsis [START_REF] Xu | RceIF5A, encoding an eukaryotic translation initiation factor 5A in Rosa chinensis, can enhance thermotolerance, oxidative and osmotic stress resistance of Arabidopsis thaliana[END_REF][START_REF] Yan | Analysis of the grape (Vitis vinifera L.) thaumatin-like protein (TLP) gene family and demonstration that TLP29 contributes to disease resistance[END_REF].

Heat stress alters key proteins driving berry development and ripening

Heat stress impacts the secondary metabolism in developing berry

The strong effects of HT observed on transcripts assigned to the "secondary metabolism" functional category (291 DEGs across the six conditions, [START_REF] Lecourieux | Dissecting the Biochemical and Transcriptomic Effects of a Locally Applied Heat Treatment on Developing Cabernet Sauvignon Grape Berries[END_REF]) were confirmed at the protein level (31 out of the 592 DAPs), albeit to a lesser extent (Supplementary Table S8). These 31 DAPs (11 up, 20 down) mainly belonged to the subcategories: "isoprenoids" (7 DAPs), "phenylpropanoidlignin" (12) and "flavonoids" (7).

HT altered expression of numerous genes related to aromatic potential, including genes encoding biosynthetic enzymes for terpenes and methoxypyrazines [START_REF] Lecourieux | Dissecting the Biochemical and Transcriptomic Effects of a Locally Applied Heat Treatment on Developing Cabernet Sauvignon Grape Berries[END_REF]), but the proteomic data from the current study suggested that these changes in transcript abundance were not translated through to the protein level. A few proteins related to isoprenoid subfamily were down-accumulated under HT including a 3-ketoacyl-CoA synthase (KCS10, VIT_04s0008g02250), which is involved in the synthesis of very long chain fatty acids that are incorporated into plant lipids (precursors of cuticular waxes), and several enzymes related to the synthesis of precursors of terpenoids (Supplementary Table S8). By contrast, a carotenoid cleavage dioxygenase (CCD1, VIT_13s0064g00810) was found more abundant in stressed berries at veraison. Expression of various VvCCD genes was reported to increase towards berry ripening and enzymes in this family were demonstrated to promote desirable flavor and aroma compounds in grapes and wine [START_REF] Mathieu | A carotenoid cleavage dioxygenase from Vitis vinifera L.: functional characterization and expression during grape berry development in relation to C13-norisoprenoid accumulation[END_REF]Rienth et al. 2014a;[START_REF] Alem | Impact of agronomic practices on grape aroma composition: a review[END_REF]. The putative role of CCD proteins in heat responses is still unknown but these proteins were suggested to enhance tolerance to various abiotic stresses through their enzymatic products (e.g. apocarotenoids) acting as stress signals [START_REF] Havaux | Carotenoid oxidation products as stress signals in plants[END_REF]. For instance, some members of the CCD family are involved in generating carotenoid-derived phytohormones, such as strigolactones known to be involved in various plant developmental and adaptation processes [START_REF] Jia | From carotenoids to strigolactones[END_REF].

It is well known that extreme high temperatures significantly affect the phenylpropanoid content of grape berries, with the impact being dependent on the intensity and duration of the HS as well as the developmental stage of the fruit [START_REF] Gouot | Modelling photosynthetic responses to temperature of grapevine (Vitis vinifera cv. Semillon) leaves on vines grown in a hot climate[END_REF]. Consistent with changes observed at the transcript level [START_REF] Lecourieux | Dissecting the Biochemical and Transcriptomic Effects of a Locally Applied Heat Treatment on Developing Cabernet Sauvignon Grape Berries[END_REF] S2 &S8). This lower amount of key enzymes probably contributes to the reduction of flavonoid synthesis in grape berries exposed to heat. However, other concomitant processes may explain the decrease of flavonoid contents in warmed berries, including reduced biosynthetic enzyme activities due to non-optimal temperature, enzymatic oxidation or chemical degradation after ROS scavenging [START_REF] Gouot | Modelling photosynthetic responses to temperature of grapevine (Vitis vinifera cv. Semillon) leaves on vines grown in a hot climate[END_REF]. Upon their biosynthesis in the cytosol, flavonoids are rapidly accumulated into vacuoles or other cellular compartments through vesicle trafficking, membrane transporters or glutathione S-transferase (GST)-mediated transport (Zhao 2015). In addition to transport of flavonoids into vacuoles mediated by ATP-binding cassette (ABC) transporters [START_REF] Francisco | ABCC1, an ATP binding cassette protein from grape berry, transports anthocyanidin 3-O-Glucosides[END_REF], there is also H + -dependent transport mechanism in grapevine [START_REF] Gomez | In vivo grapevine anthocyanin transport involves vesicle-mediated trafficking and the contribution of anthoMATE transporters and GST[END_REF][START_REF] Kuang | Quantitative Proteome Analysis Reveals Changes in the Protein Landscape During Grape Berry Development With a Focus on Vacuolar Transport Proteins[END_REF]. Vacuolar transport of anthocyanins and other metabolites, such as malate, directly or indirectly depends on the transmembrane pH gradient generated by both tonoplast H + -ATPases and H + -pyrophosphatases (V-PPase) [START_REF] Shiratake | Transporters in fruit vacuoles[END_REF]. Three V-ATPase subunits (VIT_03s0038g00790, VIT_18s0001g01020, VIT_04s0008g02460) and two V-PPases (VIT_11s0118g00350, VIT_09s0002g07880) showed strong decrease in protein abundance in HT berries, and this may impair the efficient transport of anthocyanins into the vacuole. Finally, the translational repression of several GST proteins (VIT_01s0026g01340, VIT_01s0011g01900, VIT_08s0040g03100, VIT_19s0093g00310, VIT_07s0104g01800) observed in heat-stressed berries during ripening could have an impact on fruit secondary metabolism as well [START_REF] Perez-Diaz | Differential Roles for VviGST1, VviGST3, and VviGST4 in Proanthocyanidin and Anthocyanin Transport in Vitis vinifera[END_REF].

Harmful effects of heat stress on carbohydrate and energy berry metabolism

A drastic negative effect of HT was observed for proteins assigned to primary carbohydrate metabolism (Supplementary Table S9). Almost all the DAPs related to the following processes were under-accumulated after HT: major CHO metabolism (6 out of 8 DAPs), glycolysis (8 out of 9

DAPs), fermentation (4 out of 5 DAPs), gluconeogenesis (3 out of 4 DAPs), oxidative pentose phosphate pathway (6 DAPs) and TCA cycle (13 out of 14 DAPs). To better understand the consequences of this HT repressive effect, and to expand beyond the previously reported metabolite changes [START_REF] Lecourieux | Dissecting the Biochemical and Transcriptomic Effects of a Locally Applied Heat Treatment on Developing Cabernet Sauvignon Grape Berries[END_REF], we measured an additional 31 metabolites that are involved in sugar or organic acid metabolism, or respiration. To put the measured metabolites into a metabolic context, a schematic representation of plant central carbon metabolism was created to display the metabolite profiles under HT compared to control conditions, for each developmental stage and each stress duration (Figure 3, Supplementary Table S10). A significant effect of the developmental stage (twoway ANOVA, p < 0.05) was observed for most quantified metabolites (23 out of 31), in agreement with the previously reported metabolite profiles in developing berries from Cabernet Sauvignon fruitbearing cuttings [START_REF] Dai | Metabolic profiling reveals coordinated switches in primary carbohydrate metabolism in grape berry (Vitis vinifera L.), a non-climacteric fleshy fruit[END_REF]. HT significantly influenced the content of 14 metabolites including shikimate, glycerate, glycerol-3-P (G-3-P), several glycolytic intermediates (fructose 6-phosphate (Fru6P), 3-phosphoglycerate (3-PGA) and pyruvate), fumarate, and several sugar phosphates (glucose 6-P (G6P), trehalose 6-P (T6P), sucrose 6´-P (S6P) and mannose 6-P (M6P)). The well-known accelerated decrease of malate under elevated temperature was also observed although the changes were below the level of significance in an ANOVA test. However, when considered over the 14-day time frame of the experiments, the decrease in malate was statistically significant according to a t-test (Figure 3). This agrees with the significantly lower malate content detected previously in HT berries at harvest [START_REF] Lecourieux | Dissecting the Biochemical and Transcriptomic Effects of a Locally Applied Heat Treatment on Developing Cabernet Sauvignon Grape Berries[END_REF].

