Physiological Basis Of Sugar Accumulation

The ripening grape berry is a strong sink for dry matter transported from current photosynthesis and wood reserves (Coombe 1989). Sucrose derived from leaf photosynthesis is exported via the phloem to the berries. From verai-son and throughout ripening the berries accumulate roughly equal amounts of glucose and fructose, reaching over 1M of each hexose (Coombe 1987). This implies that phloem transported sucrose is hydrolyzed at some step during its transport from the sieve tube to the vacuole of the mesocarp cell (Fig. 1). The remarkable sink strength of the berry is well illustrated by the fact that its dry mass increases four-fold during a 6 week-period, with little change observed in the dry mass of other plant parts (Conradie 1980).

In situ measurements carried out at different times on the same berry showed that glucose and fructose accumulation begins suddenly on the same day as berry softening begins. It seems that, once begun, sugar accumulation in grapes is undeviating and massive. The limitation to sink strength within individual berries is set by sink activity, not berry size. Indeed, after it is triggered, the accumulation of hexoses is linear with time, although the increase in berry volume proceeds at a variable rate (Coombe 1989).

The fleshy portion of the berry originates from the ovary wall that develops to form the pericarp, mesocarp and endocarp. The developing berry is fed with assimilates transported through the carpellary vascular bundles which are divided into the peripheral and central bundles. A dorsal bundle network extends at the periphery of the fruit, and central vascular bundles are connected to the seeds and irrigate the central flesh. A detailed analysis of the unloading pathway in the ripening berry was conducted in a hybrid grape (Vitis vinifera x Vitis labrusca; Zhang et al. 2006). A variety of techniques, including electron microscopy, determination of enzymatic activities, and transport studies with carboxy fluorescein (a symplastic tracer) and with companion cell expressed and tagged viral movement proteins, were used. Until veraison, both carboxy-fluorescein and the tagged viral movement protein could be released from the functional phloem strands, whereas at the late stage of ripening they remained confined in the strands.

This shift from a symplasmic to an apoplasmic unloading pathway, which occurs at or just prior the onset of ripening, is accompanied by a concomitant increase of the expression and activity of cell wall invertases (cwlNV) (Zhang et al. 2006). As a result, apoplastic sugar concentration and osmotic pressure in crease, which may enhance sugar uptake via stimulation of the proton-pumping ATPase activity (Li and Delrot 1987). Interestingly, a loss of symplastic connections also occurs at the inception of tomato fruit ripening (Ruan and Patrick 1995), with cwINV activity and sugar uptake increasing throughout ripening (Baxter et al. 2005).

Sugar Transport Plants

Fig. 1. Simplified scheme of long-distance sugar transport in plants. (A) Pathways for phloem loading: Sucrose (S) is synthesized in mesophyll cells through photosynthesis. S is loaded into the sieve elements/companion cell complex (SE/CC) via the apoplast. Apoplastic loading involves the retrieval of S leaking from the mesophyll or the vascular parenchyma (mechanism yet uncertain) and may occur along the phloem path. Hydrostatic pressure drives phloem sap movement toward sink tissue. (B) Pathways of phloem unloading: S enters the receiving cell by the symplastic route before véraison, using plasmodesmata, or the apoplastic pathway after véraison. The latter predominates in the ripening fruit and requires the activity of membrane transporters mediating the transport of S, and of the hexoses (Hx) resulting from S hydrolysis by metabolic enzymes (invertases, sucrose synthases). Hx are accumulated in the vacuole. Water fluxes respond to sugar concentration gradient. (1) S/H+ symporter; (2) Hx/H+ symporter; (3) S/H+ antiporter; (4) Hx/H+ antiporter; (5) S efflux transporter; *^invertase;^>'sucrose synthase; 1—x water flux.

