Sugar uptake by the grape berry: A note on the absorption pathway

Summary ³H-glucose was fed to excised Sultana grape berries via their pedicels for up to 5 hours. Autoradigraphy showed that the label was distributed throughout the fruit within 1 hour. Microautoradiography of tissue sections taken at a number of points showed that within the pedicel the walls of cortical cells had become heavily labelled, suggesting that the cortical cell walls offered a diffusion pathway for the solutes entering the vascular system from the external aqueous solution. Transport along the pedicel was confined to the central vascular tissue with little radioactivity occurring in the cortical cells. Within the pericarp, the vascular bundles and walls of nearby parenchyma cells had become heavily labelled, indicating that the labelled solute was present within the vicinity of cell walls. The general pattern of ³H-glucose accumulation by excised berries was similar to the deposition pattern of ²⁴C-labelled photosynthate within attached fruit.     

Functional xylem in the post-veraison grape berry

A number of studies have shown a transition from a primarily xylem to a primarily phloem flow of water as fleshy fruits develop, and the current hypothesis to explain this transition, particularly in grape (Vitis vinifera L.) berries, is that the vascular tissue (tracheids) become non-functional as a result of post-veraison berry growth. In most studies, pedicels have been dipped in a vial containing an apoplastic dye, which was taken up into the entire peripheral and axial xylem vasculature of pre-veraison, but not post-veraison berries. The pressure plate/pressure membrane apparatus that is commonly used to study soil moisture characteristics was adapted and the pre- to post-veraison change in xylem functionality in grape berries was re-evaluated by establishing a hydrostatic (tension) gradient between the pedicel and a cut surface at the stylar end of the berry. Under the influence of this applied hydrostatic gradient, movement of the apoplastic tracer dye, basic fuchsin, was found in the pedicel and throughout the axial and peripheral xylem of the berry mesocarp. A similar movement of dye could be obtained by simply adjoining the stylar cut surface to a dry, hydrophilic wicking material. Since both pre- and post-veraison berries hydrate when the pedicel is dipped in water, it is hypothesized that the absence of dye movement into the vasculature of post-veraison berries indicates not a loss of xylem function, but rather the loss of an appropriate driving force (hydrostatic gradient) in the berry apoplast. Based on this hypothesis, and the substantial decrease in xylem flows that occur in intact grape berries at veraison, it is suggested that there may be significant changes in the pattern of solute partitioning between the fruit symplast and apoplast at veraison. It is further suggested that diurnal patterns in symplast/apoplast solute partitioning in grapes and other fleshy fruit, may explain the observed minimal xylem contribution to the water budgets of these fruits.     

Developmental changes in the diurnal water budget of the grape berry exposed to water deficits

The diurnal water budget of developing grape (Vitis vinifera L.) berries was evaluated before and after the onset of fruit ripening (veraison). The diameter of individual berries of potted ‘Zinfandel’ and ‘Cabernet Sauvignon’ grapevines was measured continuously with electronic displacement transducers over 24 h periods under controlled environmental conditions, and leaf water status was determined by the pressure chamber technique. For well-watered vines, daytime contraction was much less during ripening (after veraison) than before ripening. Daytime contraction was reduced by restricting berry or shoot transpiration, with the larger effect being shoot transpiration pre-veraison and berry transpiration post-veraison. The contributions of the pedicel xylem and phloem as well as berry transpiration to the net diurnal water budget of the fruit were estimated by eliminating phloem or phloem and xylem pathways. Berry transpiration was significant and comprised the bulk of water outflow for the berry both before and after veraison. A nearly exclusive role for the xylem in water transport into the berry was evident during pre-veraison development, but the phloem was clearly dominant in the post-veraison water budget. Daytime contraction was very sensitive to plant water status before veraison but was remarkably insensitive to changes in plant water status after veraison. This transition is attributed to an increased phloem inflow and a partial discontinuity in berry xylem during ripening.     

