# Developmental, molecular and genetic studies on grapevine response to temperature open breeding strategies for adaptation to warming

Laurent Torregrosa, Antoine Bigard, Agnes Doligez, David Lecourieux, Markus Rienth, Nathalie Luchaire, Philippe Pieri, Ratthaphon Chatbanyong, Rezth Shahood, Marc Farnos, et al.

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## To cite this version:

Laurent Torregrosa, Antoine Bigard, Agnes Doligez, David Lecourieux, Markus Rienth, et al., Developmental, molecular and genetic studies on grapevine response to temperature open breeding strategies for adaptation to warming. OENO One, 2017, 51 (2), pp.155-165. 10.20870/oeno-one.2016.0.0.1587. hal-01607969

HAL Id: hal-01607969

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Submitted on 26 May 2020

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<figure><img src="image_2.png" /></figure>
# Developmental, molecular and genetic studies on grapevine response to temperature open breeding strategies for adaptation to warming

Laurent Torregrosa¹¹, Antoine Bigard¹,²,³, Agnès Doligez², David. Lecourieux⁴, Markus Rienth¹,²,⁵,⁶,
Nathalie Luchaire¹,⁸, Philippe Pieri⁴, Ratthaphon Chatbanyong¹,², Rezth Shahood¹,², Marc Farnos²,
Catherine Roux², Angélique Adiveze², Jérémie Pillet⁴, Yannick Sire³, Emmanuelle Zumstein³,
Mélanie Veyret³, Loic Le Cunff⁷, Fatma Lecourieux⁴, Nicolas Saurin³, Bertrand Muller⁹, Hernán Ojeda³,
Cléa Houel¹,², Jean-Pierre Péros², Patrice This², Anne Pellegrino⁸ and Charles Romieu²

¹UMR AGAP & UMT Génovigne, Montpellier SupAgro, 2 place P Viala, 34060 Montpellier, France

²UMR AGAP & UMT Génovigne, INRA, 2 place P Viala, 34060 Montpellier, France

³Unité Expérimentale de Pech-Rouge, INRA, 11430 Gruissan, France

⁴UMR EGFV, Bordeaux university, 210 chemin de Leysotte, 33883 Villenave d'Ornon, France

⁵Fondation Jean Poupelain, 30 Rue Gâte Chien, 16100 Javrezac, France

⁶Haute école de viticulture et d'œnologie, Changins, 1260 Nyon, Suisse

⁷UMT Génovigne, IFV, 2 place P Viala, 34060 Montpellier, France

⁸UMR LEPSE, Montpellier SupAgro, 2 place P Viala, 34060 Montpellier, France

⁹UMR LEPSE, LEPSE, INRA, 2 place P Viala, 34060 Montpellier, France

This article is published in cooperation
with the ClimWine international conference held in Bordeaux 11-13 April 2016.

Guest editor: Nathalie Ollat

## Abstract

**Aim:** In the long term, genetic improvement is one of the major strategies to support sustainable wine production in a changing climate. Over the past 5 years, we have developed an interdisciplinary research program that aimed to: i) characterize the impact of temperature increase sensed by the entire plant or individual bunches on the development and functioning of the plant, ii) identify the physiological and molecular mechanisms regulating the response of vegetative and reproductive development to heat stress and iii) develop tools to map quantitative trait loci (QTLs) of plant and berry development in duly controlled, stable, and contrasting environmental conditions.

**Methods and results:** Performing high-throughput genomic analyses combined with the use of innovative experimental designs (fruiting cuttings, microvines, single berry sampling) was critical to decipher the ecophysiological and molecular mechanisms involved in the vine response to high temperature.

**Conclusion:** Warming promotes vegetative growth and hampers plant carbon balance, disturbing flower set and young berry development. High temperatures modify primary and secondary fruit metabolisms, desynchronizing sugar and organic acid metabolisms and delaying sugar and polyphenol accumulation during ripening. The study of day and night transcriptomic and proteomic signatures associated with heat highlighted key players of the response to temperature in the fruit.

**Significance and impact of the study:** Capitalizing on this knowledge, a new program is being proposed for the selection of cultivars limiting the accumulation of sugars in the berry while maintaining other qualitative compounds.

