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Planta (2002) 215: 210219 DOI 10.

1007/s00425-002-0744-2

O R I GI N A L A R T IC L E

Olga A. Koroleva A. Deri Tomos John Farrar Christopher J. Pollock

Changes in osmotic and turgor pressure in response to sugar accumulation in barley source leaves
Received: 18 June 2001 / Accepted: 3 January 2002 / Published online: 13 March 2002 Springer-Verlag 2002

Abstract Pressure-probe measurements and single-cell sampling and analysis techniques were used to determine the eect of photosynthetic production and accumulation of sugars on osmotic and turgor pressures of individual cells of barley (Hordeum vulgare L.) source leaves. In control plants, the changes in osmotic pressure in individual cells during the photoperiod were dierent for mesophyll (increase of 276 mOsmol/kg), parenchymatous bundle sheath (PBS; increase of 100 mOsmol/kg) and epidermis (remains constant). There was also an increase in osmotic pressure at the tissue level. Cooling of roots and the shoot apical meristem restricted the export of sugars from leaves, and the resulting changes in osmotic and turgor pressure were monitored. In contrast to the control leaves, mesophyll, PBS, and epidermal cells showed a similar increase in osmotic pressure (up to 500 mOsmol/kg). Cooling also increased the turgor pressure in epidermal and (to a greater extent) PBS cells. The dierence in turgor pressure between epidermal and PBS cells is consistent with the presence of a water potential gradient within the leaf, from the vascular bundles towards the leaf surface. Keywords Hordeum (water relations) Single-cell monitoring Sugar accumulation Turgor and osmotic pressure Transpiration

Abbreviations p: osmotic pressure P: turgor pressure Y: water potential PBS: parenchymatous bundle sheath subscripts: cell, intracellular (protoplast); wall, extracellular (apoplast)

Introduction
Source leaves of barley produce sucrose, which is either exported to the rest of the plant, accumulated in the leaf, or used for fructan synthesis. The Mu nch hypothesis for the mechanism of phloem transport postulates a pressure-driven mass ow of assimilates from source to sink (Mu nch 1930). In this model, there is a close relationship between the long-distance export of sucrose from source leaves and the ux of water. However, the mechanisms, and even the precise pathways of assimilate distribution within the leaf, upstream of the phloem are not known. Pressure gradients could control ow through the symplast and inuence the rate of photoassimilate export from mesophyll and parenchymatous bundle sheath (PBS) cells. Cell turgor may regulate membrane transport of solutes (Wyse et al. 1986). The water potential gradients in the leaves reect the environmental conditions and they could be the primary signals perceived by plants. In addition to chemical signalling by an increased concentration of abscisic acid (ABA), an independent eect of leaf water status on leaf elongation in maize was demonstrated (Salah and Tardieu 1997), and the authors suggested a role for hydraulic signals in control of leaf expansion rate. Studying the appearance of water potential gradients may provide important information for investigation of signalling events and feedback to metabolism in source leaves. In roots, water relations have been described by a composite transport model, which demonstrates the importance of the parallel arrangement of apoplastic and cell-to-cell paths for regulation of water and solute uptake according to the demands from the shoot (Steudle and Peterson 1998). Much less is known about the interaction of water and solute transport through

