Sieve element unloading: cellular pathway, mechanism and control

The transport and distribution of phloem – mobile solutes is predominantly determined by transport processes located at the sink end of the source – transport – sink system. Transport across the sieve element boundary, sieve element unloading, is the first of a series of sink transport processes. Unloading of solutes from the sieve elements may follow an apo- or symplastic route. It is speculated that the unloading pathway is integrated with sink function and that apoplastic unloading is restricted to situations in which movement through the symplast is not compatible with sink function. These situations include axial transport and storage of osmotically active solutes against concentration and turgor gradients between the sieve elements and sink cells. Coupled with alteration in sink function, the cellular pathway of unloading can switch in stems and possibly other sinks. Experimental systems and approaches used to elucidate the mechanism of sieve element unloading are reviewed. Unloading fluxes to the apoplast can largely be accounted for by membrane diffusion in axial sinks. However, the higher fluxes in storage sinks suggests dependence on some form of facilitated transport. Proton sucrose symport is assessed to be a possible mechanism for facilitated efflux of solutes across the sieve element plasma membrane to the sink apoplast. Unloading through the symplast may occur by diffusion or mass flow. The latter mechanism serves to dissipate phloem water and hence prevent the potential elevation of sieve element turgor that would otherwise slow phloem import into the sink. The possibility of energised plasmodesmatal transport is raised. Sieve element unloading must be integrated with subsequent compartmentation and metabolism of the unloaded solute. Solute levels are an obvious basis for control of sieve element unloading, but are found to offer limited scope for a mass action mechanism. Apoplastic, cellular pathway, sieve element, solute transport, symplastic. Translated into a turgor signal, solute levels could regulate the rate of unloading, metabolism and compartmentation forming part of a turgor homeostat irrespective of the pathway of unloading.     

Scaling phloem transport: water potential equilibrium and osmoregulatory flow

In this work, the common assumption that phloem sap is in water potential equilibrium with the surrounding apoplast was examined. With a dimensionless model of phloem translocation that scales with just two dimensionless parameters (R?and F?), a ‘map’ of phloem behaviour as a function of these parameters was produced, which shows that the water potential equilibrium assumption (R?F? >> 1) is valid for essentially all realistic values of the relevant scales. When in water potential equilibrium, a further parameter reduction is possible that limits model dependence to a single parameter (F?), which describes the ratio of the solution's osmotic strength to its axial pressure drop. Due to the locally autonomous nature of individual sieve element/companion cell complexes, it is argued that long‐distance integrative control is most efficient when F? is large (that is, when the pressure drop is relatively small), permitting the sieve tube to regulate solute loading in response to global changes in turgor. This mode of transport has been called ‘osmoregulatory flow.’ Limitations on the pressure drop within the transport phloem could require that sieve tubes be shorter than the long axis of the plant, and thus arranged in series and hydraulically isolated from one another.     

On the volume-flow mechanism of phloem transport

Summary A steady-state model of solution flow in a tubular semipermeable membrane is developed for an arbitrary distribution of solute sources and sinks along the translocation path. It is demonstrated that the volume-flow mechanism of phloem transport depends only on the two assumptions: 1. that the plasmalemma of the sieve tube is a differentially permeable membrane, and 2. that sugars are actively secreted into and absorbed from the lumen of the sieve tube. It is shown that in the absence of a pressure gradient, there is a negligible concentration gradient over most of the translocation path. However, in the presence of a pressure gradient a small concentration gradient develops as a result of the continually changing chemical potential of water along the direction of solution flow. For Poiseuille flow the concentration gradient is approximately proportional to the mean stream velocity.     

