SOLUTE ACCUMULATION AND APPARENT HYDRAULIC ISOLATION OF FLESHY FRUIT

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: COMPLETE
• Funding Source: NRI COMPETITIVE GRANT
• Reporting Frequency: Annual
• Accession No.: 0207976
• Grant No.: 2006-35100-17440
• Proposal No.: 2006-02096
• Project Start Date: Sep 15, 2006
• Project End Date: Sep 14, 2008
• Grant Year: 2006
• Program Code: [22.1]- Agricultural Plants and Environmental Adaptation

Recipient Organization
UNIVERSITY OF CALIFORNIA, DAVIS
410 MRAK HALL
DAVIS,CA 95616-8671

Performing Department
VITICULTURE AND ENOLOGY

Non Technical Summary
Fleshy fruit are important for human health, but growers have little control over ripening and softening of some fruit such as grapes. A better understanding of how fruit cell's disconnect from the parent plant and how water is transported into and out of fleshy fruit cells will lead to insight into how to control the ripening processes and extend markets. This project will investigate how the hydraulic connections between fruit cells and the parent plant are regulated during fruit development. A component of the work will determine whether water deficits that arise as a consequence of reduced connection to the parent plant contribute in a mechanistic way to the onset and progression of fleshy fruit ripening.

Animal Health Component

20%

Research Effort Categories

Basic

80%

Applied

20%

Developmental

(N/A)

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
203	1131	1020	60%
203	1131	1030	20%
203	1131	1040	20%

Knowledge Area
203 - Plant Biological Efficiency and Abiotic Stresses Affecting Plants;

Subject Of Investigation
1131 - Wine grapes;

Field Of Science
1040 - Molecular biology; 1030 - Cellular biology; 1020 - Physiology;

Keywords

water relations

water potential

osmotic potential

turgor

firmness

apoplastic solutes

aquaporins

hydraulic conductivity

ripening

Goals / Objectives
Given the existence of an unrestricted xylem pathway after veraison, the principle objectives of this proposal are to (a) explain why mesocarp cells do not develop high turgor pressure with the accumulation of solutes, and (b) determine why a reduction in xylem transport occurs, particularly whether or not there is tension present in the berry xylem. These objectives will be tested with the following hypotheses: Hypothesis 1: That mesocarp cell turgor decreases rather than increases during cell solute accumulation because the movement of solutes into the apoplast ameliorates the potential gradient for cell water uptake. Here we will investigate the role of apoplastic solutes in the regulation of turgor pressure in the mesocarp. Changes in turgor pressure will be monitored with the cell pressure probe over the course of development. Concurrently, the gradient in solute potential between the symplast and apoplast will be measured by a number of methods. Symplastic solute potential will be measured by extracting sap with the cell pressure probe, and apoplastic solute potential will be measured with the pressure plate apparatus and energy dispersive x-ray microanalysis (EDX). Hypothesis 2a: The predicted accumulation of apoplastic solutes derives from incipient cell death and membrane dysfunction. Hypothesis 2b: The predicted accumulation of apoplastic solutes results from the regulated exchange of solutes between mesocarp cells and their apoplastic environment. We will differentiate between two mechanisms of apoplastic solute accumulation: (a) the passive release of solutes from the symplast into the apoplast by cell death or (b) active regulation of an apoplast/symplast solute potential gradient by living cells. This will be done by quantifying changes in cell viability across development with FDA. We will also determine developmental changes in mesocarp plasma membrane integrity using the cell pressure probe. Hypothesis 3a: That tension in the xylem of the parent plant is not effectively transmitted to the apoplast of the mesocarp after veraison. Hypothesis 3b: That tension is transmitted to the berry after veraison, but diurnal partitioning of solutes between the symplast and the apoplast acts to reduce the driving gradient between the berry and the stem. At this point there are no direct measurements of xylem tension in developing fruit. We propose to measure xylem tension directly at a series of points along the pathway between the stem and the fruit. From this we will determine if xylem tension persists in the berry after veraison and if it does not, at what point in the pathway does the relaxation of tension occur. Possible restrictions in the xylem pathway will be investigated using cryo-scanning electron microscopy. If xylem tension is found to persist in the berry after veraison it is possible that an active regulation of aploplstic solutes would provide a mechanism allowing the berry cells to compensate for diurnal fluctuations in xylem tension. Apoplastic solute concentrations will be measured with the pressure plate apparatus and EDX.

