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.     

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.     

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     

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.     

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     

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.     

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.     

Apoplastic sugar may be lost from grape berries and retrieved in pedicels

In ripening grape (Vitis sp.) berries, the combination of rapid sugar import, apoplastic phloem unloading, and water discharge via the xylem creates a potential risk for apoplastic sugar to be lost from the berries. We investigated the likelihood of such sugar loss and a possible sugar retrieval mechanism in the pedicels of different Vitis genotypes. Infusion of D-glucose-1-¹³C or L-glucose-1-¹³C to the stylar end of attached berries demonstrated that both sugars can be leached from the berries, but only the nontransport sugar L-glucose moved beyond the pedicels. No ¹³C enrichment was found in peduncles and leaves. Genes encoding 10 sugar transporters were expressed in the pedicels throughout grape ripening. Using an immunofluorescence technique, we localized the sucrose transporter SUC27 to pedicel xylem parenchyma cells. These results indicate that pedicels possess the molecular machinery for sugar retrieval from the apoplast. Plasmodesmata were observed between vascular parenchyma cells in pedicels, and movement of the symplastically mobile dye carboxyfluorescein demonstrated that the symplastic connection is physiologically functional. Taken together, the chemical, molecular, and anatomical evidence gathered here supports the idea that some apoplastic sugar can be leached from grape berries and is effectively retrieved in a two-step process in the pedicels. First, sugar transporters may actively retrieve leached sugar from the xylem. Second, retrieved sugar may move symplastically to the pedicel parenchyma for local use or storage, or to the phloem for recycling back to the berry.

Grape berry pedicels may retrieve sugar that is lost via the xylem following apoplastic phloem unloading in the berries.     

Vascular functioning and the water balance of ripening kiwifruit (Actinidia chinensis) berries

Indirect evidence suggests that water supply to fleshy fruits during the final stages of development occurs through the phloem, with the xylem providing little water, or acting as a pathway for water loss back to the plant. This inference was tested by examining the water balance and vascular functioning of ripening kiwifruit berries (Actinidia chinensis var. chinensis 'Hort16A') exhibiting a pre-harvest 'shrivel' disorder in California, and normal development in New Zealand. Dye labelling and mass balance experiments indicated that the xylem and phloem were both functional and contributed approximately equally to the fruit water supply during this stage of development. The modelled fruit water balance was dominated by transpiration, with net water loss under high vapour pressure deficit (D(a)) conditions in California, but a net gain under cooler New Zealand conditions. Direct measurement of pedicel sap flow under controlled conditions confirmed inward flows in both the phloem and xylem under conditions of both low and high D(a). Phloem flows were required for growth, with gradual recovery after a step increase in D(a). Xylem flows alone were unable to support growth, but did supply transpiration and were responsive to D(a)-induced pressure fluctuations. The results suggest that the shrivel disorder was a consequence of a high fruit transpiration rate, and that the perception of complete loss or reversal of inward xylem flows in ripening fruits should be re-examined.     

Field evaluation of water transport in grape berries during water deficits

The net flow in vascular and transpirational components of the grape berry water budget was evaluated during water deficits imposed at different stages of fruit development. Diurnal fluctuations in berry diameter were measured on field-grown grapevines ( Vitis vinifera L. cv. Cabernet Sauvignon) by using electronic displacement transducers. Water deficits were imposed by withholding irrigation, and water potentials of mid-shoot leaves, basal stem xylem and clusters were determined with a pressure chamber. The relative net flows through pedicel xylem and phloem and through berry transpiration were estimated pre-veraison and post-veraison. The xylem functioned nearly exclusively in providing net inflow pre-veraison, while the phloem was clearly dominant post-veraison. Accordingly, the amplitude of diurnal contraction was markedly smaller post-veraison than pre-veraison. The amplitude of diurnal contraction increased dramatically with decreasing plant water status pre-veraison, yet exhibited little sensitivity to low vine water status post-veraison. Measurements of the difference in water potential between clusters and source stems did not provide evidence of a gradient that would elicit significant water movement from the cluster to the stem at any time of the day. This was true for both irrigated and non-irrigated vines, although the non-irrigated vines exhibited a smaller gradient favoring inflow throughout much of the day. The gradient for xylem water transport to the cluster was considerably smaller post-veraison than pre-veraison. The results showed that berry transpiration functioned as the primary pathway for water loss both pre- and post-veraison.     

