Water uptake along the length of grapevine fine roots: developmental anatomy, tissue-specific aquaporin expression, and pathways of water transport.

To better understand water uptake patterns in root systems of woody perennial crops, we detailed the developmental anatomy and hydraulic physiology along the length of grapevine (Vitis berlandieri × Vitis rupestris) fine roots from the tip to secondary growth zones. Our characterization included the localization of suberized structures and aquaporin gene expression and the determination of hydraulic conductivity (Lpr) and aquaporin protein activity (via chemical inhibition) in different root zones under both osmotic and hydrostatic pressure gradients. Tissue-specific messenger RNA levels of the plasma membrane aquaporin isogenes (VvPIPs) were quantified using laser-capture microdissection and quantitative polymerase chain reaction. Our results highlight dramatic changes in structure and function along the length of grapevine fine roots. Although the root tip lacked suberization altogether, a suberized exodermis and endodermis developed in the maturation zone, which gave way to the secondary growth zone containing a multilayer suberized periderm. Longitudinally, VvPIP isogenes exhibited strong peaks of expression in the root tip that decreased precipitously along the root length in a pattern similar to Arabidopsis (Arabidopsis thaliana) roots. In the radial orientation, expression was always greatest in interior tissues (i.e. stele, endodermis, and/or vascular tissues) for all root zones. High Lpr and aquaporin protein activity were associated with peak VvPIP expression levels in the root tip. This suggests that aquaporins play a limited role in controlling water uptake in secondary growth zones, which contradicts existing theoretical predictions. Despite having significantly lower Lpr, woody roots can constitute the vast majority of the root system surface area in mature vines and thus provide for significant water uptake potential.

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.

Establishing a Reference Baseline for Midday Stem Water Potential in Olive and Its Use for Plant-Based Irrigation Management.

Midday stem water potential (SWP) is rapidly becoming adopted as a standard tool for plant-based irrigation management in many woody perennial crops. A reference or "baseline" SWP has been used in some crops (almond, prune, grape, and walnut) to account for the climatic influence of air vapor pressure deficit (VPD) on SWP under non-limiting soil moisture conditions. The baseline can be determined empirically for field trees maintained under such non-limiting conditions, but such conditions are difficult to achieve for an entire season. We present the results of an alternative survey-based approach, using a large set of SWP and VPD data collected over multiple years, from irrigation experiments in olive orchards located in multiple countries [Spain, United States (California), Italy, and Argentina]. The relation of SWP to midday VPD across the entire data set was consistent with an upper limit SWP which declined with VPD, with the upper limit being similar to that found in Prunus. A best fit linear regression estimate for this upper limit (baseline) was found by selecting the maximum R 2 and minimum probability for various upper fractions of the SWP/VPD relation. In addition to being surprisingly similar to the Prunus baseline, the olive baseline was also similar (within 0.1 MPa) to a recently published mechanistic olive soil-plant-atmosphere-continuum (SPAC) model for "super high density" orchard systems. Despite similarities in the baseline, the overall physiological range of SWP exhibited by olive extends to about -8 MPa, compared to about -4 MPa for economically producing almond. This may indicate that, despite species differences in physiological responses to low water availability (drought), there may be convergent adaptations/acclimations across species to high levels of water availability. Similar to its use in other crops, the olive baseline will enable more accurate and reproducible plant-based irrigation management for both full and deficit irrigation practices, and we present tentative SWP guidelines for this purpose.

Measurement of vulnerability to water stress-induced cavitation in grapevine: a comparison of four techniques applied to a long-vesseled species.