**Keywords:** Global warming, temperature, grapevine, biology, adaptation, genetic improvement

Received : 10 July 2016; Accepted : 18 October 2016

DOI: 10.20870/oeno-one.2016.0.0.1587
**Introduction**

Grapevine performance, including productivity and
wine quality, is highly dependent on climate. High
temperatures (T°) hamper carbon assimilation
beyond a 30 °C threshold (Greer and Weedon, 2012)
and limit the yield by reducing the number of berries
per vine (Rogiers *et al.*, 2011). The composition of
the wines also depends on thermal conditions (Spayd
*et al.*, 2002; Carbonneau *et al.*, 2015) because T°
increases sugar concentration in the berry at the
expense of malic acid and secondary metabolites,
leading to unbalanced wines.

In Europe, wine production relies on close
interactions between the variety, the environment and
the viticultural practices. Climate change disrupts this
balance; hence varietal adaptation is required to
maintain traditional and qualitative viticulture in
these regions (Torregrosa et al., 2011; Ollat et al.,
2014 and 2015). In the long term, genetic
improvement is one of the best strategies to support
sustainable wine production systems under global
warming. Various data confirm that genetic diversity
within Vitis may be exploited for photosynthesis
adaptation to temperature. For example,
photosynthesis was not reduced at T°<40 °C in
response to heat stress treatments in V. amurensis
(Luo et al., 2011), and most wild species and hybrids
between V. labrusca and V. vinifera also displayed a
better heat tolerance than V. vinifera (Xu et al., 2014).
Recently, Hochberg et al. (2015) observed metabolic
adaptations resulting in greater growth inhibition of
leaves at 35 °C for cv. Syrah compared to Cabernet
Sauvignon. Unfortunately, the lack of physiological
and genetic knowledge on the mechanisms of
adaptation of grapevine to T° limits the development
of non-empirical breeding programs (Torregrosa et
al., 2011), insofar as fruit quality, rather than carbon
fixation, is considered as the principal selection
target. In the last years, much effort has been carried
out under vineyard conditions to identify regulatory
mechanisms of primary and secondary metabolisms
in berries (Deluc et al., 2008; Boss and Davies,
2009; Hichri et al., 2011; Lecourieux et al., 2014)
and to identify transcriptomic changes induced by
thermal stress applied to the berry (Pillet et al., 2012)
or to the whole plant (Carbonell-Bejerano et al.,
2013; Rienth et al., 2014b; Rienth et al., 2016).

Despite these recent successes, we are far from a
comprehensive picture of the regulatory mechanisms
involved in the adaptation of the grape to heat stress.
To date, there is no genetic tool or resource suitable
to support breeding programs dealing with T°
resilience. Over the last 5 years, we have developed a

research program aiming to: i) characterize the
effects of T° increase, applied at the whole plant or
fruit level, on the development and functioning of the
plant (organogenesis, biomass partitioning,
metabolism of the berry), ii) identify the specific roles
of carbon balance and molecular (transcription)
mechanisms in regulating yield and quality
development under high T° and iii) develop resources
to study the genetic structure and stability of
developmental and qualitative berry characters
against thermal fluctuations.

As with other perennial plants, the biological
properties of grapevine cause methodological and
experimental difficulties for studying the response to
environmental changes and genetic diversity. Indeed,
its size, its long juvenile period and its discontinuous
reproductive cycle lengthen and complicate the study
of such issues. Therefore, this project relied on two
alternative models: the microvine (Chaib et al., 2010;
Rienth et al., 2012; Torregrosa et al., 2016), which
presents a dwarf stature and continuous fruiting, and
fruiting cuttings (Mullins, 1966), which allow to get
grapes in the off-season and grow them under strictly
controlled conditions (Luchaire et al., 2013; Rienth et
al., 2013).

This work reveals the central role of carbon balance
in the plant response to thermal stress, with critical
effects on the distribution of biomass within the plant
and also changes in the primary (sugars, organic
acids) and secondary (phenolic compounds) berry
metabolism. This work also highlights day and night
transcriptomic and proteomic signatures associated
with heat stresses in grapes. During the program, new
tools have been developed for grapevine phenotyping
and breeding. The most significant were: i) a spatio-
temporal analysis framework of microvine vegetative
and reproductive development, ii) stable quantitative
trait loci (QTLs) of development *vs* T° and iii)
methodologies for RNA purification, proline
quantification assays and microvine embryo rescue.