Metabolism undergoes profound reprogramming during fleshy fruit development (Beauvoit et al. 2018). The contents of primary and secondary metabolites have been investigated in developing grape berries [START_REF] Dai | Metabolic profiling reveals coordinated switches in primary carbohydrate metabolism in grape berry (Vitis vinifera L.), a non-climacteric fleshy fruit[END_REF][START_REF] De Jesus-Pires | Metabolite and transcript profiling of berry skin during fruit development elucidates differential regulation between Cabernet Sauvignon and Shiraz cultivars at branching points in the polyphenol pathway[END_REF][START_REF] Cuadros-Inostroza | GC-MS metabolic profiling of Cabernet Sauvignon and Merlot cultivars during grapevine berry development and network analysis reveals a stage-and cultivardependent connectivity of primary metabolites[END_REF][START_REF] Wang | System-Level and Granger Network Analysis of Integrated Proteomic and Metabolomic Dynamics Identifies Key Points of Grape Berry Development at the Interface of Primary and Secondary Metabolism[END_REF]. Using

Cabernet Sauvignon fruit-bearing cuttings as in the present work, [START_REF] Dai | Metabolic profiling reveals coordinated switches in primary carbohydrate metabolism in grape berry (Vitis vinifera L.), a non-climacteric fleshy fruit[END_REF] conducted a deep analysis of metabolites from central carbon metabolism using an LC-MS/MS approach. They reported the timing of important switches in primary carbohydrate metabolism during grape berry development and discussed the relationship between these metabolite changes and transcriptional and translational modifications within the fruit. Similarly, [START_REF] Wang | System-Level and Granger Network Analysis of Integrated Proteomic and Metabolomic Dynamics Identifies Key Points of Grape Berry Development at the Interface of Primary and Secondary Metabolism[END_REF] reported close correlations between the metabolome and the proteome at the interface of primary and secondary metabolism in developing V.

vinifera Early Campbell fruits. Down-regulation of carbohydrate production and protein synthesis is a conserved response of plants to abiotic stresses [START_REF] Cramer | Effects of abiotic stress on plants: a systems biology perspective[END_REF]). This may be a way for plants to save energy, and reflects a shift from plant growth to mechanisms of protection. However, with the exception of organic acids, the effects of high temperature on berry primary metabolism in general are still poorly described [START_REF] Serrano | Omics Approaches for Understanding Grapevine Berry Development: Regulatory Networks Associated with Endogenous Processes and Environmental Responses[END_REF]. It is well known that warm climatic conditions enhance the degradation of malate in grapevine fruits, leading to a decrease in total acidity [START_REF] Sweetman | Metabolic effects of elevated temperature on organic acid degradation in ripening Vitis vinifera fruit[END_REF]).

Sugar metabolism is desynchronized from organic acid metabolism in ripening berries at high temperatures [START_REF] Rienth | Temperature desynchronizes sugar and organic acid metabolism in ripening grapevine fruits and remodels their transcriptome[END_REF]). The micro-environment plays an active role in controlling the dynamics of primary metabolite accumulation that affect fruit composition. For instance, heating strongly alters the berry concentration of various amino acids, including phenylalanine and γaminobutyric acid, and decreases malate content [START_REF] Lecourieux | Dissecting the Biochemical and Transcriptomic Effects of a Locally Applied Heat Treatment on Developing Cabernet Sauvignon Grape Berries[END_REF]. Integrating microclimatic parameters and metabolomic analysis, [START_REF] Reshef | Sunlight Modulates Fruit Metabolic Profile and Shapes the Spatial Pattern of Compound Accumulation within the Grape Cluster[END_REF] reported that solar irradiance affected the overall levels and patterns of accumulation of sugars, organic acids, amino acids and phenylpropanoids, across the grape cluster. The relationship between the diurnal microclimatic changes (radiation and temperature) and the berry metabolic dynamics was recently described [START_REF] Reshef | Sunlight Modulates Fruit Metabolic Profile and Shapes the Spatial Pattern of Compound Accumulation within the Grape Cluster[END_REF]. The present study provides additional evidence about the harmful effects of heat on berry primary metabolism through the quantification of 31 metabolite intermediates related to sugar accumulation, glycolysis, and the TCA cycle, as well as through the identification of numerous proteins related to the corresponding metabolic pathways whose abundance is affected by HS. Among the 31 metabolites quantified, 14 were significantly impacted upon HT, with reduced contents in warmed berries for all of these (Figure 3, Supplementary Table S10). Some intermediates were more affected when the HT was applied during the green stage (glycerate, fumarate), and others during ripening (T6P and S6P), whereas the remaining ones were negatively impacted at all three developmental stages (pyruvate, shikimate, G3P, 3-PGA, G6P, Man6P, F6P). Reduced malate contents were also observed under HS, including the green stage (Figure 3). These primary metabolite profiles coincide with a dramatic down-regulation of numerous enzymes related to carbohydrate and energy metabolism in heat-treated berries (48 out of 56 DAPs, Supplementary Table S9), and more than 70% of these DAPs were repressed during the ripening period. Using a Cabernet Sauvignon cell suspension culture subjected to HS (42°C), [START_REF] George | Quantitative proteomic analysis of cabernet sauvignon grape cells exposed to thermal stresses reveals alterations in sugar and phenylpropanoid metabolism. Proteomics: Giribaldi M[END_REF] observed a similar decline in proteins related to sugar metabolism. Our results highlighted changes in many metabolic pathways in grape berries exposed to heat, possibly due to the inhibitory effect of elevated temperature on metabolic enzyme activities from these pathways.

Several enzymes from the pathways of sugar and sugar-phosphate metabolism were less abundant in heated berries, including a fructokinase (FRK, VIT_14s0006g01410) and two hexokinases (HXK, VIT_18s0001g14230, VIT_11s0016g03070). The decrease in these enzymes may contribute to the lower concentration in sugar phosphates (T6P, S6P, G6P and F6P) under heat, since the hexoses (glucose and fructose) resulting from cleavage of imported sucrose can be converted to hexose phosphates by FRK and HXK in the cytosol [START_REF] Stein | Plant Fructokinases: Evolutionary, Developmental, and Metabolic Aspects in Sink Tissues[END_REF]. In plants, trehalose 6-phosphate (T6P) acts as a signal of sucrose availability connecting plant growth and development to its metabolic status, and it was reported that T6P levels can exhibit dynamic responses to environmental cues (Figueroa and Lunn 2016). An increase in T6P content was observed in berries from veraison on, but this increase was lower under heat (Figure 3, Supplementary Table S10). In some plants, T6P inhibits the SnRK1 Ser/Thr protein kinase, in turn contributing to a reduction in anthocyanin content [START_REF] Baena-Gonzalez | A central integrator of transcription networks in plant stress and energy signalling[END_REF]. It is thus tempting to speculate that the increase in T6P concentration from veraison onwards contributes to anthocyanin accumulation through repression of SnRK1, and that this regulatory process is less effective under heat. However, the roles of T6P as a signalling molecule and the SnRK1/T6P pathway during the berry ripening process and in HT response remain to be demonstrated. In sink organs such as developing berries, sucrose cleavage is carried out either by invertase (INV) to yield glucose and fructose, or by sucrose synthase (SUS) to yield UDPG and fructose. Our proteomic analysis revealed a strong accumulation of a SUS isoform after HT at veraison (SUS2, VIT_05s0077g01930). However, glucose, fructose and sucrose contents were not differentially altered by heat treatment (Figure 3, Supplementary Table S10), whereas a delay in the increase in total soluble solids (TSS) content was observed in developing berries after heat exposure during the green stage [START_REF] Lecourieux | Dissecting the Biochemical and Transcriptomic Effects of a Locally Applied Heat Treatment on Developing Cabernet Sauvignon Grape Berries[END_REF]). Accumulation of SUS may be a sign of hypoxia resulting from increased respiration rate under high temperature conditions, thus consuming oxygen faster than it can diffuse into the bulky grape berry tissue. Such as AtSUS1 and AtSUS4 in Arabidopsis, SUS genes are described as hypoxic responsive genes and a role for sucrose synthase as part of the acclimation mechanism to anoxia in dicots was proposed (Santaniello et al., 2014).