Water Flow Across the Sieve Tube Boundary: Estimating Turgor and Some Implications for Phloem Loading and Unloading. IV. Root Tips and Seed Coats

In the present paper, the theory developed in Part I of thisseries is applied to seed coats of Phaseolus vulgaris and somecombined data on root tips of Hordeum distichum and Hordeumvulgare. Because of the large back-pressures implied, it isconcluded that phloem transport into these primary sinks wouldbe physiologically impossible in the absence of a symplasticpathway for the unloading of water from sieve elements. In thiscase, unloading of water and sucrose will occur predominantlyas a pressure-driven flow of solution through plasmodesmata,although diffusion can contribute significantly to the plasmodesmatalsucrose flux. At least 20% of the plasmodesmata connecting sieveelements and adjacent cells must be unobstructed if large changesin turgor and osmotic pressure are to be avoided. Dependingon the membrane area available for water fluxes, it is possiblethat the difference in water potential across the sieve-tubeplasmalemma can lead to significant errors when axial turgorgradients are estimated from gradients of osmotic pressure andexternal water potential. The magnitude and even the sign ofthese errors is uncertain, but it is possible that sieve-tubeturgor pressures will be significantly underestimated in primarysinks Phloem, turgor, osmotic pressure, plasmodesmata, Munch hypothesis, Phloem unloading     

Linking phloem function to structure: Analysis with a coupled xylem-phloem transport model

We carried out a theoretical analysis of phloem transport based on Münch hypothesis by developing a coupled xylem-phloem transport model. Results showed that the maximum sugar transport rate of the phloem was limited by solution viscosity and that transport requirements were strongly affected by prevailing xylem water potential. The minimum number of xylem and phloem conduits required to sustain transpiration and assimilation, respectively, were calculated. At its maximum sugar transport rate, the phloem functioned with a high turgor pressure difference between the sugar sources and sinks but the turgor pressure difference was reduced if additional parallel conduits were added or solute relays were introduced. Solute relays were shown to decrease the number of parallel sieve tubes needed for phloem transport, leading to a more uniform turgor pressure and allowing faster information transmission within the phloem. Because xylem water potential affected both xylem and phloem transport, the conductance of the two systems was found to be coupled such that large structural investments in the xylem reduced the need for investment in the phloem and vice versa.     

Theoretical study of flow,pressure and solute concentration in double membrane systems

A model system consisting of two rigidly held membranes in series was investigated through the application of the Kedem and Katchalsky thermodynamic single membrane flow equations. This analysis results in predictions of the steady state flow properties as well as values for the solute concentration and pressure of the internal compartment when the system is under the influence of a constant solute concentration or hydrostatic pressure gradient. It is demonstrated that although the flow properties and internal compartment pressure are complicated functions of the membrane permeability coefficients and driving gradient across the system, the relationships are greatly simplified by the explicit appearance of the internal compartment steady state solute concentration in the equations. It is shown that the steady state volume flow rate depends on the absolute value of the solute concentration in the external compartments, as well as the solute concentration gradient across the system. The properties of non-linear dependence of volume flow on concentration gradient, and rectification of volume flow are discussed and shown to be independent properties of the system. For the system under the influence of a solute concentration gradient, the internal compartment pressure can be greater or less than the ambient pressure, and depends mainly on the order in which the membranes are encountered by the volume flow. These properties are qualitatively correlated with certain available experimental observations in biological systems.     

Measurement of turgor pressure and its gradient in the Phloem of oak

A direct method is described for measuring the pressure in secondary phloem sieve tubes of oak trees. One end of a 26-gauge stainless steel tube was shaped such that when it penetrated the outer bark and transected a few sieve elements, it was stopped by the xylem so that small openings in the end allowed phloem sap to enter the tube. The other end of the stainless tube (phloem needle) was joined to a long glass capillary sealed at its other end to form a manometer for measuring phloem sap pressure. A method for measuring the average osmotic and turgor pressures in cells of leaves is also described. Phloem turgor pressures varied greatly in a series of phloem punctures around the trunk at 1.5 and at 6.3 meters. The variation in turgor pressure was always greater than the variation in osmotic pressure. In a series of turgor pressures arranged in descending order, the values in a sequence for the upper level was usually a little (0-3 atm) larger than the values for the lower level. These results may suggest that translocation of assimilate is favored by a small turgor pressure gradient, but they do more to emphasize the complications in measuring gradients in an elastic low resistance distribution system composed of contiguous longitudinal conduits. The results also imply that the sieve tubes are inflated with assimilate fluid under high pressure which can readily move longitudinally and with less pressure drop than would be necessary if the sieve tubes were rigid.     