**Materials and methods**

1. Vine responses to T° applied at whole plant

Temperature effects on biomass partitioning
between vegetative and reproductive organs and
on carbon balance in microvine

Six experiments (Exp.1 to 6) were performed on two-
year-old own-rooted potted microvines (line ML1,
Chaïb et al., 2010). Plants (6 to 12 per experiment)
were thinned to one main axis and laterals were
removed as soon as they appeared. Irrigation was
supplied to fit maximal evapotranspiration. In Exp. 1
to 2 (2011) and Exp. 3 to 5 (2013), contrasted
temperature treatments were applied over a 1- to 2-
month period after the first inflorescences started to
ripe. Control day/night air T° was set to 25 °C/15 °C.
Contrasted T° were applied both above and below
control T°, ranging from 22 °C/12 °C in Exp. 1 to
30 °C/20 °C in Exp. 2, 30 °C/15 °C in Exp. 4 and
30 °C/25 °C in Exp. 5. In Exp. 6, several couples of
day/night T° from 20 °C/15 °C to 35 °C/30 °C were
applied for 2 days. In all experiments, a 14-h
photoperiod was imposed to reach a daily PAR
(photosynthetically active radiation) close to 19.0 mol
m²d⁻¹. The daily mean VPD (vapor pressure deficit)
was maintained close to 1 kPa in Exp. 1 to 5 and
close to 2 kPa in Exp. 6.

Spatial changes in biomass partitioning in above-
ground organs

The number of unfolded leaves and the rank of the
most apical phytomer at flowering (50 % of opened
flowers) were recorded for Exp. 1 to 5 at the
beginning of the experiment. Three plants were
harvested at T0 for Exp. 3 to 5. Berries, leaves and
internodes were then harvested and weighed (dry
matter) to determine the biomass gain between T0
and final harvest.

Whole plant gas exchange measurements

Exp. 6 was dedicated to whole plant gas exchange
measurement. As described in Bédiée *et al.* (2015),
net photosynthesis and respiration of whole aerial
organs were recorded for 24 h on 3 plants at several
day/night T° couples.

Biochemical/transcriptomic berry responses

In order to study the T° effects on microvine fruit
development, additional experiments were added to

those described above. A first monitoring of
microvine fruit development was conducted at
30 °C/20 °C (day/night T°). Biochemical and
transcriptomic signatures were studied at day- and
nighttime as described in Rienth *et al.* (2014a).
Subsequently, short heat stress experiments were
conducted at 3 fruit development stages. Plants were
acclimatized for 10 days at constant day/night T°
(22 °C/12 °C), then heat stressed (37 °C) for 2 h
(Rienth *et al.*, 2014b). Long-term T° effects on whole
berry development were studied in Exp. 1 and 2. In
Exp. 3 and 5, only the *véraison* and ripening stages
were studied. The green growth stage of the berry
was analyzed in Exp. 4 adding an additional
experiment (Exp. 7) using 20 °C/15 °C (day/night
T°).

For Exp. 1 and 2, all major biochemical compounds
were analyzed during the whole fruit cycle without
any transcriptomic characterization. For Exp. 3, 4, 5
and 7, berry RNA was extracted as described in
Rienth et al. (2014c) and berry transcriptome was
studied using a NimbleGen microarray 090818 Vitis
exp HX12, which contains 29,549 predicted genes
representing 98.6 % of the 12X grapevine gene
prediction version V1, and RNA-seq using Illumina
paired-end sequencing (Rienth et al., 2016). For
stages close to véraison, berries were sorted based on
their individual sugar and acid composition, in order
to homogenize the sampling for transcriptomics.

