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The accumulation of berry solutes
Since water transport is passive in plants, the berry needs first to accumulate solutes and develop an osmotic pressure in its vacuoles, in order to accumulate water, growth and development. Cell walls and peel has to be extensible to allow vacuolar enlargement. Phloem and xylem are the two main vascular tissues able to perform this continuous unload. Xylem is important for water conduction during green growth phase (Greenspan et al. 1994) and seems to be not functional in post-veraison (Bondada et al., 2005). Phloem is transporting photo-assimilates, mainly sucrose in grape-vine (Van Bel, 2003). Xylem and phloem are physically linked and exchange water, sugars, minerals and hormones (Van Bel, 1990; Metzner et al., 2010), each berry is linked to the plant by 5-6 peripheral and central vascular bundles. Exchanges between source and sink organs can proceed in two ways (cf. Figure 4). First one is the symplastic pathway that is characterised by a mass influx through plasmodesmata ensuring a cytoplasmic continuity between the phloem conductive bundles and the sink cells (Lalonde et al., 2003). The second road is the Apoplastic pathway, which is dominant after softening (Lalonde et al., 2003; Afoufa-Bastien et al., 2010). Assimilates are transported through plasma membranes and circulate in the apoplast. Sugars either follow their concentration gradient or are accumulated against it at energy cost, depending on different transporters.
Figure 4: Comparison of the symplastic and apoplastic phloem pathway for sugar transport in plants (adapted from Lemoine et al., 2013).
As the major component of fleshy fruits, water is primordial for berry growth and attributes concentration (Conde et al., 2007; Vicens, 2007; Keller et al., 2015). This water comes mainly from root (99 %) and circulates via both phloem and xylem (Greenspan et al., 1994, 1996; Ollat et al., 2002; Matthews & Shackel, 2005). Its quantity in fruits is highly dependent on climatic conditions (Jakab et al., 2013). During first growth period, water is mainly coming from xylem (88 %) (Greenspan et al., 1994, 1996; Choat et al. 2009) and then after softening, xylem flux decreases but remains functional (Keller et al., 2006; Rogiers et al., 2006; Tilbrook & Tyerman, 2009; Clearwater et al., 2012; Cuéllar et al., 2013; Keller et al., 2015; Zhang & Keller, 2017) so water import becomes mainly phloemian as represented in figure 5 (Chatelet et al. 2008 a, b; Choat et al. 2009; Dai et al. 2010; Keller et al. 2014). Keller et al. (2015) suggested that this decrease is due to the rapid increase of turgor, changing pressure gradient (ΔPX) between extremities of the xylem.
Figure 5: Water flow for each component of water budget in berries of Cabernet-Sauvignon without water limitation (Greenspan et al., 1994).
This xylemian gradient pressure (ΔPX) between plant and berry constitutes the driving force for xylem hydraulic conductivity and depends on climatic conditions and plant water status (Keller et al., 2006; Tilbrook & Tyerman, 2008, 2009), on berry vacuolar osmotic pressure (Лv) (sugars concentrations in phloem and berry) and phloemian water recycling via the xylem (Becker & Knoche, 2011; Zhang & Keller, 2017). ΔPX affects berry growth during day/night cycle with a contraction during the day due to transpiration and water uptake by plant. As water stress tends to contract even more (Greenspan et al., 1996). During ripening, a considerable osmotic pressure develops in berry vacuoles, due to the accumulation of hexoses, which should make the berry the biggest sink for water in the plant (Greenspan et al., 1994). Keller et al. (2014) even showed that green berry shrivelled as a consequence of severe water stress start to grow again at the onset of ripening even before irrigation, showing the power of the sink for water uptake.
During ripening, when water flux becomes mainly phloemian, unloading in mesocarp cells go through from symplastic to apoplastic pathway (Zhang et al., 2006), resulting in an increase in apoplasmic osmotic pressure (Лa) facilitating water and mass influx (Ruan & Patrick, 1995; Patrick, 1997; Keller & Shrestha, 2014; Keller et al., 2015). Keller et al. (2015) inferred from phloem sugar concentration measurements and berry water budget that only 20 % of the water from phloem would be used for berry growth and transpiration (which is climate dependant; Dreier et al., 2000) with the remaining water being recycled by xylem, and suggested that if berry still accumulate sugars after maximum volume, it means that water is going back to the xylem, but calculations 12 tends to invalid this hypothesis (Shahood, 2017). This back flux would be crucial for berry solutes unload to obtain normal berries and depends on the fruit solutes demand, leaf photosynthesis, plant water status and atmospheric vapour pressure deficit (VPD). Whatever, Shahood (2017) showed that berry growth and sugars accumulation stopped simultaneously, undermining the previous theory. After physiological maturity (maximum berry volume), berry shrivel due to transpiration and/or higher xylemian reflux than phloemian influx (Greer & Rogiers, 2009).