O.A. Koroleva (&)1 A.D. Tomos J. Farrar School of Biological Sciences, University of Wales Bangor, Bangor, Gwynedd, Wales, LL57 2UW, UK C.J. Pollock IGER, Plas Gogerddan, Aberystwyth, Ceredigion, SY23 3EB, Wales, UK Present address: John Innes Centre, Colney, Norwich, NR4 7UH, UK e-mail: olga.koroleva@bbsrc.ac.uk
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leaf tissue and water relations of a leaf. Only diurnal osmotic adjustment in guard cells has received substantial attention (Talbott and Zeiger 1998). Due to their osmotic nature, the transport of solutes in leaves is always linked to water relations. These, in turn, are inuenced in two ways by processes related to photosynthesis. Firstly, osmotic pressure changes due to the photosynthetic accumulation of sugars. Typical net assimilation rates for barley leaves under controlled environmental conditions are approximately 15 mmol CO2 m2 s1. This is equivalent to a rate of sucrose synthesis of 3 mg sucrose (g FW)1 h1 (Koroleva et al. 1997), i.e. the sucrose concentration in the leaf tissue would increase by about 10 mM every hour during the photoperiod if there were no export. Secondly, leaf water relations are aected by transpiration, which is dependent on the behaviour of stomata (Nonami and Schulze 1989). Changes in guard cell aperture (and hence stomatal conductance) will alter both water relations and CO2 xation. Transpiration can also exert a feedback control aecting conductance itself (Jones 1998). Stomatal conductance changes could be triggered by the accumulation of sucrose (Lu et al. 1997; Ewert et al. 2000) or malate (Hedrich et al. 1994) in guard-cell walls or by changes in the hydrostatic pressure of the epidermis as a whole (Spannungsphase; Lo sch and Schenk 1978). Transpiration causes sub-atmospheric hydrostatic pressures in the leaf xylem (Wei et al. 1999). Cooling of sink organs, such as roots and the shoot apical meristem, is often used to generate accumulation of carbohydrate in source leaves by reducing export (Smouter and Simpson 1991; Plum and Farrar 1996; Koroleva et al. 1997, 1998). Cooling of roots and the shoot apical meristem has short- and long-term eects on water relations. The immediate consequence of root cooling is a reduction in the hydraulic conductance of root cell membranes (Malone 1993) with a resultant drop in the turgor pressure of leaf cells, leading to stomatal closure and a slow recovery of turgor (Pardossi et al. 1994). Reduction in the hydraulic conductance of cell membranes at low temperature has been observed in Tradescantia epidermal cells (Tomos et al. 1981) and in the leaf extension zone of Lolium temulentum (Thomas et al. 1989). Cooling of the roots decreases leaf water status and causes cessation of leaf growth (Malone 1993). In the long term, as metabolism in the chilled tissues is slowed, the demand for nutrients is reduced and export of photosynthetically produced carbohydrate from the leaf is decreased, so sugars accumulate in the source leaves (Smouter and Simpson 1991; Koroleva et al. 1997, 1998). Without compensating changes, the increase in soluble carbohydrate concentration in the leaf will alter total osmotic balance, but little is known of any tissue-specic changes. In contrast to mesophyll and PBS cells, epidermal cells contain a very low concentration of carbohydrate under both normal and cooled conditions (Fricke et al. 1994; Koroleva et al. 1997, 1998). Preferential carbohydrate allocation to particular cell types could, therefore, cause the appearance of

gradients of osmotic pressure within leaves. However, it is possible that accumulation of alternative solutes such as malate (Koroleva et al. 2000) occurs to balance differences in sugar concentrations between leaf cells. In our study we investigated the impact of photosynthetic production and accumulation of sugars on the osmotic and turgor pressure of individual cells of barley source leaves. Three cell types, epidermal, mesophyll and PBS, diering in their patterns of sugar accumulation, were assessed. The cell pressure probe and single-cell sampling and analysis (SiCSA) technique allow the measurement of the turgor and osmotic pressure in neighbouring cells (Tomos et al. 1994; Tomos and Leigh 1999). Combination of the cell pressure probe and micro-osmometer allows accurate measurements of cell turgor and osmotic pressure, and therefore cell water potential may also be calculated (Shackel 1987; Frensch and Schulze 1988; Nonami and Schulze 1989). The experimental design was aimed at determining the consequences for water relations at the single-cell level of short-term (during the normal diel cycle) and long-term (following reduction of net export from leaves) accumulation of sugars in barley leaf cells. The following hypotheses were addressed: 1. Photosynthetically produced sugars will increase osmotic pressure in the source leaf tissue, especially when their export from the leaf is decreased. 2. Photosynthesis and accumulation of sugars will have a dierential impact on osmotic and turgor pressure of the dierent cell types of barley source leaves. 3. Long-term accumulation of sugars (caused by reduced export from leaves) in leaf cells will enhance dierences in osmotic and turgor pressure between cell types when compared with short-term (diel) variation.