The present study also revealed the lower abundance of many glycolytic enzymes (9 DAPs) in warmed berries, mostly during ripening, together with the reduced content of some glycolytic intermediates (Fru6P, 3-PGA, pyruvate). A similar decrease in abundance of glycolytic enzymes or glycolytic intermediates was also reported for some grape varieties including Cabernet Sauvignon, when berries ripen under normal growth conditions (Giribaldi et al. 2007;[START_REF] Dai | Metabolic profiling reveals coordinated switches in primary carbohydrate metabolism in grape berry (Vitis vinifera L.), a non-climacteric fleshy fruit[END_REF][START_REF] Martinez-Esteso | iTRAQ-based protein profiling provides insights into the central metabolism changes driving grape berry development and ripening[END_REF]). Thus, the consequence of heat stress on the glycolytic pathway remains unclear since the glycolytic flux might have been maintained of even increased at high temperature although the decrease of some glycolytic-related DAPs. Disturbing the glycolytic pathway may impair the generation of energy (ATP), reducing equivalents (NADH) and various intermediates required for the production of amino acid, lipids and secondary metabolites. For example, it has been reported that cell energy, reducing power and α-ketoglutarate availability are potential drivers for flavonoid biosynthesis in grape cells [START_REF] Soubeyrand | Constraint-Based Modeling Highlights Cell Energy, Redox Status and alpha-Ketoglutarate Availability as Metabolic Drivers for Anthocyanin Accumulation in Grape Cells Under Nitrogen Limitation[END_REF].

The TCA cycle is a central metabolic hub since it generates energy, reducing power and carbon skeletons. Several enzymes from the TCA cycle were repressed in heated berries but consequences on the corresponding TCA intermediates were not obvious within the time frame of the analysis. The transcript levels of MDH, which catalyzes a reversible reaction between oxaloacetate and malate, and ME which oxidizes malate to pyruvate, were reported to increase during grape berry ripening. This higher expression might contribute to the decline of malate concentration after veraison (Martinez-Esteso et al., 2011;[START_REF] Wang | System-Level and Granger Network Analysis of Integrated Proteomic and Metabolomic Dynamics Identifies Key Points of Grape Berry Development at the Interface of Primary and Secondary Metabolism[END_REF]. In our study, however, heat exposure reduced protein amount of both enzymes in ripening fruits (MDH: VIT_10s0003g01000, VIT_19s0014g01640, VIT_00s0373g00040; ME: VIT_15s0046g03670, VIT_11s0016g03210), therefore questioning their precise role in hastening the malate decrease under heat. The amount of a phosphoenolpyruvate carboxylase (PEPC, VIT_19s0014g01390) was also reduced by HT at the green stage. This may contribute to lower the amount of malate under heat before veraison (Figure 3), since the first step of anaplerotic malate synthesis involves the carboxylation of PEP by PEPC [START_REF] Sweetman | Regulation of malate metabolism in grape berry and other developing fruits[END_REF]).

However, the loss of malate during ripening has been attributed to increased degradation rather than decreased synthesis pre-veraison [START_REF] Ruffner | Temperature and enzymic control of malate metabolism in berries of Vitis vinifera[END_REF][START_REF] Sweetman | Metabolic effects of elevated temperature on organic acid degradation in ripening Vitis vinifera fruit[END_REF]). Accordingly, PEPC activity decreases in pre-veraison berries with day heating, and malate content did not correlate with changes in PEPC activity [START_REF] Sweetman | Metabolic effects of elevated temperature on organic acid degradation in ripening Vitis vinifera fruit[END_REF]. The PEPC activity is highly regulated by reversible protein phosphorylation and mono-ubiquitination, which affects its sensitivity to feedback inhibition by malate [START_REF] Ruiz-Ballesta | New insights into the post-translational modification of multiple phosphoenolpyruvate carboxylase isoenzymes by phosphorylation and monoubiquitination during sorghum seed development and germination[END_REF]). Thus, changes in the phosphorylation and/or the ubiquitination status of PEPC might be more important than changes in total PEPC protein abundance. Energyconsuming processes such as gluconeogenesis may also be impacted in ripening berries exposed to HT, with three down-regulated enzymes (MDH, PEPCK and isocitrate lyase). Lower MDH amounts would reduce the accumulation of oxaloacetate (OAA), required for PEP formation through phosphoenolpyruvate carboxykinase activity (PEPCK). In normal growth conditions, both PEPCK activity and transcript abundance are increased in berries after veraison [START_REF] Walker | Phosphoenolpyruvate carboxykinase and gluconeogenesis in grape pericarp[END_REF]. In ripening grapes, PEPCK may function in gluconeogenesis when malate released from the vacuole exceeds the demand from other malate-consuming processes [START_REF] Walker | Phosphoenolpyruvate carboxykinase and gluconeogenesis in grape pericarp[END_REF].