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.     

Effects of Water-Deficit Irrigation on Hormonal Content and Nitrogen Compounds in Developing Berries of Vitis vinifera L. cv. Tempranillo

Water-deficit irrigation to grapevines reduces plant growth, yield, and berry growth, altering the ripening process, all of which may influence fruit composition and wine quality. Therefore, the goals of this study were (1) to investigate the influence of the main endogenous berry hormones, abscisic acid (ABA), indole-3-acetic acid (IAA), salicylic acid (SA), and jasmonic acid (JA), on berry growth and ripening under water-deficit conditions and (2) to analyze changes in fruit composition, specifically N compounds, under water deprivation. The study was carried out using container-grown Tempranillo grapevines grown under controlled conditions in a greenhouse. Two irrigation treatments were imposed: control (well-watered) and sustained deficit irrigation (SDI). Water deficit decreased leaf area and the source-to-sink ratio, reduced yield and berry size, and decreased concentrations of the main phenolic compounds. SDI also modified berry hormonal status. At the pea-size stage, SDI berries had lower IAA and higher JA and SA than nonstressed berries. At veraison (onset of ripening), accumulation of ABA was less accentuated in SDI than in control berries. At harvest, the content of amino acids and free ammonium was low in both treatments but SDI-treated berries showed a significant accumulation of amines. Results suggest that water restrictions to grapevines might be playing a physiological role in reducing berry growth through affecting hormone dynamics, phenolic synthesis, and the berry amino acid content and composition, which could compromise fruit quality. Possible roles of endogenous IAA controlling berry size and endogenous ABA and SA controlling levels of anthocyanins and flavonols at harvest are discussed.     

A shift of Phloem unloading from symplasmic to apoplasmic pathway is involved in developmental onset of ripening in grape berry

It remains unclear whether the phloem unloading pathway alters to adapt to developmental transition in fleshy fruits that accumulate high level of soluble sugars. Using a combination of electron microscopy, transport of the phloem-mobile symplasmic tracer carboxyfluorescein, movement of the companion cell-expressed and the green fluorescent protein-tagged viral movement protein, and assays of the sucrose cleavage enzymes, the pathway of phloem unloading was studied in the berries of a hybrid grape (Vitis vinifera x Vitis labrusca). Structural investigations showed that the sieve element-companion cell complex is apparently symplasmically connected through plasmodesmata with surrounding parenchyma cells throughout fruit development, though a small portion of plasmodesmata are apparently blocked in the ripening stage. Both carboxyfluorescein and the green fluorescent protein-tagged viral movement protein were released from the functional phloem strands during the early and middle stages of fruit development, whereas the two symplasmic tracers were confined to the phloem strands during the late stage. This reveals a shift of phloem unloading from symplasmic to apoplasmic pathway during fruit development. The turning point of the phloem unloading pathways was further shown to be at or just before onset of ripening, an important developmental checkpoint of grape berry. In addition, the levels of both the expression and activities of cell wall acid invertase increased around the onset of ripening and reached a high level in the late stage, providing further evidence for an operation of the apoplasmic unloading pathway after onset of ripening. These data demonstrate clearly the occurrence of an adaptive shift of phloem unloading pathway to developmental transition from growing phase to ripening in grape berry.     

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.     

