Theoretical and experimental errors for in situ measurements of plant water potential

Errors in psychrometrically determined values of leaf water potential caused by tissue resistance to water vapor exchange and by lack of thermal equilibrium were evaluated using commercial in situ psychrometers (Wescor Inc., Logan, UT) on leaves of Tradescantia virginiana (L.). Theoretical errors in the dewpoint method of operation for these sensors were demonstrated. After correction for these errors, in situ measurements of leaf water potential indicated substantial errors caused by tissue resistance to water vapor exchange (4 to 6% reduction in apparent water potential per second of cooling time used) resulting from humidity depletions in the psychrometer chamber during the Peltier condensation process. These errors were avoided by use of a modified procedure for dewpoint measurement. Large changes in apparent water potential were caused by leaf and psychrometer exposure to moderate levels of irradiance. These changes were correlated with relatively small shifts in psychrometer zero offsets (−0.6 to −1.0 megapascals per microvolt), indicating substantial errors caused by nonisothermal conditions between the leaf and the psychrometer. Explicit correction for these errors is not possible with the current psychrometer design.     

Seed coat cell turgor in chickpea is independent of changes in plant and pod water potential

Turgor pressure in cells of the pod wall and the seed coat of chickpea (Cicer arietinum L.) were measured directly with a pressure probe on intact plants under initially dry soil conditions, and after the plants were irrigated. The turgor pressure in cells of the pod wall was initially 0.25 MPa, and began to increase within a few minutes of irrigation. By 2-4 h after irrigation, pod wall cell turgor had increased to 0.97 MPa. This increase in turgor was matched closely by increases in the total water potential of both the pod and the stem, as measured by a pressure chamber. However, turgor pressure in cells of the seed coat was relatively low (0.10 MPa) and was essentially unchanged up to 24 h after irrigation (0.13 MPa). These data demonstrate that water exchange is relatively efficient throughout most of the plant body, but not between the pod and the seed. Since both the pod and the seed coat are vascularized tissues of maternal origin, this indicates that at least for chickpea, isolation of the water relations of the embryo from the maternal plant does not depend on the absence of vascular or symplastic connections between the embryo and the maternal plant.     

Seed coat cell turgor responds rapidly to air humidity in chickpea and faba bean

The turgor pressure in cells of chickpea (Cicer arietinum L.) and faba bean (Vicia faba L.) seed coats was measured with a pressure probe. Measurements were made under in situ conditions by removing a section of wall from a pod, which remained attached to the plant, and exposing the intact seed. If the pod wall was removed and the turgor measurements made under ambient laboratory conditions of 50% to 70% relative humidity (RH), cell turgor pressure declined over time, typically reaching 0 MPa. If the pod wall was removed and the turgor measurements made under conditions of 100% RH, however, cell turgor pressure was stable over time, relatively uniform within the seed coat tissue, and was found to be 0.1-0.3 MPa for chickpea, and 0.1-0.2 MPa for faba bean. In both species there was a marked decline in cell turgor, beginning within about 60 s, when humidification was discontinued. The decline in cell turgor occurred regardless of the depth of the cell within the seed coat tissue, and this decline could be stopped, but not entirely reversed, when humidification was restored. An increase in cell turgor could also be caused by wetting of the seed. These responses indicate that a very rapid water exchange can occur within the seed coat tissue in situ. The rapid and, in some cases, relatively permanent loss of seed coat cell turgor in the absence of humidification raises serious concerns regarding desiccation artefacts which may be involved in the empty seed coat technique, often used to study seed carbon and water relations in grain legumes.     

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.     

Dynamic Relation between Expansion and Cellular Turgor in Growing Grape (Vitis vinifera L.) Leaves

Measurements of the growth and water relations of expanding grape (Vitis vinifera L.) leaves have been used to determine the relationship between leaf expansion rate and leaf cell turgor. Direct measurement of turgor on the small (approximately 15 micrometer diameter) epidermal cells over the midvein of expanding grape leaves was made possible by improvements in the pressure probe technique. Leaf expansion rate and leaf water status were perturbed by environmentally induced changes in plant transpiration. After establishing a steady state growth rate, a step decrease in plant transpiration resulted in a rapid and large increase in leaf cell turgor (0.25 megapascal in 5 minutes), and leaf expansion rate. Subsequently, leaf expansion rate returned to the original steady state rate with no change in cell turgor. These results indicate that the expansion rate of leaves may not be strongly related to the turgor of the leaf cells, and that substantial control of leaf expansion rate, despite changes in turgor, may be part of normal plant function. It is suggested that a strictly physical interpretation of the parameters most commonly used to describe the relationship between turgor and growth in plant cells (cell wall extensibility and yield threshold) may be inappropriate when considering the process of plant cell expansion.     