Sugars are important for vine plants and produced by leafs photosynthesis, and then transported inside the phloem mainly in the sucrose form (Swanson & Shishiny, 1958; Conde et al., 2007). The majority of sugars imported inside berry during green growth will be metabolised and sugars will never be higher than 150 mM during this phase, to rise up at 1 M after ripening (Wu et al., 2011; Davies et al., 2012; Houel et al. 2015). At veraison, ratio of main sugars (glucose/fructose) is between 2 and 10 at veraison stage to finish around 1 at maturity (Varandas et al., 2004). Sucrose is so representing maximum 2% of these sugars except in some table grape varieties or varieties derived from Vitis labrusca , but it seems that this trait, linked with a lower vacuolar invertase activity, is recessive (Shiraishi et al., 2012). These sugars are of primary importance for both wine and table grapes (Davies et al., 2012). During green growth, sucrose imported in the berry and cleaved by different invertase enzymes, creating a sucrose gradient that favours its entry in the vacuole (Fillion et al., 1999). Change in phloem inflow at veraison would result in an increase of sugars accumulation rate in berries (Zhang et al., 2006).
Sugar accumulation involves specific transporters (Hedrich et al., 2015). Transporters are needed for all assimilates and water transport through biological membranes. Sucrose, after a movement due to mass flux will be loaded inside the berry by both phloem pathways possible (apoplastic and mostly symplastic) (Zhang et al., 2006; Turgeon & Wolf, 2009). Then, changes in activity of the invertase and phloem pathway at veraison will lead to a higher capacity to import sucrose into berry but sucrose can’t pass alone through phospholipidic membranes. Sucrose will then requires transporters to pass or endocitosis (Figure 6) (Fontes et al., 2011).
Figure 6: Sugars transport into the berry (Davies et al, 2012), with 1: Apoplastic pathway depending on pH; 2: Apoplastic pathway with couple H+/sucrose; 3: Monosaccharide transporters type VvHT; 4: Sucrose transporters; 5: Endocytisis; 6: SWEETs; 7: Monosaccharide transporters; 8: Sucrose transporters.
Cell membranes have a hydrophobic double lipidic layer that permits to keep solutes gradient between cytoplasmic and extra-cellular environments. 3 groups of sucrose transporter may reside at the plasma membrane interface: Sucrose Carrier (SUC), Sucrose transporter (SUT) and Sucrose Facilitator (SUF) (SUC and SUT with VvSUC2, 11, 12, 27 in grapevine) (Davies et al., 1999; Manning et al., 2001; Afoufa-Bastien et al., 2010), the expression of VvSUC 27 gene being higher during green stage and that of VvSUC11, 12 during ripening. Sucrose can also be cleaved in glucose and fructose by the activity of the three different vacuolar (Inv-V), cell wall (Inv-CW) et neutral (Inv-N) invertases. Zhang et al. (2006) showed that vacuolar invertase activity decreased while cell wall invertase increased at the onset of ripening, an argument in favour of the induction of the apoplastic pathway at this stage (Figure 7).
Figure 7: Vacuolar and cell wall invertase activities and immunoreactivity during berry growth (adapted from Zhang et al., 2006).