The peripheral xylem of grapevine (Vitis vinifera). 1. Structural integrity in post-veraison berries

During the development of many fleshy fruits, water flow becomes progressively more phloemic and less xylemic. In grape (Vitis vinifera L.), the current hypothesis to explain this change is that the tracheary elements of the peripheral xylem break as a result of berry growth, rendering the xylem structurally discontinuous and hence non-functional. Recent work, however, has shown via apoplastic dye movement through the xylem of post-veraison berries that the xylem should remain structurally intact throughout berry development. To corroborate this, peripheral xylem structure in developing Chardonnay berries was investigated via maceration and plastic sectioning. Macerations revealed that, contrary to current belief, the xylem was comprised mostly of vessels with few tracheids. In cross-section, the tracheary elements of the vascular bundles formed almost parallel radial files, with later formed elements toward the epidermis and earlier formed elements toward the centre of the berry. Most tracheary elements remained intact throughout berry maturation, consistent with recent reports of vascular dye movement in post-veraison berries.     

Sugar demand of ripening grape berries leads to recycling of surplus phloem water via the xylem

We tested the common assumption that fleshy fruits become dependent on phloem water supply because xylem inflow declines at the onset of ripening. Using two distinct grape genotypes exposed to drought stress, we found that a sink‐driven rise in phloem inflow at the beginning of ripening was sufficient to reverse drought‐induced berry shrinkage. Rewatering accelerated berry growth and sugar accumulation concurrently with leaf photosynthetic recovery. Interrupting phloem flow through the peduncle prevented the increase in berry growth after rewatering, but interrupting xylem flow did not. Nevertheless, xylem flow in ripening berries, but not berry size, remained responsive to root or shoot pressurization. A mass balance analysis on ripening berries sampled in the field suggested that phloem water inflow may exceed growth and transpiration water demands. Collecting apoplastic sap from ripening berries showed that osmotic pressure increased at distinct rates in berry vacuoles and apoplast. Our results indicate that the decrease in xylem inflow at the onset of ripening may be a consequence of the sink‐driven increase in phloem inflow. We propose a conceptual model in which surplus phloem water bypasses the fruit cells and partly evaporates from the berry surface and partly moves apoplastically to the xylem for outflow.     

Xylem, Phloem and Transpiration Flows in a Grape: Application of a Technique for Measuring the Volume of Attached Fruits to High Resolution Using Archimedes' Principle

The minute changes in volume of a grape berry which occur fromhour to hour were measured non-destructively in the field usingreadily available and cheap laboratory equipment and a modernelectronic balance. The method, applicable even to small (approximately10 g) fruits, is based on Archimedes' principle and gave a resolutionof about 1 part in 1 000 by measuring the buoyant upthrust experiencedby a berry when immersed in water. Volume data from control,pedicel-steamed, and detached berries were used to calculatethe magnitudes and directions of the fluid flows which tookplace through the stalk of the phloem and xylem streams andthrough the skin in the transpiration stream. In the latter stages of fruit development, after the onset ofripening, net volume growth more or less ceases in grapes althoughtheir rate of sugar import is at its strongest. Cessation ofvolume growth comes about because the strong inflow of sugarywater in the phloem is closely balanced in part by transpirationalwater loss through the skin and in part by the backflow of xylemwater to the parent vine. This xylem backflow appears to persistthroughout the diurnal cycle. The net backflow direction of the xylem stream, together withthe inability of the phloem stream to carry certain ions (notablycalcium), may explain how some mineral imbalance disorders arisein the later stages of fruit development. The intense manner in which fruiting sinks compete with vegetativesinks in Vitis finds its explanation in the breakdown of apoplast:symplast compartmentation in the berry which occurs around thetime of onset of ripening. The breakdown exposes the terminalsieve tubes of the berry to a highly negative water potentialenvironment, serving to increase both the speed and the concentrationof the translocation stream. Key words: Archimedes' principle, volume measurement, mineral nutrition, xylem, phloem, assimilate partitioning, fruit splitting     

Evidence for substantial maintenance of membrane integrity and cell viability in normally developing grape (Vitis vinifera L.) berries throughout development