Materials and methods


Growth of plants Plants of barley (Hordeum vulgare L. cv. Klaxon) were grown hydroponically in controlled-environment cabinets (Sanyo Gallenkamp) at 20 C, 16 h light/8 h dark period with a photon uence rate of 500 lmol m2 s1 and an atmospheric CO2 concentration of 350 ll l1 (Koroleva et al. 1997). Half-strength Long Ashton nutrient solution (Hewitt 1966) was changed twice a week. All samples were taken from the middle parts of third leaves 15 days after full expansion of the leaf blade. Cooling experiments Low temperature was used to induce accumulation of carbohydrate (Koroleva et al. 1997, 1998). The plant roots, together with the lower 3 cm of the shoot (where the shoot apex is situated) were submerged in nutrient solution cooled to 10 C, while the leaves were kept at 20 C. Cooling was started on the rst day after the third leaf had expanded fully. Cooling was continued for 4 days and the solution was then warmed to 20 C for the rest of the experiment. Carbohydrate measurements in bulk leaf tissue Samples of approximately 50 mg fresh leaf tissue were xed in 5 ml of boiling ethanol (80% v/v), then two subsequent extractions with

212 80% ethanol were made at 60 C. Carbohydrate concentrations were measured directly in the ethanol extracts using a phenol-sulphuric acid technique (Dubois et al. 1956). Extraction of sap from individual cells Using the single-cell sampling technique (Tomos et al. 1994), cell sap samples were extracted from individual mesophyll, PBS and epidermal cells of barley leaves using a microcapillary. Borosilicate glass capillaries (outer and inner diameters 1.0 and 0.58 mm, respectively) without lament (Clark Electromedical Instruments, Pangbourne, UK) were used on a commercial capillary puller to make microcapillaries with tip diameter of several microns. The microcapillaries were lled with paran oil and used for sampling. The capillary was inserted through a stomatal pore to sample from mesophyll and PBS cells, to avoid contamination by epidermal cell sap. A set of criteria was used as follows to distinguish between mesophyll and bundle sheath cells: position in the leaf, total volume and viscosity of the cell sap entering the microcapillary after puncturing the cell, and the relative amount of chloroplasts in the cell sap. All sampling was performed on intact leaves attached to plants. Measurement of osmotic pressure in leaf tissue and single cells Osmotic pressure in bulk leaf tissue was measured using a vapour pressure osmometer (model 5100; Wescor, Logan, Utah, USA) in tissue sap extracted by freezing and thawing followed by centrifugation. The osmotic pressure (pcell) in the individual cell sap samples was measured using a picoliter osmometer (Malone et al. 1989; Tomos et al. 1994). The technique is based on measuring the melting point of the frozen solution after supercooling to 40 C to induce freezing. Small droplets (10100 pl) of samples and standards were placed under a droplet of paran oil on a piece of coverslip glass, attached by high-heat-conductance paste on top of the cooling stage of the osmometer (details are given in Malone and Tomos 1992). In comparing values of osmotic and hydrostatic pressure, we have used a conversion factor of 400 mOsmol=1 MPa (calculated using the vant Ho relationship, Nobel 1991). Measurement of turgor pressure in cells The cell turgor pressure (Pcell) was measured using the cell pressureprobe technique (Hu sken et al. 1978), on intact leaves attached to the plants. Turgor pressure measurements were performed under normal air conditions (transpiring leaves) or in leaves submerged for 2030 min in distilled water (non-transpiring leaves). Plants were also assumed to be non-transpiring when they were measured in the dark after 8 h of darkness. Estimation of water potential The plasma membrane of plant cells is generally assumed to behave as a semipermeable membrane, with a reection coecient for cell sap solutes that approaches unity and a high conductivity to water ow (Zimmermann and Steudle 1978; Tomos 1988). Under these conditions the water potential (Y=Pp) on both sides of the cell membrane will be very close to equality (Ywall=Ycell). By measuring Pcell and pcell, a value for Ycell is obtained. Provided that water ux through the system is low, this will also represent the value of water potential of the surrounding cell wall (apoplast), Ywall. We have attempted to estimate the change in the magnitude of the apoplastic water potential caused by transpiration, by comparing Ywall under transpiring and non-transpiring conditions. Statistical treatment of the data Hypotheses that the slopes of regression lines signicantly dier from zero were tested using Systat 8.0 software (SPSS Inc, Chicago, Ill., USA). Linear regression equations were compared on the Fig. 1 Time courses of osmotic pressure and potassium concentration during 12 h of photoperiod in tissue of barley (Hordeum vulgare) third leaf. Each point is an average for three plants SD. Slopes of both regression lines are signicantly dierent from 0 at the 99% condence interval basis of hypotheses about equality of two population regression coecients using the Students t-test (Zar 1996).

