Differential response of the accumulation of primary and secondary metabolites to leaf-to-fruit ratio and exogenous abscisic acid

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Organic acids metabolism during berry development and ripening

Second to sugar, organic acids are the most abundant soluble components present in grape berry (Dharmadhikari, 1994). The ratio of sugar to organic acids is an important ripening index for deciding the harvest date (i.e. the so-called “technical maturity”). The main organic acids found in grapes are tartaric and malic acids, accounting for more than 90% of the total organic acids, the 10% remaining being citric, succinic acid and oxalic acids (Dharmadhikari, 1994, Yinshan et al., 2017). About 50% of organic acids in green berries are accumulated through photosynthesis (Conde et al., 2007). The concentrations of both acids increase during the green phase of berry growth and reach their peaks just prior to véraison, and then gradually decrease during the third stage, most particularly malic acid, tartaric acid being fairly stable. From véraison onwards, malic acid levels reduce greatly, which leads the ratio of tartaric-to-malic acid to increase until ripeness (Dharmadhikari, 1994).
Tartaric acid biosynthesis starts with L-ascorbic acid. L-idonate dehydrogenase (L-IdnDH) catalyses the conversion of L-idonate to 5-keto D-gluconic acid, which is then cleaved between the position C4/C5 to produce tartaric acid and a two-carbon compound (Figrue 1.5, Conde et al., 2007). The expression of IdnDH was detected since flowering and increased in young green fruits, then decreased at the beginning of véraison (10% ripe berries) (Cholet et al., 2016). However, the content of tartaric acid in berries may be also related to other, yet unknown enzymes activities and/or other metabolic processes since the content of IdnDH protein exhibited little variation during berry development (Cholet et al., 2016). Thus, more detailed studies of this pathway are needed to further decipher the tartaric acid biosynthetic pathway in grape.

The impact of climate change on berry metabolism

The quality of berry at harvest greatly determines the quality of the wine. Optimal grape maturity is essential for wine quality, but is becoming more and more difficult to reach in the context of global climate change, which is modifying vine physiology and especially the biochemical composition of grape berries at harvest (Drappier et al., 2019). Global climate change is intensifying, and the occurrence of regional extreme weather events is increasing, both in frequency and intensity, while the yield and quality of grapes are very sensitive to climate and weather (Jones and Webb, 2010). The major threats from climate change for maintenance of optimal grape quality and current vineyard geographical distribution are the elevated temperature, modified rainfall patterns and incoming radiations, in particular UV-Bs (Martínez-Lüscher, 2014, Martínez-Lüscher et al., 2016, Van Leeuwen and Darriet, 2016). Among these abiotic factors, temperature is the most critical one influencing grapevine phenology, altering grape ripening dynamics and ultimately grape composition at harvest (Drappier et al., 2019).