Water relations of turgor recovery and restiffening of wilted cabbage leaves in the absence of water uptake

A novel phenomenon in which wilted cabbage leaves appeared to regain positive turgor pressures without additional water uptake has been previously reported (J Levitt [1986] Plant Physiol 82: 147-153). These experiments were replicated and the biophysical nature of turgor recovery characterized. Leaf water potential and its components were assayed in hydrated, wilted, and desiccated leaves which appeared to regain turgor after wilting. The hypotheses that turgor recovery was due to an increased volumetric elastic modulus (ε), or alternatively the result of solute redistribution were tested. Quantitative evidence that turgor recovery occurs in excised leaves was found. Leaf turgor pressure in hydrated leaves (~0.6 megapascal) decreased to zero upon wilting. After continued desiccation, turgor pressure returned to approximately 0.3 megapascal even though leaf relative water content declined. The ε of hydrated leaves was large and there was no evidence of an increased ε in the turgor-recovered leaves. Solute mobilization occurred during desiccation. The apoplastic osmotic potential decreased from −0.15 to −0.44 megapascal in hydrated and turgor-recovered leaves, respectively, and solutes were transported from the lamina to the midrib tissue. Solute redistribution coupled with the high ε may have resulted in localized turgor recovery in specific cells in the desiccated leaves.     

Quantifying Apoplastic Flux through Red Pine Root Systems Using Trisodium, 3-hydroxy-5,8,10-pyrenetrisulfonate

The fluorescent compound trisodium, 3-hydroxy-5,8,10-pyrenetrisulfonate (PTS) was used to quantify the apoplastic flux through red pine (Pinus resinosa Ait.) root systems—that portion of the total water flux reaching the xylem without ever crossing a semipermeable membrane. Flow was induced by pressure through detopped root systems, and by transpiration through intact seedlings. Apoplastic flux was determined by multiplying total flux by the ratio of PTS concentration in the xylem exudate to PTS concentration in the bathing medium.

Under aeration, apoplastic flux was less than 1% of total flux. Under anaerobic conditions, up to 50% of total flux was apoplastic suggesting that anaerobic conditions change the pathway of water flow into root xylem. The change under anaerobic conditions was reversible. Detopped root systems under pressure and intact seedlings under transpiration gave similar results. In detopped root systems, the magnitude of the pressure gradient may alter the apoplastic contribution to total flux.

     

Differential effects of turgor on sucrose and potassium transport at the tonoplast and plasma membrane of sugar beet storage root tissue

When turgor was increased, by decreasing the concentration of mannitol bathing discs of sugar beet storage root tissue, the rates of sucrose and potassium uptake into the vacuole were decreased. At all external mannitol concentrations the rate of sucrose and potassium uptake across the plasma membrane was an order of magnitude greater than the rate of quasi-steady uptake into the vacuole, implying a very large efflux. Efflux of both sucrose and potassium was increased at high turgor. However, while increasing turgor decreased the rate of K⁺ uptake, the rate of sucrose uptake at the plasma membrane increased with time. Compartmental analysis of tracer exchange kinetics was used to determine unidirectional K⁺ fluxes. From these results, it was estimated that the increase in K⁺ efflux accompanying a 1.5 MPa increase in turgor could lead to a net increase of 140mol^?3h^?1 in the external potassium concentration. It is suggested that the turgor-imposed increase in solute efflux is a means of regulating intracellular osmotic pressure and/or turgor in sugar beet storage roots, but that sucrose is preferentially retrieved from the apoplast, even under conditions of excessively high turgor. However, much of this sucrose is probably lost from the cell, implying a ‘futile’ sucrose transport cycle at the plasma membrane. The turgor-stimulated leak of potassium could play a major role in the regulation of turgor pressure in sugar beet storage root tissue.     