Fluorescein diacetate (FDA) was used as a vital stain to assaymembrane integrity (cell viability) in mesocarp tissue of thedeveloping grape (Vitis vinifera L.) berry in order to testthe hypothesis that there is a substantial loss of compartmentationin these cells during ripening. This technique was also usedto determine whether loss of viability was associated with symptomsof a ripening disorder known as berry shrivel. FDA fluorescenceof berry cells was rapid, bright, and stable for over 1 h atroom temperature. Confocal microscopy detected FDA stainingthrough two to three intact surface cell layers (300–400µm) of bisected berries, and showed that the fluorescencewas confined to the cytoplasm, indicating the maintenance ofintegrity in both cytoplasmic as well as vacuolar membranes,and the presence of active cytoplasmic esterases. FDA clearlydiscriminated between living cells and freeze-killed cells,and exhibited little, if any, non-specific staining. Propidiumiodide and DAPI, both widely used to assess cell viability,were unable to discriminate between living and freeze-killedcells, and did not specifically stain the nuclei of dead cells.For normally developing berries under field conditions therewas no evidence of viability loss until about 40 d after veraison,and the majority (80%) of mesocarp cells remained viable pastcommercial harvest (26 °Brix). These results are inconsistentwith current models of grape berry development which hypothesizethat veraison is associated with a general loss of compartmentationin mesocarp cells. The observed viability loss was primarilyin the locule area around the seeds, suggesting that a localizedloss of viability and compartmentation may occur as part ofnormal fruit development. The cell viability of berry shrivel-affectedberries was similar to that of normally developing berries untilthe onset of visible symptoms (i.e. shrivelling), at which timeviability declined in visibly shrivelled berries. Berries withextensive shrivelling exhibited very low cell viability (15%). Key words: Apoplast, berry shrivel, compartmentation, DAPI, FDA, fluorescence, fruit ripening, locule, propidium iodide Received 19 September 2007; Revised 16 December 2007 Accepted 26 December 2007     

Loss of rachis cell viability is associated with ripening disorders in grapes

Rachises of grape (Vitis vinifera L.) clusters that appeared healthy or displayed symptoms of the ripening disorders berry shrivel (BS) or bunch-stem necrosis (BSN) were treated with the cellular viability stain fluorescein diacetate and examined by confocal microscopy. Clusters with BS and BSN symptoms experienced a decrease of cell viability throughout the rachis, and their berries contained 70-80% less sugar than healthy berries. The xylem-mobile dye basic fuchsin, infiltrated via the cut base of shoots with one healthy and one BS cluster, moved to all berries on the healthy cluster but generally failed to move into the peduncle of the BS cluster. Peduncle girdling did not interrupt dye movement in the xylem, but stopped solute accumulation in berries and led to berry shrinkage. In contrast, surgically destroying the peduncle xylem at the onset of ripening did not affect berry growth and solute accumulation. These results indicate that cessation of sugar and water accumulation in BS and BSN is associated with phloem death in the rachis. Although xylem flow to the berries may also cease, a functional xylem connection to the shoot may not be required for normal ripening, while water loss from berries by transpiration and xylem efflux may explain the characteristic berry shrinkage that is associated with these ripening disorders. The similarity of internal tissue breakdown in BS and BSN rachises and the correlation observed here between the proportion of shrinking berries on a cluster and the severity of rachis necrosis suggest that there may be a gradual transition between the two ripening disorders. Seeds from healthy and BS clusters showed no differences in colour, morphology, weight, viability, and ability to germinate, which indicates that the disorder may not appear until seeds are mature.     