The vacuolar invertase activity is correlated to hexose and sucrose relative quantity inside berry vacuole, but not to the total sugars quantity accumulated (Takayanagi & Yokotsuka, 1997; Davies & Robinson, 1996). Neutral invertase would be used if sucrose comes directly to cytoplasm, but not much information are recorded on it (Davies et al., 2012). In grapevine, 9 neutral (VvNIs; VvNI 1-5 being weak during ripening), 2 vacuolar (GIN1 & GIN2; diminution of activity after the onset of ripening) and 1 cell wall (VvcwINV; increase slowly during green growth and decrease during ripening period) invertases were characterised (Davies & Robinson, 1996; Dreier et al., 1998; Nonis et al., 2008). VvcwINV being induced in parallel with VvHT5 hexose transporter in some specific conditions (Lecourieux et al., 2014). After conversion, the transport of glucose and fructose formed in the apoplasm requires plasma membrane transporters too. It exists 7 classes of monosaccharide transporters: Sugar Transport Protein (STP), Vacuolar Glucose Transporter-like (VGT-like), Tonoplast Sugar Transporter (TST), Plastidic Glucose Transporter/Suppressor of G protein Beta1 (pGleT/SGB1), Early-Responsive to Dehydration-like (ERD6-like), Polyol Tranporter (PLT) and Inositol Transporter (INT) (Büttner, 2007). In grape vine, hexose transporters (VvHT1, 2, 3, 4, 5 (STP on plasmic membranes), 6 (TST) & 7) and VvGLT were identified (Fillion et al., 1999; Vignault et al., 2005; Conde et al., 2006; Hayes et al., 2007; Davies et al., 2012; Lecourieux et al., 2014). VvHT1 compared to VvHT2 decrease in activity after the onset of ripening and is located in intermediary cells. Others transporters as SWEETs (Sugars Will Eventually be Exported Transporters) may play a primordial role in sugars unload by facilitating sucrose, glucose and 15 fructose loading through the tonoplasm (Chong et al., 2014). Seventeen SWEETs genes where found in grape-vine with different expressions in ripe berry (VvSWEET4 (for glucose and located on plasmic membrane), 7, 10, 11, 15 and 17d) and in flowers (VvSWEET3, 4, 5a, 5b, 7, 10 and 11). For a review of all transporters of the literature see Shahood (2017).
Proton pumps are also important to energize sugar transport by H+ symporters at the plasma membrane and H+ antiporters at the vacuolar one. H+ pumps use the P~P link energy to transfer H+ against its concentration gradient. To promote a concentration gradient force for sucrose and acids accumulation, three different vacuolar pumps (V-pyrophosphatase (V-PPase), V-ATPase, & type P3 A/B ATPase) are known to create a gradient of protons towards the vacuole (Faraco et al. 2014). All those pumps don’t have similar H+/ATP coupling ratio (Lobit et al., 2006; Palmgren & Nissen, 2011; Etienne et al., 2013). Plasmic ATPase (PM H+-ATPase) is necessary for having pH gradient for passive ions flux through specific channels and can be useful for salt tolerance, pH regulation and cell growth (Sussman, 1994).
Sucrose from phloem can be cleaved by both invertase (Inv) and sucrose synthase (SuSy), and re-synthesized by sucrose phosphate synthase (SPS). This makes possible to define 4 substrate cycles: degradation/synthesis of cytosolic sucrose, vacuolar degradation/cytosolic synthesis, apoplasmic degradation/cytosolic synthesis and synthesis/starch amyloplastic hydrolysis (Nguyen-Quoc & Foyer, 2001). Glycolysis (Hexose + 2 ADP + 2 Pi + 2 NAD+ → 2 pyruvate +2 ATP+ 2 NADH, H+) permit synthesis of primary and secondary metabolites, or permit storage in starch. Without oxygen, NADH as to be recycled mainly by ethanol production in plants (Shahood, 2017). Metabolites are accumulated, diluted and metabolised during berry development (Conde et al., 2007). First, acids are mainly accumulated in the vacuole (Terrier & Romieu, 2001). Malic and tartaric acid are the two mains acids in grape-berry, and participate up to 90% of the juice acidity. The main difference between those two acids is that tartaric is more stable in quantity than malic acid (Lakso & Kliewer, 1975). L-(+)-tartaric acid is accumulated at the beginning of green growth phase (Champagnol, 1984). Its synthesis pathway begging with L-ascorbic acid and cleavage of one carbons pair (C2/C3 or C4/C5 depending on species) (DeBolt et al., 2006). The preferred path to synthesize tartaric acid in grapevine is using a glycolaldehyde as showed in figure 8 but the use of an oxalic acid (OxA) and a L-threonate can also be used. In 2006, the discovery of L-idonate dehydrogenase (L-IdnDH) argues in favour of the proposed path (DeBolt et al., 2006).