Effects of temperature rising on primary metabolites in berry

The general trends of the impact of mean temperatures elevation observed in past decades already impacted grapevine phenology and altered berry composition at harvest. Twenty years ago, Jones and Davis (Jones and Davis, 2000) analysed datasets collected from 1952 to 1997 in the Bordeaux area and demonstrated that Merlot and Cabernet Sauvignon varieties tended to produce berries with higher sugar-to-acids ratios, while climate tended to become warmer. The same year, Schultz et al. (Schultz, 2000) predicted that changes in the climate of the European viticultural regions in the coming decades (post year 2050) could potentially have a serious impact on wine quality by affecting the composition of the berries at harvest. Changes observed over recent years confirmed these predictions. Through an analysis of the climate and grape phenological data series from 1972 to 2003 in the Alsace region (France), Duchene and Schneider (Duchêne and Schneider, 2005) found that the developmental stages of Vitis vinifera cv. Riesling occurred earlier with the increase in mean temperature, which led the harvest dates to occurs earlier in the season. In particular, the results showed that potential alcohol levels at harvest rose by an average of 0.08% per year, meaning that the sugar content in berry also increased by year, causing the technological ripeness of the grapes (based on the sugar-to-total acidity ratio) to be reached earlier. Another analysis of climatic data between 1950 and 2006 in France indicated that the mean annual temperatures increased 1.3 °C in this period, while the mean potential evapotranspiration increased by 900 mm per year since 1999. This resulted in sugar concentrations increased leading to harvest dates already advanced by up to three weeks in French Mediterranean Hérault vineyard (Laget et al., 2008). Mean data from Languedoc (France) shows that over a 35-year time span, alcohol in wine increased from 11% to 14%, pH from 3.50 to 3.75 and total acidity decreased from 6.0 to 4.5 g.L-1 (van Leeuwen et al., 2019). Recently, a report analysed the effect of the trend of climate change between 1953 and 2013 in Italy on grape Romagna Sangiovese production area potential alcohol concentration. The results showed that the increase of potential alcohol level was largely (but not totally) explained by climatic factors during that period (Teslić et al., 2018). Similar impacts of climate change trends were also found on berry compositions in the southern hemisphere production areas. The researchers found that the date of technological ripeness (21.8°Brix) advanced at rates between-0.5 and -3.1 days/year between 1993 and 2006, and that the concentration of sugar in the berries of Cabernet Sauvignon and Shiraz still increased up to ~0.3°Brix/year at harvest in Australia (Petrie and Sadras, 2008). Based on these reports, it is concluded that increasing temperatures already had a notable effect on berry primary metabolism, leading to a general increase in sugar concentrations and lower acidity accompanied by hastened berry ripening rates, which would further result in the alcohol content in wines to increase, affecting their balance (Tilloy et al., 2014, Jordão et al., 2015). In particular, the sensory properties of wines may be affected, breaking the balance responsible of wine typicity and typicality (King et al., 2013).

Effects of temperature rising on secondary metabolites in berry

