Grape Berry Vacuole: A Complex and Heterogeneous Membrane System Specialized in the Accumulation of Solutes

Two proton pumps energize the vacuolar membrane.

Grape berry vacuoles maintain an acidic pH, ranging from pH 2.5 in the green stage to pH 3.5 during ripening. The maintenance of ion and proton concentration gradients across the tonoplast membrane is essential for acid and sugar homeostasis in the berry (Hanana et al. 2007). The low pH of the vacuole of fruit cells is the result of two processes: (1) proton pumping across the tonoplast and (2) synthesis and accumulation of organic acids in the vacuolar sap (Shiratake and Martinoia 2007). Two distinct primary proton pumps, the vacuolar ATPase (V-ATPase) and the vacuolar inorganic pyrophosphatase (V-PPase), generate a proton electromotive force, which, in turn, allows the secondary active transport of inorganic ions, sugars, and organic acids (Blumwald 1987, Maeshima, 2001, Martinoia et al. 2007, Fontes et al. 2010b). The V-PPase is generally more active than the V-ATPase in young tissues with relatively high amounts of pyrophosphate (PPi), which is a by-product of biosynthetic pathways (Martinoia et al. 2007). However, the V-PPase is the predominant vacuolar proton pump in tonoplast vesicles from mature grape berries (Terrier et al. 1998) and intact vacuoles from Cabernet Sauvignon berry suspension cultured cells (Fontes et al. 2010b). Intact vacuoles are good experimental models to monitor the mechanisms of vacuolar acidification and solute uptake (Fontes et al. 2010b).

Vacuolar compartmentation of sugars in grape berry.

Although the transport mechanisms of monosaccharides and disaccharides at the plasma membrane level are reasonably understood in several plants, including grapevine, information on tonoplast sugar transporters is still limited. However, some tonoplast monosaccharide transporters (TMT) have been recently reported as mediating a proton-coupled antiport mechanism. Three AtTMT (Arabidopsis thaliana tonoplast monosaccharide transporters) isoforms were localized at the tonoplast by fusion with the green fluorescent protein (Neuhaus 2007) and the tonoplast glucose/H⁺ antiporter AtVGT1 (At3g03090) was characterized in the same plant model (Aluri and Büttner 2007). In V. vinifera, the hexose transporter VvHT6 is presumed to be targeted to the tonoplast (reviewed by Agasse et al. 2009). The sequence of VvHT6 is similar to that of the three AtTMT, with an extended loop between the transmembrane helixes six and seven (Büttner 2007, Hayes et al. 2007), and its pattern of expression is consistent with a role in postveraison import of hexoses into the vacuole. Uptake activities of the plasma membrane hexose transporters VvHT1, VvHT4, and VvHT5 have been demonstrated by heterologous expression in the hxt-null mutant yeast, but attempts to confirm the transport activity of VvHT6 has had little success (reviewed by Agasse et al. 2009).

In addition to their role in sugar storage, vacuoles are also involved in the biosynthesis of higher saccharides from mono- or disaccharides. Vacuoles are also likely the site for glycosylation and production of various metabolites (De 2000).

Water incorporation in grape berry and the role of aquaporins.

Vine water deficit has a clear implication in wine composition and sensory attributes (Roby et al. 2004). It generally leads to smaller berries (Bravdo et al. 1985, Kennedy et al. 2002, Matthews et al. 1990), thus increasing the skin to juice ratio, which, in turn, may increase the concentration of anthocyanins and phenolics in the must and wine (reviewed by Conde et al. 2007).

Most of the berry volume gained before veraison is due to water import through the xylem, whereas most of the postveraison gain is due to water import through the phloem. This strong phloem component at veraison might explain the insensitivity of the berry to plant water deficits (Matthews et al. 1987). In addition, the shift of phloem unloading from symplastic to apoplastic pathway at veraison is associated with sugar accumulation at high levels in sink organs (Patrick 1997, Zhang et al. 2006), favoring the maintenance of a turgor pressure gradient. Moreover, the involvement of cell compartmentation of water and solutes makes it increasingly difficult for the leaves to extract water from ripening berries (Keller et al. 2006). The coexpression of some aquaporins and sugar transporters suggests a functional link between sugar and water fluxes during the processes of unloading and sugar accumulation in the vacuoles of the flesh cells (Delrot et al. 2001). Aquaporins are specialized proteins in the major intrinsic proteins (MIP) family that are implicated in water transport across biological membranes (Fouquet et al. 2008, Gomes et al. 2009).