Changes in water relations and in some physiological functions of bean under very light osmotic shock induced by polyethylene glycol

Bean plantlets ( Phaseolus vulgaris L. cv. Topcrop) were stressed at the age of 16–18 days by gradual (2–8%) or abrupt addition of 6% (w/v) polyethylene glycol Mw 6000 (PEG 6000) to Hoagland solution. Leaf conductance, photosynthesis, internal CO₂ partial pressure (C_i), relative water content (RWC), water content/dry weight (H₂O/DW), apoplastic PEG concentrations and weight of leaves, stems and roots were determined. Leaf conductance, photosynthesis and C_i were determined on non-detached primary leaves, and leaf potentials (water, osmotic and turgor potentials) were investigated in freshly detached (non-rehydrated) primary leaves, both in treated and control plants; RWC and osmotic potential were also assessed at the null turgor point. Low PEG 6000 concentrations induced early and evident decrease in leaf conductance and photosynthesis, whereas C_i decreased only moderately and tended to recover during advanced stress. There were moderate though significant decreases in RWC and H₂O/DW, no change or increases in water potential, no significant changes in osmotic potential and a moderate but significant increase in turgor potential. Even when referred to null turgor point, RWC significantly decreased and osmotic potential was unchanged. It was concluded that apoplastic PEG 6000 accumulation at evaporating sites would account for the early decrease in conductance which would also justify the unchanged or the prevalent increase in water potential and turgor potential. The subsequent PEG diffusion and concentration in the leaf apoplastic water would have induced the RWC and H₂O/DW decrease and the final turgor flexion documented.     

Water Flow Across the Sieve-Tube Boundary: Estimating Turgor and Some Implications For Phloem Loading and Unloading. I. Theory

A mathematical model of water and sucrose transport across thesieve tube boundary is presented, based on conservation of matterand the phenomenological equations for plasmodesmatal transportbetween the sieve elements and their associated cells. Plasmodesmataltransport coefficients are discussed. In parts II–IV,the equations developed here are used to assess: (i) the estimationof phloem turgor gradients from osmotic pressure gradients;(ii) plasmodesmatal transport of water and sucrose between thesieve elements and adjacent cells; and (iii) the plausibilityof symplastic and apoplastic phloem loading and unloading insome primary sources and sinks. A list of symbols is given inAppendix 1 of this paper Phloem, turgor, osmotic pressure, loading, unloading, plasmodesmata, Munch hypothesis     

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.     

Estimation of the volumetric elastic modulus and membrane hydraulic conductivity of willow sieve tubes

Severed aphid stylets were used to follow the kinetics of sieve tube turgor and osmotic pressure (π) responses following step changes in water potential applied to the cambial surface of willow (Salix exigua Nutt.) bark strips. The kinetics of the turgor response were monitored with a pressure transducer. In separate experiments, the kinetics of the π response were followed by freezing point determinations on stylet exudate. The sieve tube volumetric elastic modulus in the bark strips was about 21 bars, but may be higher in intact stems. The membrane hydraulic conductivity was about 5 × 10⁻³ centimeters per second per bar; several factors make it difficult to estimate its value accurately. Differences in the turgor pressure (P) and π responses, as well as the relatively more rapid initial turgor response to a water potential (ψ) change, suggested a time-dependent component in sieve tube wall elasticity.

Our observations were generally not supportive of the idea that sieve tubes might osmoregulate. However, the bark strip system may not be suitable for addressing that question.

Separate measurements of ψ, P, and π demonstrate that the relationship predicted by the fundamental cell water potential equation, ψ = P − π, is applicable within experimental error (± 0.4 bar) to sieve tube water relations.