2. Fruit responses to T° applied at bunch level

To characterize the response of grape to micro-
environmental thermal stress, experiments were
conducted over 3 years with fruiting cuttings of
Cabernet-Sauvignon. Cuttings were grown in small
pots, with clusters exposed to an elevated temperature
airflow produced by fan heaters (Figure 1). The other

<figure><img src="image_3.png" /><figcaption>Figure 1 - Experimental system used to investigate the effect of heat at the bunch level.
(a) Cabernet Sauvignon fruiting cuttings were used to expose clusters to an elevated T° airflow produced by fan heaters. (b) The other plant organs were all protected from the warmed airflow by polystyrene foam deflectors.</figcaption></figure>
<figure><img src="image_4.png" /><figcaption>Figure 2 - Biomass gain during thermal experiments along the shoot expressed in g of DW per day (a) and in g per organ type (b). Only the phytomers that expanded during the T° treatment and the ones that had not reached the mid-flowering stage at T0 were considered. Each value is the average of 6-12 plants, depending on the experiment considered (bar corresponds to standard deviation).</figcaption></figure>

plant organs were all protected from the warmed
airflow by polystyrene deflectors. High T° was
applied at 3 berry developmental stages (middle-
green, *véraison*, and mid-ripening) and maintained
daily (7:00 am to 7:00 pm) until harvesting. An
average daytime fruit T° of 26 °C was set for the
control and compared to a 34 °C “hot” treatment.
This led to a mean T° difference of about 8 °C
between heat stressed and control clusters, i. e. close
to the range observed in vineyards between sun-
exposed and shaded berries (Pieri and Fermaud,
2004). In order to analyze short- and long-term
responses to T°, control and heat stressed berries
were collected at 3 different time points (1, 7, and 14
days) and used for biochemical, transcriptomic and
proteomic analyses. Transcriptomic analysis was
performed using the NimbleGen microarray
described above. The relative quantification of
proteins was performed using a label-free LC-
MS/MS-based shotgun proteomic approach.

**3. Genetic mapping of developmental traits**

Several crosses were performed between 2 microvine
genotypes (ML1 and ML3, heterozygous
*VvGAI1/Vvgai1* lines) and 4 *V. vinifera* cultivars
(Alicante Bouschet, Muscat de Hambourg, Savagin,
and Carignan). Crosses were performed to create
several segregating progenies, as described in Chaïb
et al. (2010). A mapping population of 129
microvines from the cross Picovine x Ugni Blanc *flb*

(Fernandez *et al.*, 2013) was phenotyped for 43 traits
in up to 9 environments (vegetative, reproductive and
berry composition at both green lag phase and
maturity), including an experiment in growth
chambers with 2 contrasted day/night T°
(30 °C/25 °C vs 20 °C/15 °C). This population was
genotyped with a 18K SNP Illumina VeraCode chip
(Le Paslier *et al.*, 2013), and 6000 informative SNPs
(single-nucleotide polymorphisms) were used to
build parental genetic maps with CarthaGene
software (De Givry *et al.*, 2005). QTLs were detected
in each environment with Rqtl (Broman *et al.*, 2003).

**Results and discussion**

1. At whole plant level, T° promotes biomass sequestration into the leaves and internodes at the expense of inflorescence rachis and berries

T° elevation significantly increased the above-ground
biomass gain per day, specifically in vegetative
organs (leaves and internodes; Figure 2a, 2b). In
contrast, biomass allocation toward the rachis and the
berries was reduced by high T° due to the abortion of
inflorescences (data not shown).

As the phyllochron was similar for all experiments
(data not shown), plants exposed to high T°
developed faster on a calendar day basis.
Surprisingly, leaf and internode growths as a function
of growing degree days were also higher under warm
T°, although they generally follow invariant patterns
both in annual (Turc and Lecoeur, 1997; Granier and Tardieu, 1998) and perennial plants (Lebon et al., 2004; Dambreville et al., 2013). The inflorescence drop under warm T° likely resulted from the incapacity of the plant to accommodate for the boost in carbon demand. It was previously reported for other species that carbon acquisition is lower and biomass “diluted” when T° increases (Vasseur et al., 2011). Elevated T° were also found to lower the carbon storage in woody reserves in grapevine, with negative impact on fruitfulness and berry composition (Sadras and Moran, 2013; Sadras et al., 2013; Rogiers et al., 2011).

