Ernst Steudle

Diurnal variations in hydraulic conductivity and root pressure can be correlated with the expression of putative aquaporins in the roots of Lotus japonicus

Abstract. The hydraulic conductivity of excised roots (Lpr) of the legume Lotus japonicus (Regel) K. Larsen grown in mist (aeroponic) and sand cultures, was found to vary over a 5-fold range during a day/night cycle. This behaviour was seen when Lpr was measured in roots exuding, either under root pressure (osmotic driving force), or under an applied hydrostatic pressure of 0.4 MPa which produced a rate of water flow similar to that in a transpiring plant. A similar daily pattern of variation was seen in plants grown in natural daylight or in controlled-environment rooms, in plants transpiring at ambient rates or at greatly reduced rates, and in plants grown in either aeroponic or sand culture. When detached root systems were connected to a root pressure probe, a marked diurnal variation was seen in the root pressure generated. After excision, this circadian rhythm continued for some days. The hydraulic conductivity of the plasma membrane of individual root cells was measured during the diurnal cycle using a cell pressure probe. Measurements were made on the first four cell layers of the cortex, but no evidence of any diurnal fluctuation could be found. It was concluded that the conductance of membranes of endodermal and stelar cells may be responsible for the observed diurnal rhythm in root Lpr. When mRNAs from roots were probed with cDNA from the Arabidopsis aquaporin AthPIP1a gene, an abundant transcript was found to vary in abundance diurnally under high-stringency conditions. The pattern of fluctuations resembled closely the diurnal pattern of variation in root Lpr. The plasma membranes of root cells were found to contain an abundant hydrophobic protein with a molecular weight of about 31 kDa which cross-reacted strongly to an antibody raised against the evolutionarily conserved N-terminal amino acid sequence of AthPIP1a.

Abscisic acid and hydraulic conductivity of maize roots: a study using cell- and root-pressure probes.

Abstract. Using root- and cell-pressure probes, the effects of the stress hormone abscisic acid (ABA) on the water-transport properties of maize roots (Zea mays L.) were examined in order to work out dose and time responses for root hydraulic conductivity. Abscisic acid applied at concentrations of 100–1,000 nM increased the hydraulic conductivity of excised maize roots both at the organ (root Lpr: factor of 3–4) and the root cell level (cell Lp: factor of 7–27). Effects on the root cortical cells were more pronounced than at the organ level. From the results it was concluded that ABA acts at the plasmalemma, presumably by an interaction with water channels. Abscisic acid therefore facilitated the cell-to-cell component of transport of water across the root cylinder. Effects on cell Lp were transient and highly specific for the undissociated (+)-cis-trans-ABA. The stress hormone ABA facilitates water uptake into roots as soils start drying, especially under non-transpiring conditions, when the apoplastic path of water transport is largely excluded.

Water transport in barley roots

Radial transport of water in excised barley (Hordeum distichon, cv. Villa) roots was measured using a new method based on the pressure-probe technique. After attaching excised roots to the probe, root pressures of 0.9 to 2.9 bar were developed. They could be altered either by changing the root pressure artificially (with the aid of the probe) or by changing the osmotic pressure of the medium in order to induce water flows across the root. The hydraulic conductivity of the barley roots (per cm2 of outer root surface) was obtained in different types of experiments (initial water flow, pressure relaxations, constant water flow) and was (0.3–4.3)·10-7 cm s-1 bar-1. The hydraulic conductivity of the root was by an order of magnitude smaller than the hydraulic conductivity of the cell membranes of cortical and epidermal cells (0.8–2.2)·10-6 cm s-1 bar-1. The half-times of water exchange of these cells was 1–21 s and two orders of magnitude smaller than that of entire excised roots (100–770 s). Their volumetric elastic modulus was 15–305 bar and increased with increasing turgor. Within the root cortex, turgor was independent of the position of the cell within a certain layer and turgor ranged between 3 and 5 bar. The large difference between the hydraulic conductivity of the root and that of the cell membranes indicates that there is substantial cell-to-cell (transcellular plus symplasmic) transport of water in the root. When it is assumed that 10–12 membrane layers (plasmalemma plus tonoplast) in the epidermis, cortex and endodermis form the hydraulic resistance to water flow, a value for the hydraulic conductivity of the root can be calculated which is similar to the measured value. This picture for water transport in the root contradicts current models which favour apoplasmic water transport in the cortex.