Heat stress promotes changes in lipid metabolism and cell wall remodelling

The functional category "Lipid metabolism" was particularly affected in heat-exposed berries since all the lipid-related proteins (20 DAPs) identified in our work were negatively regulated after HT (Supplementary Table S2). Moreover, we also observed that warmed berries contain lower amount of glycerol 3-phosphate, precursor of the membrane glycerolipids (Figure 3). These lipid-related DAPs are involved in fatty acid (FA) synthesis, FA elongation or FA degradation, among which were the following enzymes: acetyl-CoA carboxylase (ACC, VIT_18s0001g04980, VIT_11s0065g00360), glycerol-3-phosphate acyltransferase (GPAT, VIT_15s0046g02400), beta-ketoacyl-CoA synthase (KCS, VIT_13s0067g03890) and phosphoinositide phosphatase (SAC, VIT_04s0044g00030). ACC proteins catalyse the carboxylation of acetyl-CoA to form malonyl-CoA during fatty acid synthesis [START_REF] Sasaki | Plant acetyl-CoA carboxylase: structure, biosynthesis, regulation, and gene manipulation for plant breeding[END_REF]. The cytosolic ACCase contributes to the synthesis of very-long chain fatty acids, with a role in membrane stability and biosynthesis of flavonoids. Its lower abundance in warmed berries may negatively impact the flavonoid accumulation upon veraison. The heat-repressive effects observed at the protein level may also lead to a deep remodelling of the berry protection system. Particularly, the down-regulation of some proteins such as GPAT and KCS may disturb the berry protective barrier, namely the cuticle, since these two proteins belong to families involved in cutin and wax biosynthesis, respectively [START_REF] Guo | Evolution of the KCS gene family in plants: the history of gene duplication, sub/neofunctionalization and redundancy[END_REF][START_REF] Petit | The Glycerol-3-Phosphate Acyltransferase GPAT6 from Tomato Plays a Central Role in Fruit Cutin Biosynthesis[END_REF]). The strongest translational repression was observed for a protein from the SAC (suppressor of actin) family (log 2 FC: -6.22) when the HS was applied during the green stage. SAC proteins are phosphatases that hydrolyze polyphosphoinositides, and roles in stress responses, cell wall formation, vacuolar trafficking and vacuolar morphology and function have been demonstrated in plants [START_REF] Novakova | SAC phosphoinositide phosphatases at the tonoplast mediate vacuolar function in Arabidopsis[END_REF]). Among the known effects of high temperatures, one of the most relevant is the alteration of membrane-linked processes due to modifications in membrane fluidity and permeability [START_REF] Niu | An Overview of Biomembrane Functions in Plant Responses to High-Temperature Stress[END_REF]. Heat stress decreases the total content of lipids and glycolipids [START_REF] Escandon | Metabolome Integrated Analysis of High-Temperature Response in Pinus radiata[END_REF]). To preserve membrane integrity under elevated temperatures, plants can regulate the ratio of bilayer to non-bilayerforming lipids [START_REF] Welti | Plant lipidomics: discerning biological function by profiling plant complex lipids using mass spectrometry[END_REF]. Plant thermotolerance might also be improved by reducing the membrane content of unsaturated fatty acids or by accumulating more saturated fatty acids prior to HS, thus helping to maintain optimal membrane fluidity [START_REF] Higashi | Landscape of the lipidome and transcriptome under heat stress in Arabidopsis thaliana[END_REF][START_REF] Escandon | Metabolome Integrated Analysis of High-Temperature Response in Pinus radiata[END_REF].

Accordingly, we found that two desaturase proteins (VIT_05s0094g00750, VIT_00s0499g00030)

were down-regulated in warmed berries. Membrane components may also be involved in initiating key signalling pathways that lead to the appropriate plant responses to heat [START_REF] Niu | An Overview of Biomembrane Functions in Plant Responses to High-Temperature Stress[END_REF].

Consequences of the heat-induced lipid metabolism changes for berry development and quality remain to be determined.

The cell wall forms a dynamic boundary for plant cells and plays important roles during grape berry development, governing the potential for cell expansion during fruit growth and modulating the texture of mature berries through the depolymerisation and the solubilisation of cell wall polymers (Goulao et al. 2012b). Cell wall remodelling also constitutes an important component of plant responses to HS that maintain overall function and growth, although the cell wall factors contributing to the acquisition of plant thermotolerance remain largely unknown (Wu et al. 2018). Our previous transcriptomic analysis highlighted the strong effect of HT on genes involved in cell wall homeostasis, and especially on enzymes contributing to cell wall expansion and loosening such as xyloglucan endotransglycosylase and expansins [START_REF] Lecourieux | Dissecting the Biochemical and Transcriptomic Effects of a Locally Applied Heat Treatment on Developing Cabernet Sauvignon Grape Berries[END_REF]. At the protein level, most of the 25 DAPs related to the category "cell wall" displayed lower amounts in heated berries, except for a few proteins among which were two pectin methylesterases (PME, VIT_07s0005g00730, VIT_11s0016g00290)

and two polygalacturonases (PG, VIT_02s0025g01330, VIT_08s0007g08330) that accumulate in ripening berries exposed to heat. Both PME and PG are implicated in pectin disassembly and fruit softening process, but it is difficult to assess the impact of changes in these four proteins, since both enzymes belong to large families, with 36 PMEs and 60 PGs being annotated in the reference grape genome (Goulao et al. 2012a). However, there is increasing evidence that cell wall-modifying enzymes are involved in plant responses to HS, and PMEs appear to be key players in the process (Wu et al. 2018). Similar observations were made in fruits of other species exposed to high temperature, such as strawberry [START_REF] Langer | Effects of heat treatment on enzyme activity and expression of key genes controlling cell wall remodeling in strawberry fruit[END_REF].

Identification of proteins with a potential role in berry responses to heat

Global warming represents a substantial risk for the sustainability of wine production in many regions.

Plant breeding combined with molecular plant biotechnology has the potential to deliver stable yields and quality under warmer climate conditions, but this requires the identification of key traits of tolerance to heat [START_REF] Torregrosa | Developmental, molecular and genetic studies on grapevine response to temperature open breeding strategies for adaptation to warming[END_REF]. Our proteomic study revealed 54 DAPs related to the functional category "Stress" and mainly belonging to the "abiotic/heat stress" cluster (Supplementary Table S7; 35 out of the 54 "stress" DAPs). Most of the proteins associated with the "heat stress" category were over-accumulated, mainly at veraison stage, and all were members of the heat shock protein (HSP) family. Looking for potential protective mechanisms activated in berries exposed to HT, we paid particular attention to up-regulated proteins previously described in the HS responses and thermotolerance in other plant species (Table 2; Supplementary Tables S7 andS12). We also listed some proteins that increased in abundance under HT and thus have a putative role in stress tolerance.

These proteins (Table 2) are potential markers for developing or selecting grape varieties that are better adapted to warmer climate or the extreme heat waves that are expected due to climate change.

Cabernet Sauvignon berry can improve its HS response abilities by elevating the levels of particular proteins associated with the maintenance of protein homeostasis (synthesis, folding and degradation). HSP/chaperones are considered as powerful buffers against environmental stress including HS. Their chaperone functions include not only protein folding, but also signalling and protein targeting and degradation (Bokszczanin and Fragkostefanakis 2013). Data collected over the last decades suggest that members of the HSP/chaperone network are valuable targets for engineering multiple stress resistance in crops [START_REF] Jacob | The heat-shock protein/chaperone network and multiple stress resistance[END_REF]). Among the 30 up-regulated HSPs in heatexposed berries, four of these were increased irrespective of the fruit developmental stage (HSP70, HSP90, HSP101, HSP17.6II), and all of these were described as required for thermotolerance in various plant species (Table 2). Another up-regulated protein under HS corresponds to a Bcl-2associated athanogene isoform (BAG). Plant BAGs are multifunctional proteins that regulate cell development and contribute to cytoprotection from pathogen attack and abiotic stress [START_REF] Doukhanina | Identification and functional characterization of the BAG protein family in Arabidopsis thaliana[END_REF]. In particular, AtBAG7 was described to be a key player of the heat-induced unfolded protein response (UPR) pathway that alleviates the detrimental effects of accumulation of misfolded proteins [START_REF] Li | Arabidopsis B-cell lymphoma2 (Bcl-2)-associated athanogene 7 (BAG7)-mediated heat tolerance requires translocation, sumoylation and binding to WRKY29[END_REF]. VvROF1 abundance was also increased in HS conditions. In Arabidopsis, AtROF1 encodes a peptidyl prolyl isomerase catalysing the cis/trans isomerization of peptide bonds N-terminal to proline residues within polypeptide chains. AtROF1 is transcriptionally induced by HT and is involved in acquired thermotolerance through the interaction with HSP90.1 and HSFA2 [START_REF] Meiri | Arabidopsis ROF1 (FKBP62) modulates thermotolerance by interacting with HSP90.1 and affecting the accumulation of HsfA2-regulated sHSPs[END_REF]. We also observed that two thaumatin-like proteins (VvTLPs) from the osmotin subfamily strongly accumulated in heat stressed berries. The grape genome contains 33 VvTLPs [START_REF] Yan | Analysis of the grape (Vitis vinifera L.) thaumatin-like protein (TLP) gene family and demonstration that TLP29 contributes to disease resistance[END_REF], and this family is known to be involved in biotic and abiotic stress responses including grapevine [START_REF] Liu | The superfamily of thaumatin-like proteins: its origin, evolution, and expression towards biological function[END_REF]. Osmotins have been shown to have multiple functions in enhancing stress tolerance in many plant species, although the mechanisms by which osmotins mediate plant response to abiotic stress are not well established (de Jesus-Pires et al. 2019). Finally, a strong increase in the translation initiation factor VveIF5 was detected in heated berries (Table 2). The role of eIF5 in thermotolerance was described in Arabidopsis and pea [START_REF] Suragani | The translation initiation factor, PeIF5B, from Pisum sativum displays chaperone activity[END_REF][START_REF] Yan | Analysis of the grape (Vitis vinifera L.) thaumatin-like protein (TLP) gene family and demonstration that TLP29 contributes to disease resistance[END_REF].