There are no long-term observational datasets to show that secondary metabolites have been affecting climate changes in the last decades. However, a large body of comparisons studies between different vineyards with different climates, and different climate change simulation experiments in the field or in greenhouses showed that elevated temperature conditions reduced total anthocyanin content in grape berries and alter the anthocyanins profiles at harvest (Kliewer, 1970, Buttrose et al., 1971, Kliewer and Torres, 1972, Coombe, 1987, Mori et al., 2007, Sadras and Moran, 2012, Fernandes de Oliveira et al., 2015, Martínez de Toda and Balda, 2015, Martínez-Lüscher et al., 2016, de Rosas et al., 2017). During the 2010-2012 period, total anthocyanins content of Vitis vinifera L. “Maturana Tinta de Navarrete” berries grown in warmer (up to +1.3°C) vineyards decreased, as well as the ratio of anthocyanins-to-sugars (Martínez de Toda and Balda, 2015). After a 2-3°C temperature raise under field-crop conditions, total anthocyanins content decreased by 28-41% and the relative proportion of acylated anthocyanins increased in cultivars of “Malbec” and “Bonarda” in Argentina (de Rosas et al., 2017). Additionally, high temperatures were found to also affect amino acids and aroma compounds in berries at harvest. By raising the temperature of the fruit to the expected climate conditions of the mid-21st century (c.a. +1.5°C in average) in vineyards using polycarbonate screens, anthocyanins, amino acids (especially alanine, serine, and phenylalanine), and IBMP (2-methoxy-3-isobutylpyrazine) concentrations were reduced; while total tannin content was increased before véraison followed by a decrease thereafter in Cabernet-Sauvignon berries (Wu et al., 2019). Conversely, UV-B exposure, which is linked to temperature by the fact that they both come through global solar radiation intercept by berry surface, increased the concentrations of anthocyanins in skin and GABA in pulp; while reducing some individual amino acids, such as threonine, isoleucine, methionine, serine and glycine, partially alleviating the effect of temperature (Martínez-Lüscher et al., 2014). However, after ten combinations of temperature and solar radiation exposure experiments, Tarara et al. (2008) demonstrated that temperature is among the strongest environmental determinant of anthocyanin profile in the berry skins, above a potentially low threshold of exposure to solar radiation.
The molecular mechanism by which high temperature affects the accumulation of anthocyanins in berries has been largely studied. The expression of anthocyanin biosynthetic genes is strongly affected by temperature, with high temperature causing a decrease in the transcript levels of the genes. Mori et al. (2005b) found that high night temperatures inhibited the expression of CHS, F3H, DFR, LDOX and UFGT genes at véraison, but also an increase in UFGT activity after véraison. Yamane et al. (2006) reported that high temperatures reduced the expression of VvmybA1, one of key transcription factors that positively regulate UFGT gene expression in Japanese red table grape Aki of Queen. These authors also hypothesized that a decrease in ABA content of berries grown under high temperature was responsible for the reduction of VvmybA1 transcript levels. De Rosas et al. (2017) found that the expression of VvmybA1 and UFGT genes was correlated with anthocyanin accumulation in half-ripe and harvest stage berries; and that the acyltransferase gene Vv3AT transcript levels were increased and associated with higher anthocyanin acylation in berries cultivated under high temperature. Using 13C-labelling experiments, Mori et al. (2007) showed that the content of total anthocyanins was markedly reduced compared to berries grown under lower temperatures. Furthermore, using genome-wide transcriptomic analysis, Lecourieux et al. (2017) showed that numerous genes potentially related to anthocyanin degradation increased their transcript levels in response to high temperature. Thus, it appears that high temperatures reduce anthocyanin contents by both inhibiting anthocyanins biosynthesis and accelerating their degradation processes.
In summary, under the warming trend the increase of berry sugar content and the advance of technological ripeness, as well as the decrease of anthocyanin accumulation, are likely to result in the decoupling of technological and phenolic ripeness, as predicted by Sadras and Moran (Figure 1.7, 2012).