Project Methods
In order to make the measurements required for this study with the desired level of accuracy we will modify the design of two pieces of equipment used to measure plant or soil water status: the cell pressure probe and pressure plate/membrane apparatus. We also emphasize that the majority of testing and development of these instruments has already been undertaken and the completion is not expected to account for a significant amount of time within the timetable of research. Two key methodologies to be used for the proposed studies are the cell and the xylem pressure probe. Both techniques physically measure the same thing (hydrostatic pressure, either positive or negative), but the technical challenges for each are different. The primary challenge in measuring xylem tension is that fluids under tension are metastable and will cavitate if physically disturbed. Many berry xylem conduits are located close (100 μm) to the surface of the berry, facilitating easy access to conduits for probing. However, the small diameter and uneven wall thickness of tracheids which make up the berry xylem may make measurements more difficult than in previous studies, and we propose to fabricate a cell pressure probe that should additionally serve as a xylem pressure probe. The key feature will be to use a metal bellows rather than a metal rod to regulate the oil pressure or tension in the probe. It is well known that cavitation in a liquid under tension can be caused by mechanical rubbing (friction), and that a metal bellows, which can change volume with no such friction, can be used to generate stable tensions in liquids. This approach will allow us to quickly establish tensions in the oil, without depending on a large amount of fluid flow through the capillary tip. Direct measurement of symplastic and apoplastic Ψs will be important in determining water potential gradients between the parent plant and the developing grape berry. The pressure probe can be used to sample vacuolar fluid from one or a few cells, and these small volumes measured in a freezing point nanoliter osmometer to obtain symplastic Ψs (Shackel 1987). Because of its small volume, reliably quantifying the concentrations of solutes in the apoplastic space is technically challenging, and we propose to use a number of independent methods to estimate apoplastic Ψs. We have used a pressure membrane apparatus to establish a hydrostatic Ψp gradient across a berry, and we have recently developed this system for the extraction of fluid from the apoplastic space of grape berries. The key to the use of this method was to quantify and correct for the dead volume of the system, and evidence from preliminary experiments taking this into account indicate that Ψs in the apoplast of post-veraison berries is low (high solute concentration), and similar to that of the bulk tissue (Figure 7). We have also obtained similar (although not identical) estimates of apoplastic Ψs using low speed centrifugation as the extraction method.

Progress 09/15/06 to 09/14/08

Outputs
OUTPUTS: The current hypothesis to explain the transition from green to ripening fruit, particularly in grape (Vitis vinifera L.) berries, is that the xylem vascular tissue becomes non-functional as a result of berry growth physically disrupting the conduits. To corroborate this, peripheral xylem structure in developing Chardonnay berries was investigated via maceration and plastic sectioning. Macerations revealed, contrary to current belief, the xylem was comprised mostly of vessels with few tracheids. In cross-section, 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. 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 analyzed to test for continued differentiation from 18 to 120 d after anthesis in Chardonnay berries. 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. Tracheary elements stretched by 20%, but not as much as that predicted based on the growth of the vascular diameter (40%). These results completed a comprehensive evaluation of grape berry peripheral xylem during its development and show that tracheary development continues further into berry maturation than previously thought. A corollary of the current hypothesis of the onset of ripening in grape is that flesh cells also lose function, but in this case due to a breakdown of membrane compartmentalization. Fluorescein diacetate (FDA) was used as a vital stain to assay membrane integrity (cell viability) in mesocarp tissue of the developing grape (V. vinifera L.) berry in order to test this hypothesis. Confocal microscopy detected FDA staining through 2 to 3 intact surface cell layers (300-400 mm), and showed that the fluorescence was confined to the cytoplasm, indicating the maintenance of integrity in both cytoplasmic as well as vacuolar membranes, and the presence of active cytoplasmic esterases. FDA clearly discriminated between living cells and freeze-killed cells, and exhibited little, if any, non-specific staining. Propidium iodide and DAPI, both widely used to assess cell viability, were unable to discriminate between living and freeze-killed cells, and did not specifically stain the nuclei of dead cells. For normally developing berries under field conditions there was no evidence of viability loss until about 40 d after veraison, and the majority (80%) of mesocarp cells remained viable past commercial harvest (26 Brix). Results are inconsistent with current models of grape berry development which hypothesize that veraison is associated with a general loss of compartmentation in mesocarp cells. PARTICIPANTS: Not relevant to this project. TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Not relevant to this project.