Ripening grape berries remain hydraulically connected to the shoot

Berry diameter was monitored during dry-down and rewatering cycles and pressurization of the root system of Vitis vinifera (cv. Merlot) and Vitis labruscana (cv. Concord) to test changes in xylem functionality during grape ripening. Prior to veraison (onset of ripening), berries maintained their size under declining soil moisture until the plants had used 80% of the transpirable soil water, began to shrink thereafter, and recovered rapidly after rewatering. By contrast, berry diameter declined slowly but steadily during post-veraison water stress and did not recover after rewatering; irrigation merely prevented further shrinking. Preconditioning vines with a period of water stress after flowering did not influence the berries' reaction to subsequent changes in transpirable soil water. Pressurizing the root system led to concomitant changes in berry diameter only prior to veraison, although some post-veraison Concord, but not Merlot, berries cracked under root pressurization. The xylem-mobile dye basic fuchsin, infused via the shoot base, moved throughout the berry vasculature before veraison, but became gradually confined to the brush area during ripening. When the dye was infused through the stylar end of attached berries, it readily moved back to the plant both before and after veraison. Our work demonstrated that berry-xylem conduits retain their capacity for water and solute transport during ripening. It is proposed here that apoplastic phloem unloading coupled with solute accumulation in the berry apoplast may be responsible for the decline in xylem water influx into ripening grape berries. Instead, the xylem may serve to recycle excess phloem water back to the shoot.     

The peripheral xylem of grapevine (Vitis vinifera) berries. 2. Anatomy and development

It has been hypothesized that the substantial reductions in xylemic water flow occurring at veraison are due to physical disruption (breaking) of the xylem as a result of renewed berry growth. In a companion paper, evidence was presented that the vast majority of xylem tracheary elements remained intact despite the growth of the berry, and it was proposed that existing tracheary elements stretch to accommodate growth and that additional elements may also differentiate after veraison. Measurements of the intergyre distance of tracheary elements in macerated tissue were used to test for stretching, and the numbers of tracheary elements per vascular bundle and of branch points of the peripheral xylem network were analysed to test for continued differentiation from 18 to 120 d after anthesis in Chardonnay berries. The distance between the epidermis and the vasculature increased substantially from pre- to post-veraison, potentially increasing the amount of skin available for analysis of compounds important for winemaking. Tracheary elements continued to differentiate within the existing vascular bundles throughout berry development. Additional vascular bundles also appeared until after veraison, thereby increasing the complexity of the peripheral vascular network. The results also confirmed that tracheary elements stretched by approximately 20%, but this was not as much as that predicted based on the growth of the vascular diameter (40%). These results complete a comprehensive evaluation of grape berry peripheral xylem during its development and show that tracheary development continues further into berry maturation than previously thought.     

Fruit ripening in Vitis vinifera: spatiotemporal relationships among turgor, sugar accumulation, and anthocyanin biosynthesis

This study reports the first observations indicating the spatiotemporal relationships among genetic and physiological aspects of ripening in the berry of Vitis vinifera. At the onset of ripening in the red flesh variety Alicante Bouschet, colour development began in the flesh at the stylar end of the fruit and progressed toward the pedicel end flesh and into the skin. Tissue solute potential and cell turgor also decreased first in the flesh. The decrease in flesh solute potential was due to accumulation of sugars, glucose and fructose, an accumulation that is integral to ripening. Expression of the anthocyanin biosynthesis-related genes VvMybA and VvUFGT was linearly related to the decrease in solute potential. Expression of VvMybA, and to a lesser extent VvUFGT, was correspondingly low in green tissue, higher in the red, stylar end flesh of berries beginning to ripen, and greatest in red berries. In contrast, expression of the abscisic acid biosynthesis-related genes VvNCED1 and VvNCED2 was not correlated with the other spatiotemporal aspects of the onset of ripening. These results, together with earlier work showing that sugar accumulation and acid loss also begin in the stylar flesh in other varieties, indicate that ripening in the grape berry originates in the stylar end flesh.     

Functional Characterization of Two Ripening-related Sucrose Transporters from Grape Berries

The ripening of grape (Vitis vinifera L.) berries is associatedwith a large accumulation of glucose and fructose in the vacuolesof the fruit cells. These hexoses are derived from sucrose,which is released from the phloem and may be taken up by parenchymacells prior to hydrolysis. We have expressed two putative ripening-relatedsucrose transporters from grape berries, VvSUC11 (synonymouswith VvSUT1) and VvSUC12, in an invertase deficient yeast strainto characterize their transport activities. Sucrose was takenup by yeast transformed with either transporter at an optimumpH of <4.5 and with a Michaelis constant (K_m) of 0.9–1.4m M. The uptake of sucrose through VvSUC11 and VvSUC12 was inhibitedby protonophores and by vanadate. This is consistent with anactive uptake mechanism involving proton cotransport, typicalof sucrose/H⁺symporters. The transporters from grape berrieswere functionally similar to Scr1, a sucrose transporter fromRicinus cotyledons. It is likely that in grape berries VvSUC11and VvSUC12 facilitate the loading of sucrose from the apoplastinto the parenchyma cells. Copyright 2001 Annals of Botany Company Fruit, grape berries, plasma membrane, sugars, sucrose transporters, Vitis vinifera     