Results
Changes in water relations parameters in control plants during the day Osmotic pressure and major solutes of leaf tissue The osmotic pressure of bulk leaf tissue increased over the 12 h of the light period by 237 mOsmol/kg (Fig. 1). The main osmotic constituents of barley leaves were potassium salts (Fig. 1), as described previously by Fricke et al. (1994). The tissue potassium concentration rose slowly, increasing by 59 mM over the photoperiod (Fig. 1). The increase in both osmotic pressure and potassium concentration was statistically signicant. At the same time the carbohydrate concentration in the leaf tissue also increased (Fig. 2). The main carbohydrate accumulated in temperate grass leaves held at 20 C was sucrose (Pollock 1986; Koroleva et al. 1997, 1998). Accumulation of 20 mg/g FW sucrose only during 12 h of photoperiod (Fig. 2) corresponds to an increase in the average concentration in leaf tissue of 72 millimolal based on a mean measured value (not shown) of 0.8 ml water/g FW. Osmotic coecients were taken as 0.9 for potassium and its counter-anion and 1 for sucrose (Wyn Jones and Gorham 1983). Therefore, accumulation of potassium (59 millimolal), its counter-anion (59 millimolal if monovalent) and sucrose would generate an increase of 178 mOsmol/kg in leaf osmotic pressure during 12 h of photoperiod. This corresponds well to the measured values (Fig. 1).

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Fig. 2 Time course of the content of carbohydrates in tissue samples from third leaves of barley plants (80% ethanol-soluble fraction). Each point is an average for three plants SD

Single-cell osmotic pressure Measurement of the osmotic pressure time-course for individual leaf cells showed dierences between epidermal, mesophyll and PBS cells (Fig. 3). Average osmotic pressure in mesophyll cells rose during 10 h of measurement by 276 mOsmol/kg (Fig. 3a). The variability of osmotic pressure between individual mesophyll cells, even within one leaf, was very high. In PBS cells the increase in osmotic pressure was less, about 100 mOsmol/kg (Fig. 3b). Osmotic pressure in the epidermal cells remained practically constant during the day, close to 600 mOsmol/kg (Fig. 3c). The variability in osmotic pressure between epidermal and PBS cells, both within and between dierent plants, was much less than for mesophyll cells. The slopes of regression lines for osmotic pressure in mesophyll and PBS cells (Fig. 3a, b) were signicantly dierent from zero, whilst the regression line for osmotic pressure in epidermal cells (Fig. 3c) had a slope equal to zero at the 95% condence interval. The time courses of osmotic pressure during the day shown on Fig. 3 were found to be fully representative of the 5 days following full expansion of the third leaf, and the same pattern of diurnal cycling was observed for each cell type (data not shown). Turgor pressure Diurnal time-courses for turgor pressure in epidermal and PBS cells were measured in control plants under normal conditions of transpiration (Fig. 4). For epidermal cells the highest turgor pressures (about 1.3 1.4 MPa) were observed at the end of the dark period, when the stomata were closed and transpiration was apparently close to zero. During the light period, the
Fig. 3 Time course of osmotic pressure in individual mesophyll (a), PBS (b) and epidermal (c) cells in control barley plants. Dierent plants were used for each time-point. Slopes of regression lines in a and b are signicantly dierent from 0 at the 99% condence interval; the slope of the regression line in c is not signicantly dierent from 0 at the 95% condence interval

turgor pressure initially fell to 1.11.2 MPa, then partially recovered. In contrast, the turgor pressure of the PBS cells was lowest at the end of the dark period (approx. 1.1 MPa). It increased continuously during the

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Fig. 4 Diurnal change in turgor pressure in epidermal and PBS cells of barley leaves. Each bar represents the average SD for two to ve cells from a single plant