Cell turgor changes associated with ripening in tomato pericarp tissue

The pressure microprobe was used to determine whether the turgor pressure in tomato (Lycopersicon esculentum Mill., variety “Castelmart”) pericarp cells changed during fruit ripening. The turgor pressure of cells located 200 to 500 micrometers below the fruit epidermis was uniform within the same tissue (typically ± 0.02 megapascals), and the highest turgors observed (<0.2 megapascals) were much less than expected, based on tissue osmotic potential (−0.6 to −0.7 megapascals). These low turgor values may indicate the presence of apoplastic solutes. In both intact fruit and cultured discs of pericarp tissue, a small increase in turgor preceded the onset of ripening, and a decrease in turgor occurred during ripening. Differences in the turgor of individual intact fruit occurred 2 to 4 days before parallel differences in their ripening behavior were apparent, indicating that changes in turgor may reflect physiological changes at the cell level that precede expression of ripening at the tissue level.     

High apoplastic solute concentrations in leaves alter water relations of the halophytic shrub, Sarcobatus vermiculatus

Predawn plant water potential (Psi(w)) is used to estimate soil moisture available to plants because plants are expected to equilibrate with the root-zone Psi(w). Although this equilibrium assumption provides the basis for interpreting many physiological and ecological parameters, much work suggests predawn plant Psi(w) is often more negative than root-zone soil Psi(w). For many halophytes even when soils are well-watered and night-time shoot and root water loss eliminated, predawn disequilibrium (PDD) between leaf and soil Psi(w) can exceed 0.5 MPa. A model halophyte, Sarcobatus vermiculatus, was used to test the predictions that low predawn solute potential (Psi(s)) in the leaf apoplast is a major mechanism driving PDD and that low Psi(s) is due to high Na+ and K+ concentrations in the leaf apoplast. Measurements of leaf cell turgor (Psi(p)) and solute potential (Psi(s)) of plants grown under a range of soil salinities demonstrated that predawn symplast Psi(w) was 1.7 to 2.1 MPa more negative than predawn xylem Psi(w), indicating a significant negative apoplastic Psi(s). Measurements on isolated apoplastic fluid indicated that Na+ concentrations in the leaf apoplast ranged from 80 to 230 mM, depending on salinity, while apoplastic K+ remained around 50 mM. The water relations measurements suggest that without a low apoplastic Psi(s), predawn Psi(p) may reach pressures that could cause cell damage. It is proposed that low predawn apoplastic Psi(s) may be an efficient way to regulate Psi(p) in plants that accumulate high concentrations of osmotica or when plants are subject to fluctuating patterns of soil water availability.     

Micro-injection of Lygus salivary gland proteins to simulate feeding damage in alfalfa and cotton flowers

Alfalfa and cotton flowers were pierced with small glass capillaries of an overall size and shape similar to that of Lygus stylets, and injected with small quantities (6 to 100 nL) of solutions that contained Lygus salivary enzymes. Crude and partially purified protein solutions from Lygus heads and isolated salivary glands showed substantial polygalacturonase (PG) activity, as has been previously reported. Following injection with both crude and partially purified protein solutions, as well as with pure fungal and bacterial PGs, flowers of both alfalfa and cotton exhibited damage similar to that caused by Lygus feeding. Injection with the same volume of a buffer control as well as a buffer control containing BSA at a comparable protein concentration (approximately 6 microg/mL) showed no symptoms. These results are consistent with a previously suggested hypothesis that the extensive tissue damage caused by Lygus feeding is primarily due to the action of the PG enzyme on the host tissue, rather than to mechanical damage caused by the insect stylet. Substantial genotypic variation for a PG inhibiting protein (PGIP) exists in alfalfa and cotton. We, therefore, suggest that breeding and selection for increased native PGIP levels, or transformation with genes encoding PGIP from other plant species, may be of value in obtaining alfalfa and cotton varieties that are more resistant to Lygus feeding damage.     

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.