     

Functional xylem in the post-veraison grape berry

A number of studies have shown a transition from a primarily xylem to a primarily phloem flow of water as fleshy fruits develop, and the current hypothesis to explain this transition, particularly in grape (Vitis vinifera L.) berries, is that the vascular tissue (tracheids) become non-functional as a result of post-veraison berry growth. In most studies, pedicels have been dipped in a vial containing an apoplastic dye, which was taken up into the entire peripheral and axial xylem vasculature of pre-veraison, but not post-veraison berries. The pressure plate/pressure membrane apparatus that is commonly used to study soil moisture characteristics was adapted and the pre- to post-veraison change in xylem functionality in grape berries was re-evaluated by establishing a hydrostatic (tension) gradient between the pedicel and a cut surface at the stylar end of the berry. Under the influence of this applied hydrostatic gradient, movement of the apoplastic tracer dye, basic fuchsin, was found in the pedicel and throughout the axial and peripheral xylem of the berry mesocarp. A similar movement of dye could be obtained by simply adjoining the stylar cut surface to a dry, hydrophilic wicking material. Since both pre- and post-veraison berries hydrate when the pedicel is dipped in water, it is hypothesized that the absence of dye movement into the vasculature of post-veraison berries indicates not a loss of xylem function, but rather the loss of an appropriate driving force (hydrostatic gradient) in the berry apoplast. Based on this hypothesis, and the substantial decrease in xylem flows that occur in intact grape berries at veraison, it is suggested that there may be significant changes in the pattern of solute partitioning between the fruit symplast and apoplast at veraison. It is further suggested that diurnal patterns in symplast/apoplast solute partitioning in grapes and other fleshy fruit, may explain the observed minimal xylem contribution to the water budgets of these fruits.     

Water Content-Potential Relationship in Soya Bean: Changes in Component Potentials for Mature and Immature Leaves under Field Conditions

Changes in turgor and osmotic potentials of soya bean leaves(Glycine max.) with changes in water content were measured throughouta season using the pressure-volume technique. Two distinct reponsesto water loss were found. When water was expressed from leavesin the pressure chamber their osmotic behavior was describedby a concentration effect based on the osmotic volume. The osmoticfraction of the total water content averaged 0·72 and0·84 for mature and immature leaves, respectively. Thechanges in turgor pressure in the chamber were described bya volumetric modulus of elasticity which increased linearlywith turgor pressure. The changes in total potential at highturgor pressures were almost exclusively due to changes in turgordue to the high modulus (high tissue rigidity) in that range.Responses were different, however, for leaves drying in thefield. For these, the osmotic changes were always large anddominated by solute adjustment. Diurnal changes in osmotic potentialwere as much as 5 bars (500 kPa), or around 50 per cent, andwere about the same magnitude as the changes in turgor pressurefor both mature and immature leaves. The elastic modulus atthe time of sampling showed the normal turgor dependence forimmature leaves but for mature leaves the initial modulus wasapparently constant at about 180 bars. The different behaviourin the pressure bomb and the field is interpreted in terms ofa rate dependence for turgor and osmotic response to water loss.     

The effect of mechanical impedance on root growth in pea (Pisum sativum). II. Cell expansion and wall rheology during recovery

The aim of the present work was to determine the factors limiting growth in mechanically impeded roots. Pea roots were grown in compressed and uncompressed sand cores, and then removed and transferred to hydroponics. Root elongation was slowed in impeded sand cores and did not recover to the unimpeded rates until 60 h after transfer to the hydroponics system. Root diameter was greater in impeded roots, and only after 36 h in hydroponics was new root tissue produced of the same diameter as the unimpeded controls. The turgor pressure of the growing cells was measured with a turgor probe and was the same in both treatments. The slower elongation rate of the previously impeded roots was, therefore, the result of axial tightening of the cell walls. Cell length profiles suggested that axial cell wall tightening persisted in the unrestricted hydroponics system. Production of new cells in unrestricted conditions was required before root elongation returned to the unimpeded state. Osmotic potential was decreased by approximately 0.2 MPa in previously impeded roots compared with the unimpeded ones. This corresponds to a decrease in water potential of 0.2 MPa. These data are discussed in relation to regulation of cell extension, solute unloading and the penetration of compacted soils by roots.     