Fig. 1. Simplified scheme of long-distance sugar transport in plants. (A) Pathways for phloem loading: Sucrose (S) is synthesized in mesophyll cells through photosynthesis. S is loaded into the sieve elements/companion cell complex (SE/CC) via the apoplast. Apoplastic loading involves the retrieval of S leaking from the mesophyll or the vascular parenchyma (mechanism yet uncertain) and may occur along the phloem path. Hydrostatic pressure drives phloem sap movement toward sink tissue. (B) Pathways of phloem unloading: S enters the receiving cell by the symplastic route before véraison, using plasmodesmata, or the apoplastic pathway after véraison. The latter predominates in the ripening fruit and requires the activity of membrane transporters mediating the transport of S, and of the hexoses (Hx) resulting from S hydrolysis by metabolic enzymes (invertases, sucrose synthases). Hx are accumulated in the vacuole. Water fluxes respond to sugar concentration gradient. (1) S/H+ symporter; (2) Hx/H+ symporter; (3) S/H+ antiporter; (4) Hx/H+ antiporter; (5) S efflux transporter; *^invertase;^>'sucrose synthase; 1—x water flux.

In grape berries, the analysis of different zones of the skin and flesh showed that the sugar concentrations are heterogeneous in both tissues (Coombe 1987). Concentrations in skin are generally lower than those present in the flesh tissue. Furthermore, there are longitudinal differences in the levels of glucose and fructose in the outer and central flesh tissues. Hexose concentrations in the flesh increase from low near the brush to high at the stylar end. Sucrose concentration in the flesh is low throughout ripening, but increases in the skin when the berries reach 17 -26° Brix and in the central tissues (including the brush) in overripe berries. Uptake and efflux studies with isolated skin suggested active uptake of D-glucose but not L-glucose. Surprisingly, active uptake of monosaccharides was measured in skin samples from both green unripe and ripe berries, whereas it was evident only in flesh tissues sampled from ripening berries (Coombe and Matile 1980). However, contrarily to what is observed in mesocarp tissues, a high proportion of the sugar absorbed by skin pieces is diffusive, whatever the sampling time.

The limiting steps and the molecular mechanisms leading to final trapping of high concentrations of hexoses in the vacuole of mesocarp cells are still elusive. The compartmentation and enzymatic events leading from sucrose in the sieve tubes to hexoses in mesocarp cell vacuoles involve (a) sucrose efflux from the phloem conducting complex, (b) sugar entry into the flesh cells, and (c) sugar uptake into the vacuoles of these cells. Sucrose may be cleaved in the cell wall prior to its entry into the mesocarp cells, or after compartmentation into the vacuoles. The possibility of sucrose breakdown/resynthesis and final breakdown, often referred to as sucrose futile cycle, should also be considered.

Together with sugar transporters, the sucrose metabolic enzymes invertases (INVs) and sucrose synthases (SuSys) participate in the maintenance of the sucrose gradient needed to sustain mass flow of the phloem sap. Furthermore, as they modulate the pool of available sugars, these enzymes may certainly play important roles in the context of sugar signalling (Delrot 1994, Roitsch et al. 1995, Sherson et al. 2003, Koch 2004). INVs are hydrolases cleaving sucrose into glucose and fructose. In tomato and Arabidopsis, INVs are encoded by small gene families with various expression patterns regarding the organ and the environmental and/or metabolic stimuli (Godt and Roitsch 1997, Sherson et al. 2003). These enzymes are either acidic or neutral.

Acidic INVs, such as cwINV and vINV, are localized in the cell wall and in the vacuole, respectively, while the nINV, a neutral form, is present in the cytoplasm. In grape, the cDNA sequence of a cwINV (AY538262) and the promoter region of the gene (EF122148), the complete cDNA sequence of a nINV (NIN1, EU016365), as well as 3 incomplete genomic sequences, and 2 vINVs cDNAs (VvGINl AAB47171.1 and VvGIN2 AAB47172.1) have been cloned (Davies and Robinson 1996, Hayes et al. 2007). Based on protein motif analysis (pfam, Interpro), 10 to 12 encoding putative nINVs and 10 encoding putative acidic INVs genes were found in the grape genome sequence (http:// www.genoscope.cns.fr/spip/Vitis-vinifera-whole-genome.html). Although INV activities were considered to be non-limiting (Davies and Robinson, 1996), re cent reports by Zhang et al. (2006) and Hayes et al. (2007) clearly indicate that expression and activity of cwlNV is induced just prior to veraison.