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Viticultural practices aiming to reduce carbon source: canopy management

Mature leaves are the main source of carbohydrates produced through photosynthesis and transported via the phloem to berries during ripening (Kliewer and Dokoozolian, 2005), even though in early developmental stages young bunches can also make a significant contribution to carbon assimilation (Vaillant-Gaveau et al., 2011). Hence, the sugar content in beries is largely determined by the total leaf area and the photosynthesis efficiency (Kliewer and Dokoozlian, 2005, Novello and De Palma, 2013). In general, a leaf area-to-fruit ratio around 0.8 to 1.2 m2/kg is able to produce the optimal level of total soluble solids, berry weight and berry coloration at harvest in vineyard conditions (Kliewer and Dokoozlian, 2005). To cope with the decoupling of anthocyanins and sugars accumulations under high temperature, one strategy is to control carbon source by reducing leaf-to-fruit ratio appropriately to delay berry ripening and slow down sugar accumulation rate (Martínez de Toda et al., 2014). Shoot trimming and leaves removal are the cultural practices to reduce leaf-to-fruit ratio of vines, and the application of anti-transpirant sprays can envisioned to decrease photosynthesis activity by reducing gas-exchange (Palliotti et al., 2013a).
As shoot trimming promotes lateral shoots growth below the cutting point, which can then become net exporters of carbohydrates as soon as they have two fully expanded leaves (Vasconcelos and Castagnoli, 2000), the timing and degree of shoot trimming will highly influence the effect on delaying of sugar accumulation. An early shoot trimming experiment carried out on Pinot noir at bloom improved fruit set by 25% with the Brix index increasing (Vasconcelos and Castagnoli, 2000). However, shoot trimmings at a 50% or 100% level pre-anthesis and post-anthesis stages or 50% at both times reduced fruit set and increased sugar concentration in field-grown “Barbera” and “Trebbiano” cultivars (Poni et al., 2004). In another experiment, three different shoot trimming treatments were done during a 3-year period: 1) a control with untrimmed vines; 2) vines with one trimming after fruit set; and 3) vines with two trimmings, one after fruit set and another one at véraison (Martínez de Toda and Balda, 2013). The results of this experiment showed that by harvesting at the same date, both trimming treatments delayed the véraison date by 18-20 days and decreased soluble solids by 12-14%, but the double trimming (leaf-to-fruit ratio less than 0.5 m2/kg) led to a greater reduction in total anthocyanin content compared to single trimming treatment (Martínez de Toda and Balda, 2013). Interestingly, applying the same above-mentioned treatment but harvesting berries at the same sugar concentrations, induced a delay in berry development that led to an increase in anthocyanin concentrations, which partially restored the anthocyanins-to-sugars ratio (Martínez de Toda et al., 2014). Finally, a severe shoot trimming at fruit set was found to reduce sugars levels at harvest, without affecting the accumulation of anthocyanins in Merlot (Herrera et al., 2015). Hence, it looks like that shoot trimming at fruit set stage, associated with a leaf-to-fruit ratio kept above 0.5 m2/kg and a harvest based on sugar concentrations constitute one of the optimum practices for harvesting high quality berries. Leaf removal is another commonly used canopy management practice to improve berry quality. Leaf removal in different parts of vines would have different influence on berry composition. After véraison, basal leaves are no longer the main source of photosynthetic assimilates (Poni et al., 1994), so the removal of the medial and apical part of the canopy around véraison got favoured in recent years as a field practice (Zhang et al., 2017). By removing 35% leaf area apical to the bunch zone using a leaf-plucking machine, when berry sugar content was approximately 16–17°Brix, the reach of the optimal TSS level of Sangiovese berries was delayed by 2 weeks, without significantly changing neither the concentration of total phenolic compounds in the grapes nor the chemical and chromatic characteristics of the wines (Palliotti et al., 2013b). Similarly, Poni et al. (2013) found that leaf removal above the bunch zone at pre-véraison and post-véraison developmental stages delayed technological ripeness without affecting colour and phenolics. However, Bobeica et al. (2015) reported that carbon source limitation induced by severe leaf removal at one week before véraison in Cabernet Sauvignon and Sangiovese fruiting-cuttings caused a significant accumulation of anthocyanins and sugars in berries, but the impact on anthocyanins accumulation being even greater (Bobeica et al., 2015). In addition, it was shown that the effect of leaf removal at véraison on berry composition also depends on the cultivar (Lanari et al., 2012, Gutiérrez-Gamboa et al., 2019). Removal of the most functional leaves on the upper part of the canopy of vertical shoot positioned medium-vigour ‘Sangiovese’ an ‘Montepulciano’ grapevines at post-véraison reduced juice soluble solids, anthocyanins and polyphenols concentration at harvest in ‘Montepulciano’ grapevines, but not in ‘Sangiovese’(Lanari et al., 2012). As the leaf-to-fruit ratio decreased, the accumulation of soluble solids was delayed in “Sauvignon blanc”, “Cabernet Sauvignon”, and “Syrah” cultivars, but not in “Carmenère”, and the leaf-to-fruit ratio value to reach an optimum content of soluble solids was different among these cultivars (Gutiérrez-Gamboa et al., 2019). This means that we still need to deepen our understanding of the relationship between primary metabolites (sugar, organic acids) and polyphenol accumulation in response to leaf removal around véraison, in order to find the optimal trade-off points for synchronizing phenolic ripeness with sugar ripeness in grape.

Plant material treatments and sampling

All experiments were conducted with Vitis vinifera L. cv. Cabernet Sauvignon in Bordeaux, France. Fruiting-cuttings made up of one vertical shoot with one grape cluster were prepared as described in (Mullins & Rajasekaran, 1981) and grown in a naturally lighted and semi-controlled greenhouse, with chemical disease control treatments applied every 2 weeks. In detail, after pre-rooting, fruiting-cuttings were transplanted into 0.5L volume pots containing a mixture of perlite, sand and vermiculite (1:1:1). After irrigation with water for the first 3 weeks, full strength Hoagland’s solution was supplied to each pot with a drip irrigation system 3-5 times per day to avoid any water stress throughout the experimental period. The temperatures data inside the greenhouse during each growing season was shown in Sup Fig.1 and Sup Fig.2.