Eight cDNAs encoding putative Vitis aquaporins (plasma membrane intrinsic proteins, or PIPs, and tonoplast intrinsic proteins, or TIPs) were mostly expressed in roots, eventually enhancing and regulating water permeability (Baiges et al. 2001). The aquaporin VvPIP1A mediates water transport and is mainly expressed in the berries after veraison (Picaud et al. 2003). Moreover, after the release of the grapevine genome in 2007 (Jaillon et al. 2007), 28 genes that encode putative aquaporins were identified and nine cDNAs that encode putative PIP and TIP aquaporins from grape berries at various developmental stages were isolated (Fouquet et al. 2008). Aquaporin gene expression is strongly regulated during berry development and globally decreases during ripening. The tonoplast aquaporin VvTIP2;1 and the plasma membrane aquaporin VvPIP2;1 are highly expressed in dividing and elongating cells and in cells involved in water and solutes transport (Fouquet et al. 2008). More recently, TIP2;1 was confirmed to transport water when individually expressed in Xenopus oocytes (Vandeleur et al. 2009).

Vacuolar compartmentation of malic and tartaric acids.

The final organic acid content of grape berry depends on the amount of acid synthesized, stored in the vacuole, and degraded during the ripening stages. Organic acids are produced both in leaves and fruits, but their biosynthetic mechanisms and compartmentation in grape berry cells remain poorly understood. Berry acid content varies throughout berry development and ripening. As reported above, malic acid rapidly accumulates at early stages and decreases at the onset of ripening. Tartaric acid is kept constant and its concentration declines mainly due to dilution as berry volume increases.

During the vegetative growth phase, the sugars from photosynthesis in the leaves are transformed into malic acid, which accumulates in the vacuoles of pericarp cells (Schulze et al. 2002). Also, it is believed that green berries are photosynthetically active and produce malic acid as a source of carbon and energy (Sweetman et al. 2009). Unlike many other fruits, grapes are incapable of storing significant amounts of starch. Malic acid is accumulated in the fleshy cells at the end of the first growth phase of the berry and reaches a maximal value just before veraison. At veraison, due to the severe inhibition of the glycolytic pathway, malic acid import from the large central vacuole allows energy production, lowering grape malate levels. The decrease in malic acid in the grape berry at the onset of ripening also results from reduced malate synthesis, but the reduction in the amount of acid translocated from the leaves to the berries may also play a significant role.

Malate is accumulated in vacuoles at very high concentrations (>300 mM) and the acid exchange across the tonoplast is believed to be driven by the electrochemical membrane potential difference (Martinoia et al. 2007). In Arabidopsis, malate exchange between the vacuole and the cytoplasm is mediated by AttDT, a tonoplast malate transporter (Emmerlich et al. 2003) and by AtALMT9, a tonoplast malate channel (Kovermann et al. 2007). The activity of AttDT is reportedly critical for pH homeostasis (Hurth et al. 2005).

In grape berry, four good malate transporter candidates have been identified by blast analysis of the V. vinifera genome with the AtALMT9 protein sequence (Rongala 2008). These genes are developmentally regulated. VvALMT9:1 and VvALMT9:2 showed postveraison expression, while VvALMT9:3 is expressed at high levels before veraison and VvALMT9:4 is poorly expressed. A single AttDT homologue has been identified in V. vinifera (Rongala 2008). A homologue of AttDT has been implicated in citrate efflux in citrus fruits (Shimada et al. 2006).

As previously stated, the drop in tartaric acid content, from 150 mM at veraison to 25 to 75 mM at maturity, is mostly due to the increase of the berry size. In addition, the free versus salt state of tartaric acid generally changes throughout maturation, contrary to malic acid, which generally remains as a free acid. Little is known about the biochemical mechanisms involved in tartaric acid accumulation in the vacuole of grape cells.

Vacuolar compartmentation of potassium.

Several ions, such as potassium, calcium, magnesium, and chloride, are implicated in numerous physiological processes that impact fruit quality and, ultimately, wine taste and flavor. Therefore, the study of the biochemical steps involved in their compartmentation and interaction with other solutes is of major importance for plant physiologists and viticulturists.