Impacts
The implication of the results showing that berry tracheary elements and flesh (mesocarp) cells remain functional well into fruit maturity are that the opportunity for environmental (grower) control of the ripening process is sustained longer than the current ripening paradigm would suggest. For future research into the control of ripening and earliness, the results direct attention away from degenerative cell functions and metabolism.

Publications

• Castellarin, S.D., Matthews, M.A., DiGaspero, G., and G.A. Gambetta. 2007. Water deficits accelerate ripening and induce changes in gene expression regulating flavonoid biosynthesis in grape berries. Planta 227:101-12
• Krasnow, M., M. A. Matthews, and K.A. Shackel. 2008. Evidence for substantial maintenance of membrane integrity and cell viability in normal developing grape (Vitis vinifera L.) berries throughout development. Journal of Experimental Botany 9: 849 - 859
• Chatelet, D. S., T. L. Rost, K. A. Shackel, and M. A. Matthews. 2008. The peripheral xylem of grapevine (Vitis vinifera). 1. Structural integrity in post-veraison berries. Journal of Experimental Botany 59:1987-1996.
• Chatelet, D. S., T. L. Rost, M. A. Matthews, and K. A. Shackel. 2008. The peripheral xylem of grapevine (Vitis vinifera) berries. 2. Anatomy and development. Journal of Experimental Botany 59:1997-2007.

Progress 09/15/06 to 09/14/07

Outputs
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. Grape berries are non-climacteric fruits that exhibit a double-sigmoid growth pattern, and at the point known as veraison, just before the beginning of the second period of growth, undergo an apparent loss of xylem function. A pressure plate/pressure membrane apparatus 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 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. Because the dye studies indicated a persistent hydraulic connection, the cell pressure probe was used to examine the in situ turgor (P) of cells in the mesocarp during berry development and in response to plant water deficits. Cell P at pre-dawn was about 0.25 MPa pre-veraison (PreV) and was reduced to 0.02 MPa post veraison (PostV). When water was withheld from potted vines, cell P declined about 0.2 Mpa, as pre-dawn vine water potential declined about 0.6 MPa over 12 d, whereas cell P was completely insensitive to a 1.10 MPa decrease in pre-dawn vine water potential after veraison. Rewatering resulted in a 24 h recovery of cell P before but not after veraison. The substantial decline in cell P around veraison is consistent with the decline in berry firmness that is known to occur at this time, and the PostV insensitivity of P to changes in vine water status is consistent with current hypotheses that the PostV berry is hydraulically isolated from the vine. The fact that a measurable P of about 0.02 MPa and typical cell hydraulic/osmotic behaviour were exhibited in PostV berries, however, indicates that cell membranes remain intact after veraison, contrary to hypotheses that veraison is associated with loss of membrane function and cellular compartmentation. We hypothesize that cell P is low in the PostV berry, and possibly other fleshy fruits, because of the presence of regulated quantities of apoplastic solutes.

Impacts
The implication of the pressure membrane study is that the assumption that xylem conduits become physically disrupted at veraison in grape berries must be re-evaluated. The implication of the cell pressure probe study is that preveraison water deficits can be expected to have larger consequences for fruit growth and composition than postveraison water deficits because fruit cell turgor is dramatically more responsive to plant water deficits before veraison than after veraison.

Publications

• Thomas, T. R., M. A. Matthews, and K. A. Shackel. 2006. Direct in-situ measurement of cell turgor in grape (Vitis vinifera L.) berries during development and in response to plant water deficits. Plant, Cell and Environment 29:993-1001.