L-(-)-malic acid is synthesized later in the green growing phase (Champagnol, 1984; Conde et al., 2007). This acid is the major one in many fruits (Etienne et al., 2013). This acid can be seen as a reserve of CO2 before photosynthesis, a support for respiration and neoglucogenesis, an efficient osmoticum in terms of carbon, it can also control physiological process as stoma opening (Kelly et al., 1976; Famiani et al., 2014; Sweetman et al., 2014). Malic acid is in majority synthesised inside berry from PEP (PhosphoEnolPyruvate) via the PEP-carboxylase (PEPC; inside the cytosol) and the malic dehydrogenase (MDH; inside the cytosol, glyoxysomes and mitochondria) (Taureilles-Saurel et al., 1995 a, b). PEPC and MDH activities are high during early stage of berry development and decrease just before the onset of ripening (Lakso & Kliewer, 1975; Terrier et al., 2005) and then during ripening MDH is re-increasing. Sweetman et al. (2014) showed that there is a positive linear correlation between malic content and PEPC activity. So PEPC and cytoplasmic MDH seem to be responsible for malic acid accumulation during green growth, mitochondrial MDH being mainly use for degradation by respiration. In any cases, the pathway hexose + 2CO2 <=> 2 malate2- + 4 H+ can be active in both directions, never mind the flux. Malic acid can also be produced by photosynthesis during green growth (Ollat & Gaudillere, 2000), or inside the mitochondria via main enzymes (Fumarase (not limiting), mMDH (strong activity during green growth) (Ollat & Gaudillere, 2000; Fatland et al., 2005; Sweetman et al., 2014), or via the malate synthase in the glyoxylate cycle (Terrier et al., 2005).
During ripening, neoglucogenesis will use malic acid as substrate for PEP synthesis via MDH, malic enzyme (reversible activity increasing during development)plus pyruvate ortho-phosphate dikinase (PPDK; not detected in grape), or PEP carboxykinase (PEPK) (Ruffner & Hawker, 1977; Goodenough et al., 1985; Terrier et al. 2005). During green growth, low activity of PEPK and high activity of PEPC favour malic acid synthesizing, which changes after the onset of ripening. Also, malic acid can be degraded by the respiration, which is more important in berries during green growth than ripening phase (Ollat & Gaudillere, 2000). At the onset of ripening, vacuolar released malic acid is used to produce ATP via TCA cycle. Later, its use for this cycle seems to be correlated with mMDH activity (Taureilles-Saurel et al., 1995; Etienne et al., 2013). During ripening, NADP-ME (depend on NADP+/NADPH, pH and regulators: Mn2+, Mg2+, ATP) and NAD-mtMDH seems to have a major role in malic acid degradation (Sweetman et al., 2009; Etienne et al., 2013). Pyruvate, formed by ME can be fermented in alcohol by the pyruvate decarboxylase (PDC) and the alcohol dehydrogenase (ADH) during low cytoplasmic pH and/or hypoxic environment, or in lactic acid via the lactate dehydrogenase (LDH) (Sweetman et al., 2009). Whatever, strong arguments suggest that the synthesis or degradation of malic acid is regulated by the capacity of the vacuole to accumulate it as the free acid. Terrier et al showed that tonoplast vesicles extracted from green berries were perfectly tight to H+ (and accompanying anion), allowing V-ATPase to reach thermodynamical equilibrium (pHvac = 2.7). During ripening, futile H+ recirculation cycles develops, preventing the vacuolar lumen to reach such an acidic pH (non equilibrium), so malic acid is necessarily released. Detailed investigations on the malic acid/sugar stoichiometry suggests that four hexoses are accumulated per malic acid consumed at the onset of berry ripening (Shahood, 2017), consistent with the induction of a sucrose/H+ antiporter at the tonoplast membrane, as indicated by the induction of VvHT6 transcription at this stage (Terrier et al., 2005). However, Rienth et al., 2016 showed that in cold conditions, which reduce respiration of imported photoassimilates, a noticeable accumulation of hexose can occur before the global malic acid/sugar is induced. It must be also stated that sugar loading in berries continues after malic acid is consumed.
Berry development also needs micro-elements, at least 17, considered as essential (Bashir & Kaur, 2018). It’s important to notice that inorganic cation osmotic potentials are higher than organic cations (Bonomelli & Ruiz, 2010). Potassium (K+) is the most concentrated cation in berries and its concentration depends on many factors such as fertilisation, rootstock, etc. (Deloire, 2007). Potassium is mainly absorbed by the plant between bloom and veraison by the plant with VvK1.1 gene activity and redistributed through the plant with VvK1.2 gene activity via both phloem and 18 xylem (Mpelasoka et al., 2003; Bashir & Kaur, 2018). Two potassium channels genes, VvKUP1 and VvKUP2, where found and they were highly active in the skin at the early stage of development showing it importance for berry (Cuellar et al., 2013). Expression will then decrease at veraison but stay active. At this time, other gene VvK1.2 will significantly increase in activity, promoting Shakers channels (VvCIPK04–VvCBL01 and VvCIPK03–VvCBL02) sharply increasing potassium concentration (Mpelasoka et al., 2003). Potassium is mainly accumulated in the skin where it can be 1.7-6.9 times higher than in the flesh and has a role in sugars importation.