Potassium is an essential macronutrient for grape berry growth and development and a well-known enzyme activator. It is the main cation in must and wine (~900 mg/L; reviewed by Conde et al. 2007). Potassium is the main osmoticum in skin cells, as sugar is in the flesh. Because potassium may accumulate in the vacuole, it affects several transport processes across the tonoplast membrane. Thus, potassium uptake may increase the release of malic acid, favoring its metabolism in the cytoplasm (Jackson 2008, Davies et al. 2006).

Recently, two cDNAs encoding potassium plasma membrane transporters (VvKUP1 and VvKUP2) from grape berries were isolated and their function was demonstrated by heterologous expression in an Escherichia coli mutant deficient in potassium uptake (Davies et al. 2006). The two transporters are highly expressed in the berry skin during the first phase of berry development, suggesting that, at this time, they play a role either in potassium uptake into the berries or in its compartmentation into the skin cells. However, the transcript levels of both transporters are still significant at postveraison, suggesting that VvKUP1 and VvKUP2 may therefore continue to contribute to potassium homeostasis throughout berry ripening. At the tonoplast level, a NHX-type cation/H⁺ antiporter was recently cloned and functionally characterized (Hanana et al. 2007). VvNHX1 couples the passive movement of H⁺ out of the vacuole to the active incorporation of monovalent cations (mainly K⁺ and Na⁺), playing an important role in vacuolar ion homeostasis in grape berries.

Vacuolar compartmentation of phenolic compounds.

Phenolic compounds are an extended family of secondary metabolites in grape berries (Table 1). They are involved in plant protection because they are active growth inhibitors of other living systems. They add color and flavor to the fruit, contributing to the mouthfeel, quality, and palatability of red wines. The major flavors associated with polyphenols are bitterness and astringency. Additionally, they may play important beneficial roles for human health as strong antioxidants (reviewed by Conde et al. 2007) and as activators of the human oestrogen receptor alpha (Chalopin et al. 2010). Many secondary metabolites, particularly phenolic compounds, are frequently accumulated as glycosides, which increases their solubility, transport, and storage ability (De 2000). Nonflavonoid phenolics accumulate primarily in the vacuoles of mesocarp cells, but flavonoids accumulate in the dermal cells of the skin tissue (Table 1).

Tannins or proanthocyanidins are polymers of flavan-3-ols and are the most abundant class of soluble polyphenolics in grape berries. A number of studies involving the identification of several enzymes, transcriptional regulators of proanthocyanidin biosynthesis, and transporters have illustrated the movement of proanthocyanidin precursors into the vacuole (Terrier et al. 2009). Tannins are accumulated in specific vacuoles (tannin vacuoles) and act as deterrents to herbivores and fungi. Beyond astringency, tannins also confer bitterness, which is due to the lowest molecular weight tannins.

The predominant grape berry pigments are the anthocyanins, exclusively produced by red varieties. Carotenoids, xanthophylls, and flavonols, such as quercetin, are also present in both red and white varieties, but they are more important in the color determination of white grapes (Jackson 2008). Pigments are generally confined to the vacuoles of a few cell layers immediately below the epidermis. A few cultivars, called teinturiers, such as Alicante Bouschet, also contain anthocyanins in the mesocarp cells. Whereas flavonoid pigments are deposited in cell vacuoles, carotenoids accumulate predominantly in plastids (Jackson 2008).

Water-soluble anthocyanins are synthesized at the cytosolic surface of the endoplasmic reticulum and further transported to the vacuole, where they are usually sequestered, after being glycosylated (Grotewold 2004). Some enzymes involved in anthocyanin biosynthesis may be tonoplast-bound (Fritsch and Griesbach 1975). Spherical pigmented inclusions are present in the vacuoles of grape cells (Conn et al. 2003, 2010). Sequestration of anthocyanins by anthocyanic vacuolar inclusions, loosely termed “anthocyanoplasts,” is believed to increase their stability and to reduce inhibition of certain vacuolar enzymes (Conn et al. 2003, 2010). Anthocyanoplasts start as vesicles in the cytosol and appear membrane-bound (Pecket and Small 1980, Nozzolillo and Ishikura 1988). Once in the vacuole, many factors influence the in vivo pigmentation provided by anthocyanins (Irani and Grotewold 2005). As anthocyanin synthesis and accumulation proceed, the anthocyanin content of the outer hypodermal layer(s) approaches saturation. At this time, anthocyanin combines in self-association or copigment complexes (Jackson 2008). In some cultivars, a decline in anthocyanin content is observed near or after maturity, probably because of β-glycosidases and peroxidases activity (Jackson 2008).