Fig. 5 Time-course of osmotic pressure in leaf tissue of barley plants with roots and shoot apical meristem cooled to 10 C for 4 days and in control plants. Each bar is an average SD for three plants

day, reaching a value of 1.3 MPa by the end of light period. Long-term changes in water relations parameters in plants with cooled sinks Osmotic pressure of leaf tissue The osmotic pressure in the leaf tissue was indistinguishable between control and plants with cooled sinks during the rst day of the experiment (Fig. 5). For control plants the osmotic pressure of bulk leaf tissue showed a small increase with leaf age and a diurnal cycle with a daytime increase of about 100 mOsmol/kg. In plants with cooled sinks, the osmotic pressure was slightly higher than that of control plants for the second and third days but the dierence increased sharply on the fourth day (Fig. 5). This dierence was maintained after cooling was stopped. Single-cell osmotic pressure Osmotic pressure measured in individual epidermal, mesophyll and PBS cells increased during the cooling experiment (Fig. 6); the diurnal cycle with lower values in the morning and higher values by the end of photoperiod was preserved. The total increase in osmotic pressure after 82 h of cooling approached 500 mOsmol/ kg, irrespective of the cell type (Fig. 6). The higher values of osmotic pressure were maintained during the photoperiod following the cessation of cooling. Cell turgor pressure The turgor pressure for epidermal cells (Fig. 7a) and PBS cells (Fig. 7b) was measured under transpiring and
Fig. 6 Osmotic pressure time-course in individual epidermal, mesophyll and PBS cells in barley plants with roots and shoot apical meristem cooled to 10 C for 4 days. Each bar is an average SD for three plants; two to three samples of each cell type were analysed from each plant

non-transpiring conditions. Cell turgor pressure increased gradually throughout the cooling experiment. The average value for turgor pressure in transpiring epidermal cells increased from 1.2 to 1.4 MPa, and for non-transpiring cells from 1.4 to 1.8 MPa. Slopes of regression lines in transpiring and non-transpiring conditions were signicantly dierent (Fig. 7a). The decrease in absolute value of water potential of the apoplast due to transpiration was calculated as the difference in turgor pressure between measurements made on leaves under transpiring and non-transpiring conditions (as described above). The estimated apoplastic water potential of epidermis due to transpiration

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decreased from 0.2 to 0.4 MPa during the experiment (Fig. 7a). At the beginning of the experiment the average value for turgor pressure in the PBS cells under transpiring conditions was very close to that under non-transpiring conditions, with less than 0.1 MPa dierence (Fig. 7b). Turgor rose from 1.3 to 2.1 MPa during cooling (for transpiring conditions). The transpiration eect (estimated as dierence in turgor between transpiring and non-transpiring plants) caused a decrease in apoplast water potential of no more than 0.2 MPa during this period; there was no signicant dierence in slopes of regression lines (Fig. 7b). Measurement of turgor pressure in barley mesophyll cells proved to be very dicult technically due to loss of

pressure following penetration by the pressure probe. We measured only a few cells successfully. Mesophyll cells from two transpiring control plants, for which pressure-relaxation curves to stable values were obtained, gave turgor values of 0.420.08 MPa (n=6 cells; after approx. 1 h illumination) and 0.810.11 MPa (n=3; after approx. 10 h illumination). From the initial values of the traces of leaking cells there was also an indication that mesophyll cells have a lower turgor pressure than the other cell types. Hypothetical map of leaf water-relations parameters Ideally, four measurements [Pcell(transpiring), Pcell(non- tranpcell (transpiring) and pcell (non- transpiring)] need to be made simultaneously for each of the three cell types to describe fully the components of water potential. This is technically impossible. Performing sequential measurements on cells as close as possible to each other also proved exceptionally dicult especially in the case of turgor measurements. The potential sources of error are addressed in the Discussion. Time lags and plant-toplant variation resulted in considerable scatter of the derived parameters. We have tried to interpret our data by using linear regression lines to the appropriate turgor data for epidermal and PBS cells (Fig. 7). Results from cells probed during the rst and last 3 h of the photoperiod were pooled. The former is termed morning and the latter evening. Cell osmotic pressures used are those shown in Fig. 6. The calculated data are presented as a diagram (Fig. 8). The data obtained from the epidermal cells showed the smallest variability. During the day in control plants, and in those cooled for only a few hours, p was 1.5 and P was 1.2 MPa (Fig. 8, calculated from data in Figs. 6 and 7), so the value of Y was relatively close to zero, about 0.3 MPa. Following 82 h of cooling, Y had decreased
spiring),