ABA and GA3 increase carbon allocation in different organs of grapevine plants by inducing accumulation of non‐structural carbohydrates in leaves,enhancement of phloem area and expression of sugar transporters

Grape quality for winemaking depends on sugar accumulation and metabolism in berries. Abscisic acid (ABA) and gibberellins (GAs) have been reported to control sugar allocation in economically important crops, although the mechanisms involved are still unknown. The present study tested if ABA and gibberellin A₃ (GA₃) enhance carbon allocation in fruits of grapevines by modifying phloem loading, phloem area and expression of sugar transporters in leaves and berries. Pot‐grown Vitis vinifera cv. Malbec plants were sprayed with ABA and GA₃ solutions. The amount of soluble sugars in leaves and berries related to photosynthesis were examined at three points of berry growth: pre‐veraison, full veraison and post‐veraison. Starch levels and amylase activity in leaves, gene expression of sugar transporters in leaves and berries and phloem anatomy were examined at full veraison. Accumulation of glucose and fructose in berries was hastened in ABA‐treated plants at the stage of full veraison, which was correlated with enhancement of Vitis vinifera HEXOSE TRANSPORTER 2 (VvHT2) and Vitis vinifera HEXOSE TRANSPORTER 6 (VvHT6) gene expression, increases of phloem area and sucrose content in leaves. On the other hand, GA₃ increased the quantity of photoassimilates delivered to the stem thus increasing xylem growth. In conclusion, stimulation of sugar transport by ABA and GA₃ to berries and stems, respectively, was due to build‐up of non‐structural carbohydrates in leaves, modifications in phloem tissue and modulation in gene expression of sugar transporters.     

Is desiccation tolerance and avoidance reflected in xylem and phloem anatomy of two coexisting arid‐zone coniferous trees?

Plants close their stomata during drought to avoid excessive water loss, but species differ in respect to the drought severity at which stomata close. The stomatal closure point is related to xylem anatomy and vulnerability to embolism, but it also has implications for phloem transport and possibly phloem anatomy to allow sugar transport at low water potentials. Desiccation‐tolerant plants that close their stomata at severe drought should have smaller xylem conduits and/or fewer and smaller interconduit pits to reduce vulnerability to embolism but more phloem tissue and larger phloem conduits compared with plants that avoid desiccation. These anatomical differences could be expected to increase in response to long‐term reduction in precipitation. To test these hypotheses, we used tridimensional synchroton X‐ray microtomograph and light microscope imaging of combined xylem and phloem tissues of 2 coniferous species: one‐seed juniper (Juniperus monosperma) and piñon pine (Pinus edulis) subjected to precipitation manipulation treatments. These species show different xylem vulnerability to embolism, contrasting desiccation tolerance, and stomatal closure points. Our results support the hypothesis that desiccation tolerant plants require higher phloem transport capacity than desiccation avoiding plants, but this can be gained through various anatomical adaptations in addition to changing conduit or tissue size.     

Vascular Function in Grape Berries across Development and Its Relevance to Apparent Hydraulic Isolation