How plants sense and transduce the HT signal is still an important topic to be addressed. It was reported that phytochromes function as plant thermosensors [START_REF] Jung | Phytochromes function as thermosensors in Arabidopsis[END_REF]. Transduction of the HT signal involves various players [START_REF] Li | Molecular mechanisms governing plant responses to high temperatures[END_REF], including members of the plasma membrane cyclic nucleotide gated calcium channel family (CNGC). In Arabidopsis, CNGC2 and CNGC6 were described as heat sensors, contributing to the Ca 2+ signalling induced by HT and leading to the onset of plant acquired thermotolerance [START_REF] Finka | Plasma membrane cyclic nucleotide gated calcium channels control land plant thermal sensing and acquired thermotolerance[END_REF]. Whereas CNGC6 gene is ubiquitously expressed in Arabidopsis [START_REF] Gao | A heat-activated calcium-permeable channel--Arabidopsis cyclic nucleotide-gated ion channel 6--is involved in heat shock responses[END_REF], our results revealed that the protein abundance of the grape ortholog VvCNGC2 increased after berry exposure to heat, underlining the need for de novo synthesis of this protein to achieve its role in thermotolerance.

Integration of elevated temperature, signal transduction and the appropriate stress response is mediated, at least partially, by plant hormones [START_REF] Ku | Plant Hormone Signaling Crosstalks between Biotic and Abiotic Stress Responses[END_REF]. Several proteins related to hormone homeostasis were seen to be more abundant in heat-exposed berries (Table 2). Four abscisic acid (ABA)-related DAPs were up-regulated in the present study. The first DAP is a 9-cis-epoxycarotenoid dioxygenase (NCED). This enzyme catalyses the first committed step in ABA biosynthesis [START_REF] Sweetman | Metabolic effects of elevated temperature on organic acid degradation in ripening Vitis vinifera fruit[END_REF]) and NCED overexpression improves drought tolerance in grape [START_REF] He | Overexpression of 9-cis-Epoxycarotenoid Dioxygenase Cisgene in Grapevine Increases Drought Tolerance and Results in Pleiotropic Effects[END_REF]. At the transcript level, a mixed picture has emerged for the NCED isogenes in HS grape berries [START_REF] Carbonell-Bejerano | Thermotolerance responses in ripening berries of Vitis vinifera L. cv Muscat Hamburg[END_REF]Rienth et al. 2014a;[START_REF] Lecourieux | Dissecting the Biochemical and Transcriptomic Effects of a Locally Applied Heat Treatment on Developing Cabernet Sauvignon Grape Berries[END_REF]. The second ABA-related DAP was GER5/GRE5, a protein closely related to GEM (GLABRA2 Expression Modulator) and belonging to a subfamily of the GRAM domain-containing proteins [START_REF] Mauri | GEM, a member of the GRAM domain family of proteins, is part of the ABA signaling pathway[END_REF]. In heat-stressed berries, GER5 was up-regulated at both mRNA and protein levels (Supplementary Table S11). GER5 and closely related GRAM domain genes (GER1, GEM) were proposed to be required in the reproductive development of Arabidopsis whereas a putative role in abiotic stress responses remains unclear [START_REF] Bhatta | Genome-Wide Association Study Reveals Novel Genomic Regions for Grain Yield and Yield-Related Traits in Drought-Stressed Synthetic Hexaploid Wheat[END_REF]). The third ABA-related DAP is an ABA-, stress-, and ripening-(ASR) induced protein known to play prominent roles in the protection of plants against abiotic stress [START_REF] Gonzalez | Twenty years of research on Asr (ABA-stress-ripening) genes and proteins[END_REF]. For instance, the grape ASR VvMSA was reported to be involved in responses to water deficit [START_REF] Medici | Profiling of sugar transporter genes in grapevine coping with water deficit[END_REF]). The last ABA-related DAP is RACK1 (receptor for activated C kinase 1).

In rice, this protein positively regulates seed germination by controlling endogenous levels of ABA and H 2 O 2 (Zhang et al. 2014). More broadly, plant RACK1 proteins regulate various signalling pathways ranging from developmental processes such as seed germination, flowering and leaf initiation, to immune and stress responses against pathogens and environmental stimuli [START_REF] Islas-Flores | The Receptor for Activated C Kinase in Plant Signaling: Tale of a Promiscuous Little Molecule[END_REF]. Another phytohormone-related DAP was a brassinosteroid-related protein phosphatase which accumulated in heated berries. In Arabidopsis, this protein is one of a four-member gene family (BSL, for BRI1-SUPPRESSOR1 Like) shown to participate in brassinosteroid signalling [START_REF] Maselli | Revisiting the Evolutionary History and Roles of Protein Phosphatases with Kelch-Like Domains in Plants[END_REF], and potentially involved in stress tolerance [START_REF] Gruszka | Crosstalk of the Brassinosteroid Signalosome with Phytohormonal and Stress Signaling Components Maintains a Balance between the Processes of Growth and Stress Tolerance[END_REF].

Heat stress is accompanied by oxidative stress, which may trigger adverse effects through oxidative damage of cell membranes and important macromolecules (Bokszczanin and Fragkostefanakis 2013).

Therefore, redox regulation and signalling have been recognised as crucial mechanisms for acute responses to HT [START_REF] Katano | Integration between ROS Regulatory Systems and Other Signals in the Regulation of Various Types of Heat Responses in Plants[END_REF]. Our proteomic analysis of HT berries highlighted several upregulated DAPs with antioxidant properties, including two glutathione S-transferases (GSTs), a glutathione reductase (GR, VIT_07s0141g0039), one catalase, a protein-disulfide isomerase (PDI), an aldo-keto reductase (AKR), two mannitol dehydrogenases (MTD) and an S-nitrosoglutathione reductase (GSNOR). Glutathione S-transferases belong to a well-characterized detoxification enzyme family, catalyzing the conjugation of the reduced glutathione tripeptide (GSH) to electrophilic substrates [START_REF] Kumar | Glutathione S-Transferases: Role in Combating Abiotic Stresses Including Arsenic Detoxification in Plants[END_REF]. GSTs quench reactive molecules through the addition of GSH and protect the cell from oxidative damage. Accordingly, plant GSTs play an important role in abiotic and biotic stress responses [START_REF] Nianiou-Obeidat | Plant glutathione transferase-mediated stress tolerance: functions and biotechnological applications[END_REF]). The two up-regulated GSTs were observed at veraison but an opposite pattern was seen at ripening with five GST isoforms being strongly repressed at the protein level. This GST down-regulation observed under HT may negatively impact the accumulation efficiency of flavonoids into the vacuolar compartment of ripening berries. Indeed, GSTs function as non-enzymatic carriers in intracellular transport, allowing the accumulation of flavonoid-GSH conjugates into vacuoles [START_REF] Petrussa | Plant flavonoids--biosynthesis, transport and involvement in stress responses[END_REF]). The GST-mediated transport of anthocyanins and proanthocyanidins has been established in grape as well [START_REF] Perez-Diaz | Differential Roles for VviGST1, VviGST3, and VviGST4 in Proanthocyanidin and Anthocyanin Transport in Vitis vinifera[END_REF]).