Calcium (Ca2+) is also an important element of plant development as its involved in the cell wall and membranes structure, as counter-ion for acids and anions in the vacuole (Mpelasoka et al., 2003; Bonomelli & Ruiz, 2010). This cation which can’t be transported via phloem have to traffic through xylem using an unidirectional stream (as in Phaseolus vulgaris (Steucek & Koontz, 1970)). It also can’t be mobilized from older tissue showing it dependency to xylem flow (Mpelasoka et al., 2003). In fact calcium in fruit can be separated in three pools depending on activities (soluble Ca : nitrates, chlorides, organic acids; exchangeable Ca; Ca not physiologically active : oxalate, phosphates, and carbonates). Maximum uptake occurs at the early stage of berry development. Higher cell size, firmness and less dry matter were observed on berries from plants supplemented with Ca2+.
Magnesium (Mg2+) accumulation starts early during berry development (Duchène & Chardonnay, 1992). This cation is really mobile and can be redirected (Christensen, 2000), and Steucek & Koontz (1970) studies on Phaseolus vulgaris showed that magnesium was moving in the phloem. Mg2+/H+ exchangers (AtMHX in Arabidopsis) permit it to enter inside the vacuole (Shaul, 2002). Others transporters were also found in Arabidopsis MGT6, MGT1, MGT7, MGT9, MGT2, and MGT3, which are expressed in roots, with (MGT1 and MGT6) also located in the plasma membrane and expressed in the epidermal cells (Mao et al., 2014). Studies shows higher amount in skin than flesh but a stable concentration at similar level to calcium (Conde, 2007).
Ammonium (NH4+) represents half of the nitrogen in the early stage of berry development (Christensen, 2000) and is assimilated via the glutamine synthetase (GS; EC 18.104.22.168) NADPH-glutamate synthetase (GOGAT; EC 22.214.171.124) pathway to form glutamine and glutamate in Phaseolus vulgaris L. (Hungria & Kaschuk, 2014). After being uptake by roots, long distance nitrogen is transported in the plant in nitrate, ammonium and amino acids forms (Schobert & Komor, 1992). All three forms are possibly navigating through the xylem (With really low amount 19 of NH4+ in Phaseolus vulgaris L. (Hungria & Kaschuk, 2014)), in contrast with phloem that contain only nitrogen in Ricinus communis. Then after veraison the production of amino acids will decline its concentration (Christensen, 2000). Researches on Phaseolus vulgaris L. suggested a diminution in ammonium uptake during warm conditions (Hungria & Kaschuk, 2014).
Table of contents :
I. The grapevine fruit development
I.1. The kinetic of berry development
I.2. The accumulation of berry solutes
II. Climate change and its impact on berry development
II.1. The “greenhouse” effect
II.2. The rising temperature
III. Climate change and viticulture
III.1. Effects on plant physiology
III.2. An overview of putative means to mitigate grape/wine composition
Location and varieties change
Chapter I – How to monitor grape development using simple tools?
Materials and methods
Osmolality and Dry Matter
Primary and secondary metabolites
Results and Discussion
1. Determination of the onset of ripening
2. Heterogeneity of berry development during ripening
3. Detection of the end of the growing phase
Chapter II – Vitis vinifera L. fruit diversity
II.1: Vitis vinifera L. fruit diversity to breed varieties anticipating climate changes
Materials and methods
Plant material and growing conditions
Fruit sampling methods
Berry growth and composition determination
Data Presentation and Statistical Analyses
Berry growth during ripening
Correlations between traits
Major descriptors of grapevine fruit development and composition
Phenotyping at key stages of grapevine berry development
Conflict of interest statement
II.2: The existing diversity in cations in Vitis vinifera and it segregation in a progeny of
Materials and methods
Plant material and growing conditions
Weight measurement and sample preparation
Data analysis and graphic representations
Chapter III – The low sugars content trait
III.1: Average berries population characterisation
Materials and methods
Volume and weight measurement
Primary and secondary metabolites
Results and discussion
Growth Kinetics of average berries population
Characterisation of the fruit heterogeneity and asynchronous development
III.2: The key developmental stages during berry development
Materials and Methods
Results and discussion
1. Kinetics of berry growth and metabolites accumulation
2. Berry composition at specific stages
2.1. The onset of ripening
2.2. Maximum berry volume as an indicator of physiological maturity
3. The low sugars content trait
3.1. The onset of ripening
3.2. Physiological ripe stage
3.3. Relations Pressure – Growth
Conclusions and perspectives