During the latter stages of development in fleshy fruit, water flow through the xylem declines markedly and the requirements of transpiration and further expansion are fulfilled primarily by the phloem. We evaluated the hypothesis that cessation of water transport through the xylem results from disruption or occlusion of pedicel and berry xylem conduits (hydraulic isolation). Xylem hydraulic resistance (R_h) was measured in developing fruit of grape (Vitis vinifera ‘Chardonnay’) 20 to 100 d after anthesis (DAA) and compared with observations of xylem anatomy by light and cryo-scanning electron microscopy and expression of six plasma membrane intrinsic protein (PIP) aquaporin genes (VvPIP1;1, VvPIP1;2, VvPIP1;3, VvPIP2;1, VvPIP2;2, VvPIP2;3). There was a significant increase in whole berry R_h and receptacle R_h in the latter stages of ripening (80–100 DAA), which was associated with deposition of gels or solutes in many receptacle xylem conduits. Peaks in the expression of some aquaporin isoforms corresponded to lower whole berry R_h 60 to 80 DAA, and the increase in R_h beginning at 80 DAA correlated with decreases in the expression of the two most predominantly expressed PIP genes. Although significant, the increase in berry R_h was not great enough, and occurred too late in development, to explain the decline in xylem flow that occurs at 60 to 75 DAA. The evidence suggests that the fruit is not hydraulically isolated from the parent plant by xylem occlusion but, rather, is “hydraulically buffered” by water delivered via the phloem.The development of grape (Vitis vinifera) berries is typical of many fleshy fruits, following a double sigmoid pattern of growth with three distinct phases: an initial phase of rapid cell division and expansion in green berries, a short transitory phase of very little growth, and a final phase in which growth is reinitiated and the fruit ripens. The transition to the ripening phase is accompanied by many physiological changes, such as the production of anthocyanins and fruit softening. In grape, these distinctive and highly visible physiological changes are collectively referred to as veraison. The rapid accumulation of sugars that is initiated in the berry mesocarp around the time of veraison is accompanied by a dramatic shift in the proportion of xylem and phloem transport (Lang and Thorpe, 1989; Greenspan et al., 1994, 1996). This same shift, albeit more gradual, occurs in many other fleshy fruits such as tomato (Solanum lycopersicum; Ho et al., 1987), apple (Malus domestica; Lang and Ryan, 1994; Drazeta et al., 2004), and kiwifruit (Actinidia deliciosa; Dichio et al., 2003) as well as in the flowers of tropical trees (Chapotin et al., 2003). The sudden reduction in xylem transport to the fruit is perceived as a mechanism to hydraulically isolate the fruit and buffer them from environmental stresses experienced by the parent plant.Using mass balance techniques, Greenspan et al. (1994, 1996) reported major changes in the role of the xylem and phloem in water transport to the grape berry at veraison. During the first growth phase, the xylem provides the majority of water transport into the berry. In the final growth stage, the phloem provides more than 80% of the berry''s water requirements and the contribution of the xylem becomes negligible. Berry water status also becomes apparently uncoupled from plant water status after veraison. Before veraison, diurnal contractions in berry diameter were strongly related to changes in plant (stem) water potential, while after veraison, diurnal contractions were greatly reduced and unrelated to changes in stem water potential (Matthews and Shackel, 2005). A similar lack of response was also observed for mesocarp cell turgor after veraison (Thomas et al., 2006). Thus, it is clear that some mechanism acts to decouple berry water relations from the water status of the parent plant.Over the past two decades, a general consensus has developed that the berry xylem becomes physically disrupted after veraison, effectively blocking the xylem pathway and isolating the fruit essentially as a whole from the parent plant (During et al., 1987; Findlay et al., 1987; Lang and Ryan, 1994). Evidence for this has been provided by observations of dye uptake into the berry through the xylem. When the cut pedicel of a preveraison berry is submerged in dye, the dye is taken up into peripheral and axial xylem conduits of the entire berry (Findlay et al., 1987; Creasy et al., 1993; Rogiers et al., 2001). After veraison, dye uptake is limited to the base of the berry vasculature (brush). From this evidence, together with micrographs that appeared to show stretched and ruptured xylem conduits in postveraison berries, it was inferred that the lignified tracheids present at veraison were physically torn apart by the expansion of the berry that occurred postveraison.Recent experimental work using a range of techniques suggests that the hypothesis of physical disruption may be oversimplified and that the berry xylem remains at least potentially functional after veraison (Bondada et al., 2005; Keller et al., 2006; Chatelet et al., 2008b). The results of Chatelet et al. (2008a, 2008b) demonstrate that the majority of xylem conduits in the berry remain intact after veraison and suggest that xylem development (growth of new conduits) continues well into the postveraison growth phase. Using both a modified pressure plate/membrane apparatus and a wicking technique, it was demonstrated