## 2. At whole plant level, high T° negatively impact microvine daily carbon balance

Net photosynthesis was optimum between 25-30 °C and decreased beyond 30 °C (Figure 3a). Night respiration exponentially increased within the range 15-30 °C (Figure 3b). Such patterns were consistent with those generally reported for grapevine (Zufferey et al., 2000; Huang et al., 2005). Carbon gain thus tended to slightly decrease when T° were higher, specifically at night (Figure 3c). Berry ripening (monitored through sugar accumulation) was much more delayed under warm T° on a thermal time basis than in calendar scale (Figure 4a, 4b). Thus, in contrast with vegetative organogenesis, berry

development is not linearly linked to thermal time. The impairment of sugar accumulation by high T° fits with previous observations showing that fleshly fruit berries are very sensitive to heat during ripening (Civello et al., 1997; Greer and Weston, 2010).

Taken together, our results globally fit with some previous findings on grapevine response to elevated T° such as the maintenance of biomass accumulation in vegetative organs, the reduction of carbon storage toward woody reserves and the delay of ripening. Importantly, they showed that with continuous flowering and fruiting, microvine is a suitable model to study the fine tuning of the vegetative and reproductive developmental sequence under T° fluctuations.

## 3. Effect of T° applied at whole plant on biochemical/transcriptomic berry responses

The microvine made it possible, for the first time, to characterize fleshy fruit development simultaneously during day and night under completely controlled conditions using whole genome microarrays. Up to this point, circadian cycles had only been studied on non-perennial model plants such as Arabidopsis (Schaffer et al., 2001). For 2 green and ripening stages, gene expression changes occurring along development as well as between day/night were

<figure><img src="image_5.png" /></figure>

**Figure 3** - Microvine whole plant (aerial organs only) net photosynthesis (a) and night respiration (b) responses to day T° and night T°, respectively. Each point is the average of 3 plants during one complete day or night measurement. Data were obtained during Exp. 6. Carbon gain over 24 h was calculated for the Exp. 1 to 5 thermal conditions (c). Carbon gain was estimated from photosynthesis and respiration response to T° fitting curves on (a) and (b).
<figure><img src="image_6.png" /><figcaption>Figure 4 - Effect of +8 °C on the accumulation of sugars in the microvine berry.
(a) A ML1 microvine plant during the experiment.</figcaption></figure>

Organs were spatially sampled and spatial positions converted using the phyllochron and the sum of T° above +10 °C. (b) The accumulation of sugars as a GDD (Growing degree days) function. (c). The accumulation of sugars as a calendar function.

studied. All day-detected developmentally regulated transcripts were also modulated at night, whereas, surprisingly, 1,843 supplementary genes displayed night-specific developmental regulation. Hierarchical clustering of those transcripts revealed a similar regulation pattern at day and night with secondary metabolism being more distinctly regulated during night development. A diurnal modulation throughout all stages could only be detected for 9 transcripts, indicating that circadian regulation in the berry has a high stage specificity. Regarding functional categories, mainly cellular organization and photosynthesis were day-upregulated in green berries, whereas secondary metabolism and stress-related genes were night-upregulated in ripening berries.

Short T° stresses confirmed that gene modulation
could vary to a large extent according to sampling
time. Application of a short stress at 3 different stages
at day and night yielded a total of 5,653 modulated
transcripts (two-fold change, p<0.05). Responsive
pathways differed according to time and stage. A
clear distinction of ripening stages by single berry
selection led to stage-specific detection of malic acid
metabolism-related transcripts displaying heat
activation, whereas anthocyanin regulatory genes

were repressed. Although stress application lasted
only 2 h, a heat-induced delay in ripening and sugar
accumulation could already be observed at *véraison*
at a transcriptomic level. The heat activation of
several candidate genes controlling the responses to
T° in the berry, *VvGols1* (Galactinol Synthase 1) and
*VvHsfA2* (Heat stress factor A2), could be confirmed
(Pillet *et al.*, 2012). One complementary Heat Shock
transcription Factor (HSF), *VvMbflc*, previously
described in Arabidopsis (Suzuki *et al.*, 2011), could
also be identified in the grapevine berry.