Partitioning Control by Water Potential Gradient: Evidence for Compartmentation Breakdown in Grape Berries

Xylem exudate was obtained from berries of Riesling grapes atdifferent stages of development after the onset of ripeningusing a pressure bomb technique. The osmotic potential of theexudate bore a 1:1 relationship to that of juice from the sameberries which were afterwards crushed and centrifuged. Thisresult provides the first direct evidence of compartmentationbreakdown in grape berries after the onset of ripening. Changesin berry deformability which occur at the same time and measurementsof the dynamics of exudation flow lead to the same conclusionregarding compartmentation breakdown. The breakdown in compartmentation occurs at the same time asthe rate of phloem translocation to the fruit suddenly increases.A mechanism was recently proposed to account for this increase.It required the existence of a water potential difference betweensource and sink such as would result from compartmentation breakdownin the sink tissues. The results, therefore, may be taken toindicate that this mechanism is indeed involved in the controlof assimilate partitioning in Vitis. Evidence in other publicationssuggests that the mechanism may be reasonably widespread inplants. Key words: Assimilate partitioning, phloem translocation mechanism, Vitis vinifera L., water potential gradients     

Sugar accumulation in grape berries. Cloning of two putative vacuolar invertase cDNAs and their expression in grapevine tissues.

During grape berry (Vitis vinifera L.) ripening, sucrose transported from the leaves is accumulated in the berry vacuoles as glucose and fructose. To study the involvement of invertase in grape berry ripening, we have cloned two cDNAs (GIN1 and GIN2) from berries. The cDNAs encode translation products that are 62% identical to each other and both appear to be vacuolar forms of invertase. Both genes are expressed in a variety of tissues, including berries, leaves, roots, seeds, and flowers, but the two genes have distinct patterns of expression. In grape berries, hexose accumulation began 8 weeks postflowering and continued until the fruit was ripe at 16 weeks. Invertase activity increased from flowering, was maximal 8 weeks postflowering, and remained constant on a per berry basis throughout ripening. Expression of GIN1 and GIN2 in berries, which was high early in berry development, declined greatly at the commencement of hexose accumulation. The results suggest that although vacuolar invertases are involved in hexose accumulation in grape berries, the expression of the genes and the synthesis of the enzymes precedes the onset of hexose accumulation by some weeks, so other mechanisms must be involved in regulating this process.     

An in vivo experimental system to study sugar phloem unloading in ripening grape berries during water deficiency stress

An in vivo experimental system-called the 'berry-cup' technique-was developed to study sugar phloem unloading and the accumulation of sugar in ripening grape berries. The berry-cup system consists of a single peeled grape berry immersed in a buffer solution in a cup prepared from a polypropylene syringe. A small cross-incision (2 mm in length) is made on the stylar remnant of a berry during its ripening phase, the skin of the berry then being easily peeled off, exposing the dorsal vascular bundles without damaging either these or the pulp tissue of the berry. The sites of sugar phloem unloading are thus made directly accessible and may be regulated by the buffer solution. In addition, the unloaded photoassimilates are easily transported into the buffer solution in the berry-cup. With the berry-cup technique, it takes 60 min to purge the sugar already present in the apoplast, after which the amount of sugar in the buffer solution is a direct measure of the sugar unloading from the grape berry phloem. The optimum times for sampling were 20 or 30 min, depending on the type of experiment. Sugar phloem unloading was significantly inhibited by the inclusion of either 7.5 mm NaF or 2.5 mm PCMB in the buffer solution. This study indicates that sugar phloem unloading in ripening grape berries is via the apoplastic network and that the process requires the input of energy. The system was shown to be an appropriate experimental system with which to study sugar phloem unloading in ripening grape berries, and was applied successfully to the study of berry sugar unloaded from grapevines subjected to water stress. The results showed that water deficiency inhibits sugar unloading in grape berries.     