that dye moved through the xylem of postveraison berries when a hydrostatic pressure or matric gradient was applied between the pedicel and the cut stylar surface (Bondada et al., 2005; Chatelet et al., 2008b). Keller et al. (2006) demonstrated this in reverse, showing that berry xylem was still capable of conducting a dye tracer back to the parent plant if the dye was introduced at the cut stylar end while the plant was transpiring. Thus, given a large enough pressure gradient, the xylem of postveraison berries retains the potential to transport water between the parent plant and the berry or vice versa. However, anatomical measurements and dye tracer studies can only be used to infer the degree to which fruit may become isolated from the parent plant. Knowledge of changes in hydraulic resistance (R_h) is required to determine whether xylem dysfunction is actually responsible for declining xylem flows reported with the progression of ripening. It is also important to differentiate between xylem flows and R_h, as these variables are sometimes confused in the literature; xylem flow rates can vary independently of R_h if water potential gradients along the pathway are altered.Previous studies examining changes in R_h associated with the development of fleshy fruit generally indicate that R_h increases during ripening but show differences in the timing and location of the increase. Some fruits develop an abscission zone in the pedicel or receptacle that is associated with vascular constriction and high R_h (Mackenzie, 1988; Lee, 1989; Van Ieperen et al., 2003). However, although some table grapes are believed to develop an abscission zone, there is no evidence of an abscission zone in wine grapes (Pratt, 1971). Tyerman et al. (2004) reported a substantial increase in R_h of grape berries after veraison, although this increase in resistance did not occur in the pedicel or receptacle but mainly in the distal section of the berries. Similarly, Malone and Andrews (2001) evaluated R_h in developing tomato fruits and stems and found that R_h increased in the fruit, but not proximal to the calyx. In apple, Lang and Ryan (1994) observed an increase in R_h at 80 d after anthesis (DAA) and also reported an increasing proportion of samples in which the xylem was completely occluded with age. Although they described these data as pedicel R_h, their measurements actually included the fruit vascular pathway; therefore, it is difficult to determine if the increase in R_h was manifested in the fruit or the pedicel.Increases in pedicel and receptacle R_h should be associated with changes in the dimensions or conductive state or xylem conduits. An increase in the R_h within the fruit may relate either to xylem dysfunction or to extravascular resistance beyond the xylem. Although they were not able to partition an increase in fruit R_h between the apoplast and symplast, Tyerman et al. (2004) suggested that the site of increased resistance may be the plasma membranes of vascular parenchyma cells separating xylem conduits and mesocarp cells rather than the xylem itself. A likely candidate driving hydraulic isolation at the cellular level is changes in plasma membrane R_h resulting from the differential expression and activity of aquaporins. Aquaporins are a family of transmembrane proteins considered to be largely responsible for the high permeability to water exhibited by plasma membranes. The regulation of R_h by aquaporins is now well documented in roots (Martre et al., 2001; McElrone et al., 2007; Vandeleur et al., 2009), and oxidative gating of aquaporins has been reported to reduce hydraulic conductivity by 90% in cells of the giant algae Chara (Henzler et al., 2004). The results of previous work suggest that aquaporins play an important role in the regulation of water movement during the development of flowers, seeds, and fruits (Maurel et al., 1995; Gao et al., 1999; Picaud et al., 2003; Shiota et al., 2006; Zhou et al., 2007). Changes in the expression of the plasma membrane intrinsic protein (PIP) PIP1 and PIP2 aquaporin gene families have been noted in ripening grapes (Picaud et al., 2003; Fei et al., 2004), although the effects of these changes on water transport (membrane conductivity) have not been documented. An increase of R_h between the mesocarp cells and the xylem within the fruit could provide a mechanism to restrict water movement between the parent plant and the berry if a large gradient in xylem tension existed between the two (Tyerman et al., 2004).While the concept of hydraulic isolation is generally accepted as part of the physiology of fleshy fruit development, we note that no studies have demonstrated an increase in R_h that is coincident with the decline in xylem flow. Additionally, measured variation in the R_h of the fruit and pedicel has not been quantitatively related to the water requirements of the fruit, taking into account water potential gradients between the fruit and the parent plant. In this study, we examined changes in the R_h of the berry, receptacle, and pedicel of Chardonnay grape over the course of fruit development. These measurements were compared against observations of xylem anatomy and aquaporin gene expression in order to investigate the hypotheses that (1) occlusion and/or disruption of xylem conduits results in the hydraulic isolation of ripening grape berries, and (2) an increase in the R_h of the berry is associated with changes in the expression of aquaporin genes in the mesocarp.     