In a series of long-term T° treatments using day and
night sampling of single berries corresponding to
green and ripening stages, a total of 674 millions
sequenced reads yielded 10,788 differentially
expressed transcripts in response to T°. Green berry
development accelerated as evidenced by higher rates
of organic acid accumulation and berry growth under
higher T°. This could be confirmed when considering
the transcriptional signatures of genes related to cell
expansion. Transcripts related to tannin synthesis
(CHS, PAL) and galloylation were found to be
repressed by high T°. Surprisingly, the onset of
malate breakdown was delayed to mid-ripening when
plants were grown under cold conditions. This
observation suggests that malate breakdown is not an
intrinsic part of the *véraison* program. Whole plant carbon balance could be determinant for the trigger of malate breakdown, which was considered to be non-plastic. Several ATPases and malate transporters displayed development and T° dependent expression patterns, besides less marked but significant regulation of other genes in the malate pathway.

Heat responsive genes (HSPs, HSFs) detected in the short heat stress studies were also found modulated by long-term treatments. However, several of them showed a decreased T° response indicating their role in short-term adaptation to high T°. By contrast, other transcripts maintained a high expression level even during night sampling, when night T° was kept stable besides various T° conditions.

The single berry biochemistry analysis made prior to RNA extraction not only made it possible to reduce biases in transcriptomic results (Carbonell-Bejerano *et al.*, 2016), but also highlighted that the ripening program of single berries can be faster than for whole bunches.

### 4. At bunch level, T° also dramatically changes the developmental program of the berry

With fruiting cutting experiments, metabolites (sugars, acids, phenolic compounds, amino acids) were quantified. Critical changes were observed for malic acid, anthocyanins, flavonols and some amino acids including GABA. We also noticed that accumulation of sugars and phenolic compounds was postponed by 2 to 3 weeks by heat stress applied at green stage. Transcriptomic analyses identified more than 7,500 transcripts showing differential expression under heat stress. However, most of these responses were found to be stage-specific and only 38 genes exhibited the same deregulation across all conditions.

To understand the biological significance of the differentially expressed genes, a Genome Ontology (GO) category enrichment analysis was performed and revealed both similar and different T° effects according to the developmental stage and the stress kinetics. Several significantly affected functional categories were identified, among which “abiotic-heat stress”, “secondary metabolism”, “transport”, and “signaling”. Interestingly, the category “RNA-regulation of transcription” is also highlighted through many heat responsive genes encoding putative transcription factors or epigenetic regulators. Proteomic analyses identified around 2,000 non-redundant proteins. T° led to significant remodeling of the berry proteome with up to 556 deregulated proteins. However, these responses depended on both developmental stage and stress length. The GO

category enrichment analyses indicated that the most affected processes belong to stress responses, protein metabolism, and primary and secondary metabolism. Interestingly, less than 20 % of these heat-deregulated proteins were also modulated at the transcriptional level. Taken together, these omics data contribute to explain the dramatic changes in metabolite contents observed in heat stressed berries and highlight the intrinsic capacity of this fleshly fruit to perceive heat stress and to build adaptive responses.

We initiated the functional characterization of some major candidates. Both *VvGols1* and *VvHsfA2* were found upregulated at the transcriptional level in berries under heat stress. *VvGols1* expression profile correlated positively with galactinol accumulation in heat stressed berries. Heterologous expression of *VvGols1* in *E. coli* showed that it encodes a functional galactinol synthase. Transient expression assays showed that the heat stress factor *VvHsfA2* transactivates the promoter of *VvGols1* in a heat stress dependent manner. The results also suggest that galactinol may play a signaling role in these responses (Pillet *et al.*, 2012). To extend this study, several transgenic grapevine material (microvine, hairy roots and embryonic cells) over- or under-expressing *VvHsfA2* and *VvGols1* were produced. The characterization of these transgenic lines is under progress (phenotyping, heat sensitivity, transcriptomic and metabolic modifications). The functional characterization of other heat stress responsive genes that potentially act as transcription factors (TF) or epigenetic regulators was also initiated.