Table of contents :

Chapter 1 Introduction
1.1 The grapevine fruit development and ripening
1.1.1 Sugars accumulation during berry development and maturation
1.1.2 Anthocyanins biosynthesis
1.1.3 Organic acids metabolism during berry development and ripening
1.1.4 Amino acid accumulation
1.1.5 ABA accumulation
1.2 Climate change and viticulture
1.2.1 The impact of climate change on berry metabolism
1.2.2 Adaption strategies to climate change-induced berry composition alterations .
1.3 Interplay between sugar, accumulation, anthocyanins biosynthesis and ABA signalling
1.3.1 Sugars as metabolites and signalling molecules for anthocyanins synthesis
1.3.2 Abscisic acid induction of anthocyanins biosynthesis
1.3.3 Crosstalk between abscisic acid and sugar signalling in anthocyanins biosynthesis induction
1.4 Objectives of this work
Chapter 2 Differential response of the accumulation of primary and secondary metabolites to leaf-to-fruit ratio and exogenous abscisic acid
2.1 Introduction
2.2 Materials and Methods
2.2.1 Plant material treatments and sampling
2.2.2 L/F Measurement
2.2.3 Berry processing
2.2.4 Sugars, organic acids and free amino acids analysis
2.2.5 Anthocyanins analysis
2.2.6 ABA and ABA metabolites analysis
2.2.7 Data statistical analysis
2.3 Results
2.3.1 Leaf-to-fruit ratio treatments validation
2.3.2 Berry metabolite concentrations upon various leaf-to-fruit ratio treatments .
2.4 Discussion
2.4.1 Manipulation of L/F decoupled sugar and anthocyanins at harvest
2.4.2 Effects of L/F manipulation on organic acids and free amino acids at harvest
2.4.3 Effect of exogenous ABA application
2.5 Conclusions
2.6 References
Chapter 3 Mechanistic exploration on the regulation of anthocyanins and sugar accumulation by carbon source limitation and exogenous ABA application
3.1 Introduction
3.2 Materials and methods
3.2.1 Plant material and sampling
3.2.2 Leaf area measurement
3.2.3 Berry biochemical composition
3.3 Results
3.3.1 Leaf area evolution
3.3.2 Metabolite accumulations in berries
3.3.3 ABA and its catabolism products in berries
3.3.4 Gene expression patterns in berries
3.4 Discussion
3.4.1 Source limitation resulted in the reduction of sugar and anthocyanins concentrations through differential gene expression
3.4.2 Exogenous ABA application partially restore the decoupling sugar and anthocyanins under source limitation via differential expression of the associated genes and altered endogenous ABA
3.4.3 The relative effects of carbon limitation and whole-vine transpiration on berry sugar and anthocyanins
3.5 References
Chapter 4 The roles of sugar and ABA in inducing anthocyanin synthesis at véraison in grape berries cultured in vitro
4.1 Introduction
4.2 Materials and methods
4.2.1 Berry in vitro culture, treatments and sampling
4.2.2 Sugars, organic acids and free amino acids analysis
4.2.3 Anthocyanins analysis
4.2.4 ABA and ABA metabolites analysis
4.2.5 Data statistical analysis
4.3 Result and discussion
4.3.1 Effect of NAG and DGH hexokinase inhibitors combined with different concentrations of glucose on berry composition
4.3.2 The relationship of sugar accumulation and ABA on the induction of anthocyanins accumulation
4.3.3 The effect of HXK inhibitors and exogenous ABA on berry ABA and its catabolism products
4.4 Conclusion
Chapter 5 General discussion
5.1 A decrease in ABA concentration in berry caused by reducing leaf-to-fruit ratio could be the reason for the unbalanced decrease in sugar and anthocyanins
5.2 Sugar and ABA interaction in regulating anthocyanin synthesis in berries . 179
Chapter 6 Conclusion and prospective
Chapter 7 General bibliography

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