Among the four other heat-affected proteins, GR catalyses the reduction of glutathione disulfide (GSSG) to GSH, and its role in heat tolerance was described in different plant species [START_REF] Jegadeesan | Proteomics of Heat-Stress and Ethylene-Mediated Thermotolerance Mechanisms in Tomato Pollen Grains[END_REF]. The ROS detoxifying enzyme catalase is considered as an acclimation protein required for basal thermotolerance [START_REF] Vanderauwera | Extranuclear protection of chromosomal DNA from oxidative stress[END_REF], whereas PDI encodes an oxidoreductase enzyme belonging to the thioredoxin superfamily. PDI can be considered as a folding enzyme since it catalyses the formation of correct disulfide bridges between the cysteine residues in proteins to maintain ERhomeostasis [START_REF] Park | Heat shock proteins: a review of the molecular chaperones for plant immunity[END_REF]. In potato, StPDI1 was found to interact with a sucrose transporter (StSUT1) [START_REF] Krugel | The potato sucrose transporter StSUT1 interacts with a DRM-associated protein disulfide isomerase[END_REF], and a role in abiotic stress tolerance and H 2 O 2 detoxification was suggested [START_REF] Eggert | A sucrose transporter-interacting protein disulphide isomerase affects redox homeostasis and links sucrose partitioning with abiotic stress tolerance[END_REF]. Recently, the heterologous over-expression of a Methanothermobacter thermautotrophicus PDI with a chaperone function and disulfide isomerase activity could confer thermotolerance to transgenic rice [START_REF] Wang | Overexpression of a protein disulfide isomerase gene from Methanothermobacter thermautotrophicus, enhances heat stress tolerance in rice[END_REF]. The aldo-keto reductase (AKR) superfamily contains proteins able to reduce a broad spectrum of substrates, ranging from simple sugars to potentially toxic aldehydes. Plant AKRs are involved in diverse metabolic reactions including reactive aldehyde detoxification, biosynthesis of osmolytes, secondary metabolism and membrane transport.

These proteins may also confer multiple stress tolerance [START_REF] Sengupta | Plant aldo-keto reductases (AKRs) as multitasking soldiers involved in diverse plant metabolic processes and stress defense: A structure-function update[END_REF], including heat stress tolerance as reported in rice [START_REF] Turoczy | Overproduction of a rice aldo-keto reductase increases oxidative and heat stress tolerance by malondialdehyde and methylglyoxal detoxification[END_REF]. Two mannitol dehydrogenases (MTD) increased in abundance in grape berries exposed to HT. The role of mannitol and by extension, its catabolic enzyme MTD, is well established in resistance to both biotic and abiotic stresses. Several studies suggest that mannitol protects plants from various abiotic stresses by acting as an osmoprotectant and by quenching the damaging ROS [START_REF] Patel | Mannitol in Plants, Fungi, and Plant-Fungal Interactions[END_REF]. In grapevine, VvMTD1 was described as involved in water-deficit stress tolerance [START_REF] Conde | Polyols in grape berry: transport and metabolic adjustments as a physiological strategy for water-deficit stress tolerance in grapevine[END_REF]. Finally, GSNOR has an important function in the homeostasis of NO (nitric oxide), which is considered as a broad-spectrum anti-stress molecule when acting at low concentration in plants. Whereas NO was described to accumulate in tobacco exposed to HT [START_REF] Gould | Nitric oxide production in tobacco leaf cells: a generalized stress response?[END_REF], the key role of GSNOR1-mediated de-nitrosylation in thermotolerance was revealed by the characterization of Arabidopsis hot5 (sensitive to hot temperatures) knock-out mutants [START_REF] Lee | Modulation of nitrosative stress by S-nitrosoglutathione reductase is critical for thermotolerance and plant growth in Arabidopsis[END_REF]. Taken together, these up-regulated and redoxrelated proteins may play a role in signalling as well as in preventing harmful effects of ROS and reactive nitrogen species in berries under HS conditions.

Finally, our proteomic work highlighted the heat-induced accumulation of two proteins related to vitamin metabolism, namely VvPDX1.2 and VvPANK (Table 2). The pseudoenzyme PDX1.2 (Pyridoxine biosynthesis 1) is a noncatalytic homolog of the PDX1 subunit of the vitamin B 6 biosynthesis protein machinery. Transcriptionally regulated by HsfA1, PDX1.2 serves to stabilize the catalytic PDX1s under HT, thereby maintaining vitamin B 6 homeostasis in plants [START_REF] Dell'aglio | The Pseudoenzyme PDX1.2 Sustains Vitamin B<sub>6</sub> Biosynthesis as a Function of Heat Stress[END_REF]). AtPDX1.2 was reported as essential for plant growth under heat and oxidative stress conditions [START_REF] Moccand | The pseudoenzyme PDX1. 2 boosts vitamin B6 biosynthesis under heat and oxidative stress in Arabidopsis[END_REF]. The second up-regulated protein corresponds to a pantothenate kinase (PANK).

Pantothenate (known as vitamin B5) is the key precursor of CoA and acyl carrier protein, which are essential co-factors for many metabolic enzymes [START_REF] Raman | Pantothenate synthesis in plants[END_REF]. The PANK converts pantothenate to phosphopantothenate, using ATP as phosphate donor. PANK activity plays a critical role in regulating intracellular coenzyme A (CoA) levels in bacteria and animals but little is known about the role of this enzyme in plants [START_REF] Ottenhof | Organisation of the pantothenate (vitamin B5) biosynthesis pathway in higher plants[END_REF]). It was suggested that an increased level of CoA, through an increased PANK activity, may be responsible for improved plant growth and stress resistance [START_REF] Rubio | The coenzyme a biosynthetic enzyme phosphopantetheine adenylyltransferase plays a crucial role in plant growth, salt/osmotic stress resistance, and seed lipid storage[END_REF].

Conclusion

Global warming represents a substantial risk for the sustainability of wine production in many regions.

Plant breeding combined with molecular plant biotechnology have the potential to deliver stable yields and quality under warmer climate conditions, but this requires the identification of key traits of tolerance to heat. To this end, we combined omics approaches to decipher the molecular components and processes affected in developing Cabernet Sauvignon berries exposed to heat. In the present study, 592 differentially abundant proteins were identified in heated berries among which some may play a substantial role in heat tolerance. Our data also show that there is not a straightforward correlation between the heat-modulated berry transcriptome and proteome, indicating the major involvement of post-transcriptional regulation under heat stress. These pronounced changes contribute on the one hand to generate fruit damaging effects, including disruption of carbohydrate and secondary metabolism, and on the other hand to mobilize key heat-signalling players as well as protective molecules such as antioxidants and chaperones (Figure 5). In summary, these findings help elucidate heat-triggering effects on developing grape berries and identify genes and proteins that potentially control heat acclimation in grapevine. The functional characterization of some putative candidates is in progress.

MATERIAL AND METHODS

Plant material, temperature and sampling

Elevated temperature (+ 8°C) was directed on clusters of V. vinifera Cabernet Sauvignon fruiting cuttings. The HT was applied at three different berry developmental stages and maintained throughout the light period (7:00 am to 7:00 pm) until harvesting. To separate short-and long-term responses, berries were collected after 1 and 7 days of treatment. Details of the experimental protocol and sampling were provided in [START_REF] Lecourieux | Dissecting the Biochemical and Transcriptomic Effects of a Locally Applied Heat Treatment on Developing Cabernet Sauvignon Grape Berries[END_REF]. The same pools of deseeded berries were used for both the transcriptomic analysis reported in [START_REF] Lecourieux | Dissecting the Biochemical and Transcriptomic Effects of a Locally Applied Heat Treatment on Developing Cabernet Sauvignon Grape Berries[END_REF] and the proteomic analysis reported here.