A Mathematical Treatment of Munch's Pressure-Flow Hypothesis of Phloem Translocation

The steady state solutions of two mathematical models are used to evaluate Münch's pressure-flow hypothesis of phloem translocation. The models assume a continuous active loading and unloading of translocate but differ in the site of loading and unloading and the route of water to the sieve tube. The dimensions of the translocation system taken are the average observed values for sugar beet and are intended to simulate translocation from a mature source leaf to an expanding sink leaf. The volume flow rate of solution along the sieve tube, water flow rate into the sieve tube, hydrostatic pressure, and concentration of sucrose in the sieve tube are obtained from a numerical computer solution of the models. The mass transfer rate, velocity of translocation, and osmotic and hydrostatic pressures are consistent with empirical findings. Owing to the resistance to water flow offered by the lateral membranes, the hydrostatic pressure generated by the osmotic pressure can be considerably less than would be predicted by the solute concentration. These models suggest that translocation at observed rates and velocities can be driven by a water potential difference between the sieve tube and surrounding tissue and are consistent with the pressure-flow hypothesis of translocation.     

Interpretation of an empirical model for stomatal conductance in terms of guard cell function

An empirical model for stomatal conductance (g), proposed by Leuning (1995, this issue) as a modification of Ball, Woodrow & Berry's (1987) model, is interpreted in terms of a simple, steady-state model of guard cell function. In this model, stomatal aperture is a function of the relative turgor between guard cells and epidermal cells. The correlation between g and leaf surface vapour pressure deficit in Leuning's model is interpreted in terms of stomatal sensing of the transpiration rate, via changes in the gradient of total water potential between guard cells and epidermal cells. The correlation between g, CO₂ assimilation rate and leaf surface CO₂ concentration in Leuning's model is interpreted as a relationship between the corresponding osmotic gradient, irradiance, temperature, intercellular CO₂ concentration and stomatal aperture itself. The explicit relationship between osmotic gradient and stomatal aperture (possibly describing the effect of changes in guard cell volume on the membrane permeability for ion transport) results in a decrease in the transpiration rate in sufficiently dry air. Possible extension of the guard cell model to include stomatal responses to soil water status is discussed.     

Gradients in water potential and turgor pressure along the translocation pathway during grain filling in normally watered and water-stressed wheat plants

The water relations parameters involved in assimilate flow into developing wheat (Triticum aestivum L.) grains were measured at several points from the flag leaf to the endosperm cavity in normally watered (Psi approximately -0.3 MPa) and water-stressed plants (Psi approximately -2 MPa). These included direct measurement of sieve tube turgor and several independent approaches to the measurement or calculation of water potentials in the peduncle, grain pericarp, and endosperm cavity. Sieve tube turgor measurements, osmotic concentrations, and Psi measurements using dextran microdrops showed good internal consistency (i.e. Psi = Psi(s) + Psi(p)) from 0 to -4 MPa. In normally watered plants, crease pericarp Psi and sieve tube turgor were almost 1 MPa lower than in the peduncle. This suggests a high hydraulic resistance in the sieve tubes connecting the two. However, observations concerning exudation rates indicated a low resistance. In water-stressed plants, peduncle Psi and crease pericarp Psi were similar. In both treatments, there was a variable, approximately 1-MPa drop in turgor pressure between the grain sieve tubes and vascular parenchyma cells. There was little between-treatment difference in endosperm cavity sucrose or osmotic concentrations or in the crease pericarp sucrose pool size. Our results re-emphasize the importance of the sieve tube unloading step in the control of assimilate import.