Among woody plants, grapevines are often described as highly vulnerable to water-stress induced cavitation with emboli forming at slight tensions. However, we found native embolism never exceeded 30% despite low xylem water potentials (Psi(x)) for stems of field grown vines. The discrepancy between native embolism measurements and those of previous reports led us to assess vulnerability curve generation using four separate methods and alterations (i.e. segment length and with/without flushing to remove embolism prior to measurement) of each. Centrifuge, dehydration and air-injection methods, which rely on measurement of percentage loss of hydraulic conductivity (PLC) in detached stems, were compared against non-invasive monitoring of xylem cavitation with nuclear magnetic resonance (NMR) imaging. Short segment air-injection and flushed centrifuge stems reached >90 PLC at Psi(x) of-0.5 and -1.5 MPa, respectively, whereas dehydration and long-segment air-injection measurements indicated no significant embolism at Psi(x) > -2.0 MPa. Observations from NMR agreed with the dehydration and long segment air-injection methods, showing the majority of vessels were still water-filled at Psi(x) > -1.5 MPa. Our findings show V. vinifera stems are far less vulnerable to water stress-induced cavitation than previously reported, and dehydration and long segment air-injection techniques are more appropriate for long-vesseled species and organs.

Seasonal pattern of apoplastic solute accumulation and loss of cell turgor during ripening of Vitis vinifera fruit under field conditions.

Using a novel pressure membrane (PM) apparatus for the extraction of apoplastic fluid from field-grown grape (Vitis vinifera L.) berries, our hypothesis that significant apoplast solutes accumulate at the beginning of the ripening process (i.e. veraison), and that this accumulation might contribute to progressive berry softening due to a progressive loss of mesocarp cell turgor pressure (P) was tested. It was necessary to correct the solute potential (Psi(s)) of fluid collected with the PM for dilution due to the presence of a dead volume in the apparatus, but after correction, the Psi(s) obtained with the PM agreed with that obtained by low speed centrifugation. A clear decline in fruit apoplastic solute potential (psi(S)(A)) began approximately 10 d prior to fruit coloration, and it was found to be coincident with a decline in mesocarp cell P and fruit elasticity (E). By late in fruit development when berry growth ceased (90 d after anthesis), both apoplast and fruit Psi(s) reached almost -4 MPa. These results support the hypothesis that a decrease in psi(S)(A) is responsible for the observed loss in mesocarp cell P, and is the mechanistic cause of berry softening.

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 assay membrane integrity (cell viability) in mesocarp tissue of the developing grape (Vitis vinifera L.) berry in order to test the hypothesis that there is a substantial loss of compartmentation in these cells during ripening. This technique was also used to determine whether loss of viability was associated with symptoms of a ripening disorder known as berry shrivel. FDA fluorescence of berry cells was rapid, bright, and stable for over 1 h at room temperature. Confocal microscopy detected FDA staining through two to three intact surface cell layers (300-400 mum) of bisected berries, 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 degrees Brix). These 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. The observed viability loss was primarily in the locule area around the seeds, suggesting that a localized loss of viability and compartmentation may occur as part of normal fruit development. The cell viability of berry shrivel-affected berries was similar to that of normally developing berries until the onset of visible symptoms (i.e. shrivelling), at which time viability declined in visibly shrivelled berries. Berries with extensive shrivelling exhibited very low cell viability (15%).

Fruit ripening in Vitis vinifera: apoplastic solute accumulation accounts for pre-veraison turgor loss in berries.

In Vitis vinifera L. berries, the onset of ripening (known as "veraison") involves loss of turgor (P) in the mesocarp cells. We hypothesized that P loss was associated with an accumulation of apoplastic solutes in mesocarp tissue prior to veraison. Apoplastic sap was extracted from the mesocarp by centrifugation at the appropriate gravity to measure the apoplast solute potential (Psi (s) (A) ) and assay the sap composition. The Psi (s) (A) was about -0.2 MPa early in development, decreased about 1.0 MPa by veraison, and continued to decrease during ripening to almost -4.0 MPa by the end of berry development. Potassium, malate, tartrate, proline, glucose, fructose, and sucrose were quantified in apoplastic sap. The calculated contribution of these solutes was about 50% of the total Psi (s) (A) preveraison, but increased to about 75% as fructose and glucose accumulated during ripening. The contribution of the estimated matric potential to apoplast water potential decreased during development and was only 1.5% postveraison. We conclude that high concentrations of solutes accumulated in the mesocarp apoplast prior to veraison, and that P loss was a direct result of decreased Psi (s) (A) . Because Psi (s) (A) decreased before veraison, our findings suggest that apoplast solutes play an important role in the events of cellular metabolism that lead to the onset of ripening.