Fig. 7 Change in turgor pressure in epidermal (a) and PBS (b) cells of barley leaves in plants with roots and shoot apical meristem cooled to 10 C for 4 days. Each time-point includes two to three measurements of each cell type made from each of two plants. Slopes of the two regression lines in a are signicantly dierent at the 95% condence interval. Slopes of the two regression lines in b are not signicantly dierent at the 95% condence interval. Slopes of regression lines for two cell types in non-transpiring leaves (1)a and (1)b are not signicantly dierent at the 99% condence interval; regression lines for transpiring leaves (2)a and (2)b are signicantly dierent at the 99% condence interval

Fig. 8 Schemes representing osmotic and turgor pressure in dierent cell types and corresponding water potentials in barley leaf tissue. Day 1 Data illustrate diurnal changes in control plants, Day 4 changes in cooled plants after 4 days

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to 1.1 MPa. At this point, there was a clear change in the value of p, which rose to 2.5 MPa. For PBS cells, the Y decreased very little, from 0.1 to 0.2 MPa during the cooling period. As with the epidermis, it was very close to zero in the morning, at the beginning of the cooling experiment. In contrast to the epidermis, however, its value changed much less, only by 0.1 MPa even after 82 h of cooling. Regression coecients for turgor pressure in transpiring conditions were signicantly dierent for epidermal and PBS cells (Fig. 7). Reliable measurements of turgor pressure values were obtained for only a few mesophyll cells, so we used the two groups of cells from control plants for which meaningful pressures were available. The corresponding values of pcell are 1.1 and 1.8 MPa (Fig. 3) for the cells having mean turgor pressures of 0.4 (morning) and 0.8 MPa (evening). This indicates water potential values of 0.7 and 1.0 MPa respectively.

Discussion
The accumulation of sugars produced by photosynthesis increased osmotic pressure in bulk tissue of barley source leaves. At the single-cell level, however, heterogeneity between cell types becomes evident. We found an increase in osmotic pressure during the photoperiod in mesophyll and PBS, but not in the epidermis of control barley plants. Turgor pressure of the individual leaf cells was determined not only by its osmotic pressure, but also by transpiration via its eect on the water potential of the apoplast. However, when reducing the sugar export elevated leaf sugar content, the osmotic pressure of all three cell types (epidermis, mesophyll and PBS) increased to a similar degree. Osmotic pressure rose in epidermal cells, which do not accumulate carbohydrate (Koroleva et al. 1997, 1998), so they must have accumulated alternative solutes, such as inorganic or organic ions, including malate (Koroleva et al. 2000). There appears to be cooperation between leaf cells with dierent functions. The changes can be expressed as a spatial and temporal map of the pattern of dierential behaviour of epidermal, mesophyll and PBS cells in a single photoperiod or during sugar accumulation over several days. There was an eect of transpiration on the turgor pressure of epidermal cells. In a diurnal cycle (Fig. 4), the decrease in turgor pressure in epidermal cells coincides with the illumination of the plants, which is known to induce opening of the stomata. Moreover, the articial abolition of transpiration by immersion of the leaves in water (the leaf water potential will rise towards zero under these conditions) led to an increase in the turgor pressure in the epidermis (Fig. 7a). In sugar beet plants, an inverse relationship between stomatal conductance and turgor pressure was demonstrated by Palta et al. (1987). Thurmer et al. (1999) found a similar diurnal change in turgor pressure in mesophyll cells of Tetrastigma, with the amplitude dependent on