Tissue preparation and total protein extraction

Each biological replicate contained 25 pooled berries underwent independent protein extraction using a protocol adapted from Lücker et al. (2009) and Martinez-Estezo et al. (2011). Briefly, deseeded berries were ground to a fine powder in a mortar with liquid nitrogen and 3 g of frozen ground tissue were used to prepare a total protein extract. Pectins and pigments were removed after incubation of the frozen tissues in 25 mL of a cold (-20°C) ethyl acetate: ethanol (1 : 2 (v/v) for 30 min at -20°C with repeated vortexing. The protein extract was precipitated by centrifugation for 3 min at 21000 × g at 4°C, and a second ethyl acetate: ethanol wash was performed in a similar way. The pellet was next extracted twice with cold (-20°C) 100% acetone by vortexing and centrifuging, as before. The pellet was transferred to a mortar and dried in a fume hood until complete acetone evaporation. The dried pellet was finely ground after addition of 1/3 vol of sand, and washed with 1.5 mL chilled 10% (w/v) trichloroacetic acid (TCA) in acetone. This washing step was repeated seven times. This was followed by two washes with aqueous 10% (v/v) TCA, two with chilled 80% (v/v) acetone, and drying at 4 °C.

Each washing step included incubation at -20°C for 20 min followed by centrifugation for 3 min at 21000 × g at 4°C. Subsequently the dried pellet was resuspended in 500 µL protein extraction buffer containing: 0.7 M sucrose, 0.1 M KCl, 0.5 M Tris-HCl (pH 7.5), 50 mM EDTA, 1% PVPP, 1%

DTT and protease inhibitor cocktail (Roche, Germany), and incubated at room temperature (RT) for 30 min with intermittent vortexing. Then, an equal volume of Tris-saturated phenol (pH 7.5) was added, the sample was incubated at RT for 30 min with vortexing every 5 min. After centrifugation at 15,000 × g at RT for 40 min, the phenol phase in the upper layer was transferred into a new 2-mL Eppendorf tubes and the remaining aqueous lower phase was re-extracted with another 500 µL Trissaturated phenol (pH 7.5). The two phenol phases were combined and washed twice with an equal volume of phenol washing buffer (0.7 M sucrose, 0.1 M KCl, 0.5 M Tris (pH 7.0, 50 mM EDTA, 1%

DTT, and protease inhibitor cocktail). Proteins contained in the organic phase were precipitated overnight at 4°C with 5 volumes of 0.1 M ammonium acetate in methanol. Precipitated proteins were washed three times in 0.1 M ammonium acetate in methanol and twice in ice-cold 80% (v/v) acetone.

All these precipitating and washing steps included incubation for 30 min followed by centrifugation at 15,000 × g at 4°C for 40 min. Finally, proteins were dissolved in 200 µL freshly prepared solubilization solution (7 M urea, 70 mM SDS, 20 mM DTT) and quantified according to the Bradford method (Bradford, 1976) with bovine serum albumin as standard.

Sample preparation and protein digestion

Thirty µg of each protein sample (three biological replicates for each condition; 12 conditions in total:

nLC-MS/MS analysis

Peptide mixtures were analyzed on an Ultimate 3000 nanoLC system (Dionex, Amsterdam, The Netherlands) coupled to a nanospray LTQ-Orbitrap XL mass spectrometer (ThermoFinnigan, San Jose, USA). Ten microliters of peptide mixtures were loaded onto a 300-µm-inner diameter x 5-mm C 18 PepMap TM trap column (LC Packings) at a flow rate of 30 µL/min. The peptides were eluted from the trap column onto an analytical 75-mm id x 15-cm C18 Pep-Map column (LC Packings) with a 5-40% linear gradient of solvent B in 105 min (solvent A was 0.1% formic acid in 5% ACN, and solvent B was 0.1% formic acid in 80% ACN). The separation flow rate was set at 200 nL/min. The mass spectrometer operated in positive ion mode at a 1.8-kV needle voltage and a 39-V capillary voltage.

Data were acquired in a data-dependent mode alternating a Fourier transform mass spectrometry (FTMS) scan survey over the range m/z 300-1700 and six ion trap MS/MS scans with CID (Collision Induced Dissociation) as activation mode. MS/MS spectra were acquired using a 3-m/z unit ion isolation window and a normalized collision energy of 35%. Only +2 and +3 charge-state ions were selected for fragmentation. Dynamic exclusion duration was set to 30s.

Database search and MS data processing

Data were searched using the SEQUEST algorithm (supported by the Proteome Discoverer 1.4 software; Thermo Fisher Scientific Inc.) against a subset of the 2012.01 version of UniProt database restricted to Vitis vinifera Reference Proteome Set (29,817 entries). Spectra from peptides higher than 5000 Da or lower than 350 Da were rejected. The search parameters were as follows: mass accuracy of the monoisotopic peptide precursor and peptide fragments was set to 10 ppm and 0.8 Da respectively.

Only b-and g-ions were considered for mass calculation. Oxidation of methionines (+16 Da) was considered as a variable modification. Two missed trypsin cleavages were allowed. Peptide validation was performed using the Percolator algorithm [START_REF] Käll | Semi-supervised learning for peptide identification from shotgun proteomics datasets[END_REF], and only "high confidence" peptides were retained corresponding to a 1% false positive rate at the peptide level.

Label-Free Quantitative Data Analysis

Raw LC-MS/MS data were imported into the Progenesis LC-MS 4.0 (Non Linear Dynamics) software. Data processing includes the following steps: (i) Features detection, (ii) Features alignment across the six samples for each part, (iii) Volume integration for 2-6 charge-state ions, (iv) Protein abundance changes between control and heat-exposed fruits were considered to be significant when a minimum fold change of 1.5 (log 2 FC > 0.58 or < -0.58) was reached, with a significance threshold of p < 0.05. Only proteins identified in all three biological replicates were considered for quantification. Significantly affected protein categories based on the MapMan Ontology [START_REF] Usadel | Extension of the visualization tool MapMan to allow statistical analysis of arrays, display of corresponding genes, and comparison with known responses[END_REF] were identified using a Chi-square test. MapMan mappings for the Cribi 12X grapevine genome were based on closest homologs regarding to the Arabidopsis thaliana genome. Protein annotations were taken from Grimplet et al. (2012).

Primary metabolism quantification

Deseeded berries were ground to a fine powder in liquid nitrogen and aliquots (15-20 mg) of frozen powder were extracted with chloroform/methanol as described by [START_REF] Lunn | Sugar-induced increases in trehalose 6-phosphate are correlated with redox activation of ADPglucose pyrophosphorylase and higher rates of starch synthesis in Arabidopsis thaliana[END_REF]. Primary metabolites were measured by LC-MS/MS as described by [START_REF] Lunn | Sugar-induced increases in trehalose 6-phosphate are correlated with redox activation of ADPglucose pyrophosphorylase and higher rates of starch synthesis in Arabidopsis thaliana[END_REF], with modifications as described in Figueroa et al. (2016) and [START_REF] Dai | Metabolic profiling reveals coordinated switches in primary carbohydrate metabolism in grape berry (Vitis vinifera L.), a non-climacteric fleshy fruit[END_REF].