### 5. Identification and genetic mapping of developmental traits

All traits phenotyped segregated in the Picovigne x Ugni Blanc *flb* population (Figure 5). New correlations were found between malate and total sugars at green lag phase, and between internode length and leaf area (Houel *et al.*, 2015). Broad-sense heritability was unexpectedly lower than 0.40, except for traits related to berries and acids. Dense and reliable genetic maps were built for both parents, with low inter-marker distances. Fourteen QTLs were detected in at least two environments, among which a novel QTL for berry size on chromosome 7, explaining up to 44 % of total variance. This QTL was co-localized with QTLs for the number of berries, clusters and seeds, as well as with QTLs for major berry acids at green lag phase. The dwarf stature of the microvine allowed a fine phenotyping for many traits in a T° range wider than in previously published studies. Quantifying acids at green lag phase allowed us to find the first berry acidity QTLs
in a *Vitis vinifera* intra-specific cross, and particularly
the first QTLs for tartaric acid. These QTLs were
found under several environments and then offer
opportunities for breeding.

Conclusions

1. On the development of new methods to study the abiotic stress effect

A robust analytical framework was developed to
characterize the phenotype of the microvine,
especially for inferring temporal data from spatial
data (Torregrosa *et al.*, 2016). Thus, it becomes
possible to reduce the calendar duration of the
experiments to a few days or weeks and to study
simultaneously various stages of reproductive
development (Rienth *et al.*, 2012; Luchaire *et al.*,
2015).

2. On the central role of carbon in the response to
T° fluctuation

Data revealed a relationship between the lower
carbon balance within the plant and the abortion of
reproductive organs (Luchaire, 2015). In calendar
time, while berry development remains stable, T°
boosts vegetative organogenesis disturbing
source/sink balance. As a consequence, the
differential response of reproductive vs vegetative
system occurs at the expense of berry biomass
allocation. Then, T° can change malic acid
metabolism in relation to the accumulation of sugar
(Rienth et al., 2016, Romieu et al., 2016) by acting
on the energy status of the plant. This observation
reveals a hitherto unsuspected phenomenon because
it was considered a dogma for the vine berry that the
decrease in malic acid was triggered at the start of
sugar unloading (Carbonneau et al., 2015). These
observations provide a new perspective on the
capacity of a plant, subjected to limiting carbon
assimilation conditions and/or to changes in biomass
allocation, to support fruit energy supply. However,
these data acquired with the microvine should be
validated on other genotypes.

3. On the identification of specific regulatory
elements controlling fruit response to heat

Elevated T° applied at the level of the fruit or the
entire plant delayed fruit ripening, including sugar
and secondary metabolite accumulation. We have
found specific regulatory mechanisms that are
independent of the carbon status of the plant,
showing the existence of a direct impact of T° on
fruit behavior. Thus, several regulators, metabolic
elements (e.g. *VvGols1*) or transcription factors

<figure><img src="image_7.png" /><figcaption>Figure 5 - (a) Berry weight and (b) sugar content individual distributions within the Picovigne x Ugni Blanc *flb* progeny.</figcaption></figure>

M: mean value over two copies. T° in growth room:
cool: day/night 20 °C/15 °C; hot: day/night
30 °C/25 °C.

(VvMbf1 or VvHsfA2) were highlighted, opening new
avenues for research.

4. On the identification of genetic determinants of adaptation to T°

In this study, 14 QTLs showed a stable behavior with
respect to environment fluctuations (Houel et al.,
2015). These regions include genetic traits
controlling vegetative (e.g. leaf area) and
reproductive (e.g. size and acidity of the berry) traits.
These resources open new perspectives for future
breeding programs. During our program, we have
identified interesting phenotypes that could mitigate
some negative effects of heat on berry development.
Based on these resources, a new program has started
to identify QTLs limiting sugar accumulation in the
berry, as a major step to select cultivars more suitable
for hot conditions (Figure 6).

**Acknowledgements:** This work was supported by ANR-
Genopole (project ANR-2010-GENM-004-01), Jean
<figure><img src="image_8.png" /><figcaption>Figure 6 - Scheme of a genetic introgression to combine traits of berry adaptation to warming and plant tolerance to diseases using the microvine system.</figcaption></figure>

Poupelain foundation, INRA, Montpellier SupAgro, CNIV (Comité National des Interprofessions des Vins à appellation d'origine) and CIVB (Comité Interprofessionnel des Vins de Bordeaux).

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