Mesocarp cell turgor in Vitis vinifera L. berries throughout development and its relation to firmness, growth, and the onset of ripening.

Vitis vinifera L. berries are non-climacteric fruit that exhibit a double sigmoid growth pattern and dynamic changes in gene expression, cell metabolism, and water relations at the onset of ripening. The cell-pressure probe was utilized to examine the relationships of turgor pressure (P) in mesocarp cells to growth, sugar accumulation, and fruit softening during development. In replications utilizing three different varieties, mesocarp cell P demonstrated a consistent pattern of a relative mid-range P early in development, followed by an increase to a maximum of about 0.35 MPa, and a subsequent rapid decline before ripening to less than 0.1 MPa. Fruit "apparent elastic modulus" (E, units of MPa), was introduced as a standard measure to describe ripening-related softening. E changed dynamically and synchronously with P during development and in response to water deficits for fruit grown in greenhouse and field conditions. Thus, E and P were positively and linearly related. Sugar accumulation did not increase significantly until P had declined to less than 0.1 MPa. The results suggest that P is an important determinant of fruit softening and that P decreases in conjunction with many of the physiological and gene expression changes that are known to occur at the onset of ripening.

Xylella fastidiosa infection and ethylene exposure result in xylem and water movement disruption in grapevine shoots.

It is conventionally thought that multiplication of the xylem-limited bacterium Xylella fastidiosa (Xf) within xylem vessels is the sole factor responsible for the blockage of water movement in grapevines (Vitis vinifera) affected by Pierce's disease. However, results from our studies have provided substantial support for the idea that vessel obstructions, and likely other aspects of the Pierce's disease syndrome, result from the grapevine's active responses to the presence of Xf, rather than to the direct action of the bacterium. The use of magnetic resonance imaging (MRI) to observe the distribution of water within the xylem has allowed us to follow nondestructively the development of vascular system obstructions subsequent to inoculation of grapevines with Xf. Because we have hypothesized a role for ethylene produced in vines following infection, the impact of vine ethylene exposure on obstruction development was also followed using MRI. In both infected and ethylene-exposed plants, MRI shows that an important proportion of the xylem vessels become progressively air embolized after the treatments. The loss of xylem water-transporting function, assessed by MRI, has been also correlated with a decrease in stem-specific hydraulic conductivity (K(S)) and the presence of tyloses in the lumens of obstructed water conduits. We have observed that the ethylene production of leaves from infected grapevines is greater than that from healthy vines and, therefore, propose that ethylene may be involved in a series of cellular events that coordinates the vine's response to the pathogen.

Direct in situ measurement of cell turgor in grape (Vitis vinifera L.) berries during development and in response to plant water deficits.

Vitis vinifera L. berries are non-climacteric fruits that exhibit a double-sigmoid growth pattern, and at the point known as 'veraison', which is just before the beginning of the second period of rapid fruit growth, these berries undergo several abrupt physiological changes. 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. Initial tests comparing attached and detached berries demonstrated that cell P was stable for up to 48 h after detachment from the vine, provided that water loss from the berry was prevented. Cell P at pre-dawn was on the order of 0.25 MPa pre-veraison (PreV) and was reduced by an order of magnitude to 0.02 MPa post veraison (PostV). Cell P declined slightly but significantly with depth from the berry surface PreV, but not 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 of stressed plants also 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 many current hypotheses that veraison is associated with a general loss of membrane function and cellular compartmentation in the grape berry. 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.