Effects of Osmotic Potential in Nutrient Solution on Diurnal Growth of Tomato Fruit

Tomato fruit on plants grown in circulating nutrient solutionexhibited a diurnal cycle in growth rate, measured as a changein diameter, with a maximum during thc day. The diurnal growthcycle was less evident in those fruit grown at high electricalconductivity (17 mS), or on days of reduced irradiance. Girdledfruit of low conductivity plants grew at a much reduced ratewith a diurnal cycle in reverse to that of ungirdled fruit,while girdled fruit of high conductivity plants showed no diurnalgrowth. The evidence suggests that phloem and xylem water transportinto fruit operate on opposite diurnal cycles. Partitioning of available xylem water in detached fruit betweenthe calyx and berry, as well as within the berry, was determinedby berry size and relative humidity in the air. Although berrytranspiration rate was unaffected by conductivity treatmentduring plant growth, water uptake capacity was greatly reducedin the berry from high conductivity plants, suggesting an increasedresistance in the xylem transport system within the fruit. Key words: Salinity, electrical conductivity, tomato fruit, xylem transport, transpiration     

Role of ABA and Gibberellin A<Subscript>3</Subscript> on gene expression pattern of sugar transporters and invertases in <Emphasis Type="Italic">Vitis vinifera</Emphasis> cv. Malbec during berry ripening

Sugars are key constituents that affect quality of grape berries, and consequently the grape metabolic profile relevant to wine’s industry. However, enzymes and transporter genes expression involved in sugar transport at different phenological stages are scarcely studied. In addition, little is known about the role of the plant hormones ABA and Gibberellin (GA₃) as endogenous regulators, over the expression pattern of the sugars transporters genes in grapevine. The aim of this study was to analyze the expression pattern of the most relevant sugar transporters and invertases in leaves and berries of grapevine plants cv. Malbec during berry ripening stages and its shift after ABA and GA₃ sprays. In leaves, VvHT1 was the sugar transporter highly expressed, whereas VvHT6 was the most abundant in berries throughout berry ripening. Moreover, VvSUC12 and VvSUC27 were expressed at veraison greater in leaves than in berries, suggesting an active phloem loading at the onset of ripening. Applications of ABA and GA₃ enhanced the expression of VvSUC12 and VvSUC27 in pre-veraison leaves. Furthermore, hormones increased the expression of VvHT2, VvHT3 and VvHT6 in berries at different stages of ripening favoring sugar unloading from phloem. In conclusion, ABA and GA₃ are involved in the long-distance sugar transport from leaves to berries in Vitis vinifera L. cv. Malbec, and their exogenous application could be a suitable strategy to improve the process.     

Sugar transport together with environmental conditions controls time lags between xylem and stem diameter changes

In the present study the seasonal patterns of time lags between diurnal xylem and whole stem diameter variations at the top and at the base of two Scots pine trees (Pinus sylvestris L.) were compared. The diameter variations were measured during the summers of 2001 and 2002. Time lags were determined using the cross‐correlation method. The lags were found to vary in time according to the different stages of growth. At the top the xylem lagged behind the whole stem between the beginning of stem growth and the end of shoot growth in both years. In 2001 the time lags at the base showed a similar behaviour during stem growth. That kind of seasonal pattern of the time lags would result from the changes in the sink strength due to changing growth rate at different parts of the tree and the differences in the annual rhythm of growth and water availability in the soil (based on precipitation measurements) between the years 2001 and 2002 were reflected in the patterns. The time lags of shrinking and swelling periods during high and low photosynthetic activity (measured using a shoot chamber) were also compared. It was found, for example, that in 2001 in the middle of the growing season at the top of the tree the whole stem lagged on average 15 min more behind the xylem on the days of high photosynthetic activity than on the days of low or moderate. These results show for the first time that the transportation of carbohydrates and variable sink activity could be detected during the growing season in field conditions using stem and xylem diameter variation measurements. Furthermore, these results provide evidence of the pressure gradient‐driven flow also in the phloem of gymnosperms.