Identification of putative stage-specific grapevine berry biomarkers and omics data integration into networks. Plant Physiol 154: 1439-1459 Zhang D, Chen L, Li D, Lv B, Chen Y, Chen J, XuejiaoYan, Liang J (2014) OsRACK1 is involved in abscisic acid-and H2O2-mediated signaling to regulate seed germination in rice (Oryza sativa, L.). PLoS One 9: e97120 Zhang L, Liu X, Gaikwad K, Kou X, Wang The detailed information of the common DAPs upon HT for each stage or for each exposure time was listed as in Supplementary Tables 1 and2. Category description is given on the y-axis. Numbers of down-and up-regulated proteins across the 6 experimental conditions are provided on the x-axis for each category.

  , HT triggered an enhanced abundance of proteins involved in lignin synthesis, including shikimate O-hydroxycinnamoyltransferase (HCT; VIT_09s0018g01190), caffeic acid O-methyltransferase (CCoAOMT1; VIT_16s0098g00850). A significant increase in lignin biosynthesis would confer additional strength to lignified secondary cell wall, helping berries to protect against shriveling mediated-HT. For instance, the activation of the lignin pathway is a reaction to advanced stages of dehydration in grape berry fruit(Zamboni et al. 2010). Key enzymes of the general phenylpropanoid pathway (phenylalanine ammonia-lyase PAL, VIT_06s0004g02620, VIT_08s0040g01710, VIT_13s0019g04460; cinnamate 4-hydroxylase C4H, VIT_06s0004g08150; 4-coumarate-CoA ligase 4CL; VIT_11s0052g01090), enzymes from the flavonoid biosynthesis pathway (dihydroflavonol 4-reductase DFR, well as enzymes mediating the glycosylation of flavonoids (UDP-glucosyl transferases, VIT_12s0055g00290, VIT_02s0033g00130, VIT_17s0000g04750; UDP-rhamnosyl transferase, VIT_00s0218g00150) were also affected at the protein level upon HT. Most of these proteins were less abundant in heated berries, suggesting a sharp decline of the corresponding enzymatic activities (Supplementary Tables

  three stages x two treatments x two treatment durations) were solubilized in Laemmli buffer and were separated by SDS-PAGE in a 10% (w/v) acrylamide gel. After colloidal blue staining, each lane was horizontally divided into four parts of equal length and each part was subsequently cut in 1 mm x 1 mm gel pieces. Gel pieces were destained in 25 mM ammonium bicarbonate dissolved in 50% (v/v) acetonitrile (ACN), rinsed twice in ultrapure water and dehydrated in 100% ACN for 10 min. After ACN removal, gel pieces were dried at room temperature, covered with proteolysis solution (containing 10 ng/µl trypsin dissolved in 40 mM NH 4 HCO 3 and 10% (v/v) ACN), rehydrated at 4 °C for 10 min, and finally incubated overnight at 37 °C. The gel pieces were then incubated for 15 min in 40 mM NH 4 HCO 3 and 10% (v/v) ACN at room temperature with rotary shaking. The supernatant was collected, and an H 2 O/ACN/formic acid (47.5:47.5:5 by volume) extraction solution was added and gel slices incubated for 15 min. The extraction step was repeated twice. Supernatants were pooled and concentrated in a vacuum centrifuge to a final volume of 40 µL. Digests were finally acidified by addition of 2.4 µL of 5% (v/v) formic acid and the resulting peptide mixtures stored at -20 °C until analysis.

Figure 1 .

 1 FIGURE LEGENDS

Figure 2 .

 2 Figure 2. Total number of differentially abundant proteins for each MapMan main category.

Figure 3 .

 3 Figure 3. Metabolite profiles of Cabernet Sauvignon developing berries exposed (red line) or not (blue line) to heat stress. Metabolites are shown in their respective metabolic pathways (sugar metabolism, glycolysis, and the TCA cycle) and their temporal profiles during berry treatment(µmol.g -1 FW for glucose, and fructose; and nmol.g -1 FW for the others) are presented alongside. For each profile, the x-axis shows the developing stage (G: middle-green, V: véraison and R: middleripening) and the stress duration (1, 7 and 14 days).

Figure 4 .

 4 Figure 4. Correlation between the expression of proteins and mRNAs in developing control and HT berries. DAPs and DEGs were selected using a 1.5 and 2 fold expression change, respectively, and an adjusted p < 0.05 (with FDR correction). Venn diagrams display the number of DAPs and DEGs (red: up-regulated and blue: down-regulated) according to the developing stage (A: middlegreen, B; véraison and C: middle-ripening) and the stress duration (1 and 7 days).

Figure 5 .

 5 Figure 5. Schematic overview of heat stress effects on developing Cabernet Sauvignon berries.This illustration summarizes the main findings deduced from transcriptomic, proteomic and metabolomics analyses (this work and[START_REF] Lecourieux | Dissecting the Biochemical and Transcriptomic Effects of a Locally Applied Heat Treatment on Developing Cabernet Sauvignon Grape Berries[END_REF]). Up and down arrows reflect the accumulation levels of the corresponding transcripts, proteins or metabolites in heated berries.

Figure 2 .Figure 3 .

 23 Figure 2. Total number of differentially abundant proteins for each MapMan main category.Category description is given on the y-axis. Numbers of down-and up-regulated proteins across the 6 experimental conditions are provided on the x-axis for each category.

Figure 4 .

 4 Figure 4. Correlation between the expression of proteins and mRNAs in developing control and HT berries. DAPs and DEGs were selected using a 1.5 and 2 fold expression change, respectively, and an adjusted p < 0.05 (with FDR correction). Venn diagrams display the number of DAPs and DEGs (red: up-regulated and blue: down-regulated) according to the developing stage (A: middlegreen, B; véraison and C: middle-ripening) and the stress duration (1 and 7 days).

Table 1 : Amount of identified proteins and differentially abundant proteins in control and heat berries according to the developmental stage and the stress duration

 1

	Samples	Total	Total protein	Total DAPs	Number of	Number of	Number of
		protein	quantified		DAPs	DAPs	DAPs up-
		identified	(≥ 2 peptides)		up-regulated	down-	or down-
						regulated	regulated
	All 6 conditions	3972	2279	592
				à DAPs in 1 condition (501)	139	362	--
				à DAPs in > 1 condition (91)	16	61	14
	G1D		1633	39	11	28
	G7D		1263	108	14	94
	V1D		1580	148	70	78
	V7D		1054	83	49	34
	R1D		1244	238	38	200
	R7D		857	84	13

ACKNOWLEDGMENTS

The authors would like to thank Christelle Renaud and Ghislaine Hilbert for metabolite quantifications. For the production of the fruiting cuttings and technical assistance during greenhouse experiments, we express our gratitude to Jean Pierre Petit, Nicolas Hoquard and Guillaume Pacreau.

FUNDING

This research received funding from the Agence Nationale de la Recherche for the project "DURAVITIS" (grant no. ANR-2010-GENM-004-01).

AUTHOR CONTRIBUTIONS

DL, FL and PP designed the research; DL oversaw the research; PP and DL performed the greenhouse experiments; FL, JC, and DL carried out the protein extraction; CK performed the bioinformatics analysis; RF, DL and JEL did the metabolic analysis; SC and MB did the metabolic analysis, FL, CK, and DL analysed and interpreted the data; DL and FL drafted the manuscript; SD, JEL, LW and EG critically revised the manuscript. All authors read and approved the final manuscript.

CONFLICTS OF INTEREST

The authors declare no conflicts of interest.

SUPPORTING INFORMATION

Figure S1: MapMan GO categories impacted at the transcriptomic and proteomic levels in heated grape berries Table S1: All identified proteins with calculated differentials and corresponding statistics Table S2: List and information about the differentially abundant proteins between control and heat stress conditions Table S3: Differentially abundant proteins between control and heat stress condition at green stage The detailed information of the common DAPs upon HT for each stage or for each exposure time was listed as in Supplementary Tables 1 and2.