Anthocyanins assume their distinct color after being transported to the vacuole, and this compartmentation also decreases the feedback inhibition of cytosolic biosynthetic enzymes. The presence and number of hydroxyl groups, methylation, and sugar moiety produce red, violet, and blue coloration. Anthocyanins are red when acidic, colorless at pH 4.0, and purple above pH 4.5. Under alkaline conditions, a blue color can be produced. The causes of blue color in some cultivars are not known. The blue color of other plant tissues has been attributed to anthocyanin complexes with alkaline metals or to copigmentation in anthocyanin-flavonoid complexes (Mullins et al. 1992).

The expression of the genes involved in anthocyanin biosynthesis is induced by light in seedlings and cell cultures, but the effect of light on the transcriptional activity of the anthocyanin pathway in berry skins is yet to be determined (reviewed by Boss and Davies 2009). Interestingly, light induces an alteration of anthocyanin distribution within vacuolar compartments (Irani and Grotewold 2005). Moreover, the temperature influences anthocyanin accumulation in grape berries, with higher temperatures generally decreasing total anthocyanin levels (Boss and Davies 2009).

While the biosynthesis and regulation of anthocyanins has been extensively described, little is known concerning their sequestration in the vacuole and to what extent their color is affected by storage (Irani and Grotewold 2005). Recently, and due to the sequencing of the grapevine genome (Jaillon et al. 2007), two grapevine proteins belonging to the multidrug and toxic extrusion (MATE) family, anthoMATE1 (AM1) and anthoMATE3 (AM3), have been implicated in the mediated transport of specifically acylated anthocyanin (Gomez et al. 2009). Vacuolar sequestration of anthocyanins is an important process for cell survival because anthocyanins are believed to be toxic (reviewed by Boss and Davies 2009).

Vacuolar compartmentation of aroma compounds.

Several hundred volatile compounds have been identified in ripe grapes. Aroma compounds are mostly accumulated in the exocarp (skin) tissue, as previously stated. However, some volatile compounds accumulate differentially between the exocarp and the mesocarp (Luan and Wüst 2002). The final mixture of secondary metabolites in ripe grapes depends on multiple variables, including the grape variety used, the environmental conditions during the growing season, and the management of the vineyard and harvest date (reviewed by Dunlevy et al. 2009). The major groups of aroma and flavor compounds produced in grapes are terpenoids, norisoprenoids (mainly C₁₃-norisoprenoids), aromatics and aliphatics, and also organo-sulfur compounds (with a thiol function) and methoxypyrazines (Table 2).

Most of the literature on grape berry aroma compounds does not specify the vacuole as the main storage compartment for such compounds. Instead, it has been speculated that, after veraison, the plastids that lose their chlorophyll are the site of terpenoid and norisoprenoid synthesis and storage, but in other plants, such as in Pinus species, terpenoid synthesis is carried out by the endoplasmic reticulum (De 2000). Nevertheless, these secondary metabolites are frequently accumulated as glycosides (De 2000, Terrier et al. 2009, Dunlevy et al. 2009), and a number of glycosides also exist in vacuolar sap. In addition to their general storage function, the vacuoles are also involved in the biosynthesis of higher saccharides from mono- or disaccharides and the site of glycosylation and production of various metabolites. Thus, given that glycosylation increases solubility and mobility and facilitates transport and storage processes, the vacuole may act as a reservoir of the glycosylated aroma compounds. Aromas such as terpenes, norisoprenoids, and thiols conjugated with sugars or amino acids are accumulated in the vacuoles of exocarp cells (Lund and Bohlmann 2006). In addition, the S-cysteine conjugates have been shown to accumulate in the vacuoles, where the glutathione moiety is cleaved by a peptidase, which yields a specific cysteine conjugate (Peyrot des Gachons et al. 2002).

There have been many advances in understanding how the tonoplast machinery promotes the passage of and accumulates in the vacuole lumen such a variety of compounds, including ions, water, sugars, organic acids, phenolics, aromas, alkaloids, enzyme inhibitors, and toxins, and yet excludes other compounds. This area of research is still in the early stages.