irradiance. However, there was a non-linear relation between transpiration and turgor pressure in mesophyll and epidermal cells of Tradescantia (Nonami and Schulze 1989). In contrast, in barley PBS cells over 1012 h of light, the turgor pressure increased, i.e. it changed in the opposite direction to that of the epidermis (Fig. 4). The trends in mesophyll cells were similar to those in the PBS. In both cell types, the change is largely attributable to an increase in the osmotic pressure of the protoplast. This increase in PBS turgor pressure is very dierent from the observation of Thurmer et al. (1999) who demonstrated that an increase in transpiration led to a decrease in the mesophyll cell turgor as well as xylem pressure. Considering the pathway of water ow from rhizosphere to the point of evaporation in terms of an electrical circuit model as described by Molz and Ferrier (1982), this indicates that, relative to the hydraulic resistance of the leaf (from PBS or mesophyll to the evaporation point), the hydraulic resistance to water ow from rhizosphere to leaf was considerably greater over the 9.5 m of the liana compared with the 0.20.3 m of barley. This dierence can be seen in that under transpiring conditions a tension of 0.4 MPa is set up in the Tetrastigma xylem, whereas in barley the xylem tension cannot be greater than approximately 0.1 MPa, because in our system the plants were growing in hydroponic solution with an osmotic pressure less than 0.1 MPa. This value is derived from the water potential of the PBS cells (approx. 0.1 MPa), which must have water potential values lower than that of the xylem if water is going to enter the leaf from the xylem. In barley, in contrast to the shallow water potential gradient from root to PBS (0.1 MPa/0.3 m), there exists a steeper water potential gradient over the short distance between PBS and epidermis (0.2 MPa/0.3 mm). The magnitude of the water potential gradient from PBS to the epidermis was increased approximately 3-fold under the conditions of reduced sugar export from the leaf. The turgor pressure, which diered among all cell types of the control plants during the morning, became more uniform towards the evening. This observation has implications for our understanding of plasmodesmatal function within the leaf. We speculate that they must be closed between cells of dierent pressures if bulk ow of cytoplasm from one cell into the other is to be prevented under these conditions. The consequences of this have been discussed for turgor and osmotic pressure gradients in root tissue of transpiring plants (Rygol et al. 1993). Oparka and Prior (1992), using Nicotiana leaf trichome cells, obtained direct evidence for pressure-generated closure of plasmodesmata when the pressure dierential between the neighbouring cells exceeded 0.2 MPa. In sink tissue of pea root tips, the short-term increase in symplastic phloem unloading under osmotic stress was accompanied by plasmodesmatal widening, increasing the eective passage area of plasmodesmata (Schulz 1995). The increasing evidence for multiple roles of plasmodesmata in controlling trac of nutrients, low-

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molecular-weight growth regulators and proteins by uctuations in aperture during development and spatially in dierent tissues, is discussed in a review by Zambryski and Crawford (2000). It is possible, therefore, that in the source leaves the plasmodesmata are closed during the dark period, but opened later in the photoperiod when the turgor pressures become similar. This is particularly interesting in the case of the mesophyll/PBS boundary, across which assimilate movement can be symplastic, as has been demonstrated by dyecoupling experiments (Farrar et al. 1992). At the same time it is unlikely that there is a signicant proportion of symplastic transport between the epidermis and mesophyll, as these two cell types are strikingly dierent in their solute composition (Fricke et al. 1994; Koroleva et al. 1997, 1998, 2000). Cooling of the roots plus shoot apical meristem increased the osmotic pressure of the leaf tissue, due in part to additional accumulation of sugars. The total increase in bulk leaf osmotic pressure measured at the end of the light period was approximately 250 mOsmol/ kg between the rst and fourth days of this experiment. In a dierent experiment, individual cells of all types demonstrated higher values, the osmotic pressures measured at the end of the light period increased between the rst and fourth days by about 400500 mOsmol/kg. A possible explanation for the higher values of osmotic pressure in the individual cells compared with the total tissue could be a higher degree of hydration in the vascular bundles tissues. Although it is more likely that the dierence is due to variations between separate batches of plants used. Some of the increase in osmotic pressure in mesophyll and PBS cells was caused by accumulation of sucrose and of fructans with a low degree of polymerisation. The maximum concentrations of total sugars in these cell types can be estimated from single-cell measurements (Koroleva et al. 1998). If the minimum degree of polymerisation of 3 is taken for fructan, sugars account for at most 300 mOsmol/kg. We have shown that the remainder is due to an increased accumulation of other solutes, such as malate (Koroleva et al. 2000) and potassium (our unpublished observations). The osmotic pressure in plants with cooled sinks was maintained at a higher level 24 h after the cooling was discontinued. This is consistent with the observation that the high concentration of sugars is still present in leaf tissue at that time (Koroleva et al. 1997, 1998), keeping the osmotic pressure at an elevated level. Apparently, there are two reasons for the phenomenon in the mesophyll and bundle sheath cells: rst, the accumulated amounts of sugars along with fresh photosynthate exceeded the capacity for phloem loading and translocation; second, our experimental data showed rapid hydrolysis of fructan oligomers to fructose after the cooling was stopped, which would lead to an increase in osmotic pressure. In epidermal cells, the higher osmotic pressure during sink cooling is due to accumulation of malate, which cannot be readily mobilised later (Koroleva et al. 2000).