However, although the co-regulation of cwlNV and some monosaccharide transporters in sink tissues has been confirmed in several plant species and organs, including young grape leaves, the same could not be demonstrated in grape berries (Fotopoulos et al. 2003, Weschke et al. 2003, Roitsch and Gonzalez 2004, Baxter et al. 2005, Hayes et al. 2007). Furthermore, cwlNV enzyme activity in berry represents only 4% of total INV activity (Ruffner et al. 1990, Davies and Robinson, 1996) and recent microarray results showed a constant level of both cwINV and nINV mRNAs throughout berry development (Deluc et al. 2007). This suggests that cwINV alone cannot be responsible for the increase of monosaccharide concentration in the ripening berry. Acidic vINV activity, although participating in sink activity and strength, as the major sucrolytic activity in grape berry, occurs too early to trigger by itself hexose accumulation at ripening inception (Ruffner et al. 1990, Davies and Robinson 1996, Patrick 1997, Dreier et al. 1998). VvGIN1 and VvGIN2 transcripts and protein levels accumulate before veraison and decrease during fruit ripening (Davies and Robinson, 1996, Sarry et al. 2004, Deluc et al. 2007). However, vINV activity is important to drive the import of sugars along ripening, since its natural reduction in the Steuben grapevine hybrid contributes to an increase of the relative proportion of sucrose in the maturing berry (Takayanagi and Yokotsuka 1997). Likewise, vINV activity in tomato fruit is crucial to determine both the levels and the nature of accumulated sugars in the vacuole (Klann et al. 1993, Ohyama et al. 1995, Nguyen-Quoc and Foyer 2001).

SuSys are cytosolic glycosyl transferases which, in the presence of UDP, convert sucrose into UDP-glucose (UDP-G) and fructose. In vivo, SuSys are also able to catalyse the reverse reaction, but with lower efficiency (Geigenberger and Stitt 1993). Unlike INVs, SuSy enzymes are active and transcriptionally upregu-lated under low oxygen conditions. Interestingly, SuSys that respond to low oxygen are also highly expressed under carbohydrate depletion (Zeng et al. 1999, Koch et al. 2000). In addition, SuSy activity is known to be involved in key metabolic processes such as storage, defence and cell wall synthesis since its UDP-G product is implicated in the formation of callose and in the synthesis of several cell-wall polysaccharides (Albrecht and Mustroph 2003).

In tomato, SuSy but not INV, is involved in sugar unloading and metabolism at the beginning of fruit development (Dali et al. 1992, D'Aoust et al. 1999, N'tchobo et al. 1999). Later in maturation, SuSy activity is strongly reduced but sucrose unloading rates, although low, are maintained, and might be driven by both sugar uptake and INV activities (D'Aoust et al. 1999, Nguyen-Quoc and Foyer 2001). In grape, 6 to 9 genes encoding putative SuSys are expected based on recent genome sequencing. However, SuSys activities do not vary much along ripening and are not conclusively correlated with the modifica tions on the soluble sugar concentration (Zhang et al. 2006). For instance, the expression of a SuSy gene homologue to CiTSUA (1609402_at, TC62599, http://www.plexdb.org/modules/PD_browse/experiment_browser.php? plex_na-me=GrapePLEX) increases gradually along berry development. However, whether the subsequent enzyme is involved in sugar import or cell wall expansion during berry softening is not clear. Hence, it appears that sugar accumulation in berry, and sink organs in general, would rather result from the coordinate action of several mechanisms, involving various transporters and hydrolytic enzymes (Deluc et al. 2007).

The simultaneous events of sucrose (or starch) synthesis and degradation occurring in plants cells are sometimes referred to as 'futile cycle'. Although SuSy is able to catalyze the synthesis of sucrose, in the cytosol this sugar is mainly synthesized by sucrose phosphate synthase (SPS) (Huber and Huber 1996). This holds true both for photosynthetic tissues and for non-photosynthetic storage organs. Sucrose futile cycle implicates the re-synthesis of sucrose from the hexoses present in the cytosol, its entry in the vacuole and its degradation by vINV. A continuous sugar exchange between the cytosol and the vacuole (sucrose influx, hexose efflux) has also been suggested in tomato pericarp cells (Scholes et al. 1996). Sucrose futile cycles would then favour the unloading and storage of sugars into the ripening fruit which becomes symplastically isolated (Nguyen-Quoc and Foyer 2001). In grape berry, an SPS gene is preferentially expressed in the pericarp, suggesting a probable participation in the re-synthesis of sucrose following its unloading from the phloem into the fruit (Grimplet et al. 2007).

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  • stella
    What is the physiological basis for sink strength in plants?
    6 years ago

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