Solute Accumulation by Grape Pericarp Cells: V. RELATIONSHIP TO BERRY SIZE AND THE EFFECTS OF DEFOLIATION

We have studied the accumulation of water, dry matter (DM) andglucose and fructose (G + F) in selected grape berries cv. Dolcettoof varying initial size growing on leafed and defoliated vines.The first measurements at 2 d after veraison were obtained non-destructivelyfrom correlations with linear dimensions and deformability;the final measurements were made when the berries were harvested13 d later. The increments in DM and G + F per pericarp increased with initialberry size thus discounting an hypothesis of an equal amountof solutes supplied to all berries. The increments in weightof DM and G + F per increment of pericarp volume were constantin berries of different size, supporting an hypothesis of acontrol determined by concentration in the solution availablefor accumulation in all berries. Defoliation reduced the incrementsper pericarp and per g fr. wt. by about the same proportionand its effects were consistent with the above interpretations. Key words: Grape pericarp, sugar accumulation, phloem unloading, Vitis vinifera.     

Differential incorporation of 1-deoxy-D-xylulose into (3S)-linalool and geraniol in grape berry exocarp and mesocarp

In vivo feeding experiments with [5,5-(2)H(2)]mevalonic acid lactone (MVL) and [5,5-(2)H(2)]-1-deoxy-D-xylulose (DOX) indicate that the novel mevalonate-independent 1-deoxy- D-xylulose 5-phosphate/2C-methyl- D-erythritol 4-phosphate (DOXP/MEP) pathway is the dominant metabolic route for monoterpene biosynthesis in grape berry exocarp and mesocarp and in grape leaves. The highly uneven distribution of the monoterpene alcohols (3S)-linalool and geraniol between leaves, berry exocarp and berry mesocarp can be attributed to a compartmentation of monoterpene metabolism. In grape berries incorporation of [5,5-(2)H(2)]-DOX into geraniol is mainly restricted to the exocarp, whereas (3S)-linalool biosynthesis can be detected in exocarp as well as in mesocarp tissue. The results demonstrate that grape berries exhibit an autonomic monoterpene biosynthesis via the novel DOXP/MEP route throughout the ripening process.     

Proanthocyanidin synthesis and expression of genes encoding leucoanthocyanidin reductase and anthocyanidin reductase in developing grape berries and grapevine leaves

Proanthocyanidins (PAs), also called condensed tannins, can protect plants against herbivores and are important quality components of many fruits. Two enzymes, leucoanthocyanidin reductase (LAR) and anthocyanidin reductase (ANR), can produce the flavan-3-ol monomers required for formation of PA polymers. We isolated and functionally characterized genes encoding both enzymes from grapevine (Vitis vinifera L. cv Shiraz). ANR was encoded by a single gene, but we found two highly related genes encoding LAR. We measured PA content and expression of genes encoding ANR, LAR, and leucoanthocyanidin dioxygenase in grape berries during development and in grapevine leaves, which accumulated PA throughout leaf expansion. Grape flowers had high levels of PA, and accumulation continued in skin and seeds from fruit set until the onset of ripening. VvANR was expressed throughout early flower and berry development, with expression increasing after fertilization. It was expressed in berry skin and seeds until the onset of ripening, and in expanding leaves. The genes encoding LAR were expressed in developing fruit, particularly in seeds, but had low expression in leaves. The two LAR genes had different patterns of expression in skin and seeds. During grape ripening, PA levels decreased in both skin and seeds, and expression of genes encoding ANR and LAR were no longer detected. The results indicate that PA accumulation occurs early in grape development and is completed when ripening starts. Both ANR and LAR contribute to PA synthesis in fruit, and the tissue and temporal-specific regulation of the genes encoding ANR and LAR determines PA accumulation and composition during grape berry development.