Turgor pressure of both PBS cells and epidermal cells increased during the cooling experiment. The turgor pressure of epidermal cells in transpiring leaves increased by much less than did the osmotic pressure (Fig. 6). Rapid eects of water supply on concentration of apoplastic solutes and therefore on turgor pressure of cortical cells has been demonstrated in Ricinus hypocotyl (Meshcheryakov et al. 1992). Abolition of transpiration by submersing the leaves in water could cause partial hydration and dilution of apoplastic solution. The inuence of transpiration on the water potential of both the epidermal cells and their surrounding apoplast became statistically greater during the cooling experiment. The accumulation of sucrose in the guard cell wall of Vicia faba as a joint result of photosynthesis and transpiration may have a function as a physiological signal that integrates the rates of transpiration, photosynthesis and translocation (Lu et al. 1997). Transpiration was the primary cause of the decrease in epidermal turgor pressure during a light-dark cycle, with accompanying inevitable concentration of apoplastic solution and overall decrease in water potential. Concentration of some solutes in the cell wall is possible if the rate of water evaporation exceeds its supply. In contrast to the situation in the epidermis the value of water potential of the PBS cells, based on single-cell osmotic and turgor pressure measurements, was close to zero. The eect of transpiration on water potential was not statistically dierent from zero (P>0.2). Thus there was a gradient of water potential in the apoplast, from vascular bundles to the epidermis. The relationship between osmotic and turgor pressure of PBS cells in sink-cooled plants is quite dierent from that of the epidermis. The increase in turgor pressure is directly attributable to the considerable increase in osmotic pressure due to accumulation of photosynthetically produced sugars, with no decrease in the water potential of the apoplast of the PBS. Of particular interest are mesophyll cells. High amounts of sugars are accumulated during the light period, even in control plants, resulting in large diurnal oscillations of osmotic pressure. Mesophyll cell turgor pressure was lower than that of epidermal and PBS cells. If these few pressure measurements are correct, it is unlikely that there is permanent symplastic continuity between mesophyll and either PBS or epidermal cells, as discussed above. Also, the water potential (Y) of mesophyll is lower than both that of epidermis and PBS. Lower water potentials in mesophyll cells than in epidermal cells were also measured by Nonami and Schulze (1989). As above, this can be explained by a transpiration eect, caused by evaporational water loss directly from the mesophyll into the sub-stomatal cavity (Tyree and Yianoulis 1980) and therefore lower water potential. Even if due to the problems associated with measuring mesophyll turgor pressures we underestimated the turgor values, there is still evidence of a gradient of apoplastic water potential from the epidermis to the PBS in plants cooled for 4 days.

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Conclusions
1. Accumulation of sugars during the photoperiod was accompanied by the rise in the tissue osmotic pressure. Reduction of sugar export by root cooling further increased the tissue osmotic pressure. 2. The accumulation of sugars during the day as the result of photosynthetic activity in barley leaves does increase osmotic pressure in mesophyll and PBS cells, but not in epidermal cells. The time-course of turgor pressure in epidermal cells had a diurnal rhythm dependent on transpiration. The turgor of PBS cells increased during the light period, apparently due to accumulation of solutes in these cells. 3. Following restriction of sugar export from the leaf, the osmotic pressure increased by approximately 1 MPa in parallel in all three cell types. This was not the proportional increase in each cell type that might have been expected. The turgor pressure rose in response to an increase in osmotic pressure in PBS cells and to a lesser extent in epidermal cells. 4. The water potential of epidermal cells became more negative due to transpiration during the day; the eect was more pronounced in plants with cooled sinks. At the same time, the water potential of PBS cells did not show a signicant decrease, suggesting a gradient of Y across the leaf.

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