Apoplastic transport across young maize roots: effect of the exodermis

Abstract. The uptake of water and of the fluorescent apoplastic dye PTS (trisodium 3-hydroxy-5,8,10-pyrenetrisulfonate) by root systems of young maize (Zea mays L.) seedlings (age: 11–21 d) has been studied with plants which either developed an exodermis (Casparian band in the hypodermis) or were lacking it. Steady-state techniques were used to measure water uptake across excised roots. Either hydrostatic or osmotic pressure gradients were applied to induce water flows. Roots without an exodermis were obtained from plants grown in hydroponic culture. Roots which developed an exodermis were obtained using an aeroponic (=mist) cultivation method. When the osmotic concentration of the medium was varied, the hydraulic conductivity of the root (Lpr in m3 · m−2 · MPa−1 · s−1) depended on the osmotic pressure gradient applied between root xylem and medium. Increasing the gradient (i.e. decreasing the osmotic concentration of the medium; range: zero to 40 mM of mannitol), increased the osmotic Lpr. In the presence of hydrostatic pressure gradients applied by a pressure chamber, root Lpr was constant over the entire range of pressures (0–0.4 MPa). The presence of an exodermis reduced root Lpr in hydrostatic experiments by a factor of 3.6. When the osmotic pressure of the medium was low (i.e. in the presence of a strong osmotic gradient between xylem sap and medium), the presence of an exodermis caused the same reduction of root Lpr in osmotic experiments as in hydrostatic ones. However, when the osmotic concentration of the medium was increased (i.e. the presence of low gradients of osmotic pressure), no marked effect of growth conditions on osmotic root Lpr was found. Under these conditions, the absolute value of osmotic root Lpr was lower by factors of 22 (hydroponic culture) and 9.7 (aeroponic culture) than in the corresponding experiments at low osmotic concentration. Apoplastic flow of PTS was low. In hydrostatic experiments, xylem exudate contained only 0.3% of the PTS concentration of the bathing medium. In the presence of osmotic pressure gradients, the apoplastic flow of PTS was further reduced by one order of magnitude. In both types of experiments, the development of an exodermis did not affect PTS flow. In osmotic experiments, the effect of the absolute value of the driving force cannot be explained in terms of a simple dilution effect (Fiscus model). The results indicate that the radial apoplastic flows of water and PTS across the root were affected differently by apoplastic barriers (Casparian bands) in the exodermis. It is concluded that, unlike water, the apoplastic flow of PTS is rate-limited at the endodermis rather than at the exodermis. The use of PTS as a tracer for apoplastic water should be abandoned.

Water transport across roots

Usually, roots are looked at as rather perfect osmometers with the endodermis being the ‘root membrane’ which is equivalent to the plasma membrane of cells. However, this ‘single-equivalent-membrane model’ of the root does not explain the findings of a variable hydraulic resistance of roots as well as of differences between hydraulic and osmotic water flow and of low reflection coefficients of roots. Recent work with the root pressure probe is reviewed and discussed which indicates that the simple osmometer model of the root has to be extended by incorporating its composite structure, i.e. the fact that there are different parallel pathways for water in the root, namely, the cell-to-cell and apoplasmic path. The new ‘composite transport model of the root’ readily explains the experimental findings mentioned above. Pressure probe work with roots in which the endodermis was punctured to create an additional parallel path as well as anatomical studies support the model.

Chemical composition of apoplastic transport barriers in relation to radial hydraulic conductivity of corn roots (Zea mays L.)

Abstract. The hydraulic conductivity of roots (Lpr) of 6- to 8-d-old maize seedlings has been related to the chemical composition of apoplastic transport barriers in the endodermis and hypodermis (exodermis), and to the hydraulic conductivity of root cortical cells. Roots were cultivated in two different ways. When grown in aeroponic culture, they developed an exodermis (Casparian band in the hypodermal layer), which was missing in roots from hydroponics. The development of Casparian bands and suberin lamellae was observed by staining with berberin-aniline-blue and Sudan-III. The compositions of suberin and lignin were analyzed quantitatively and qualitatively after depolymerization (BF3/methanol-transesterification, thioacidolysis) using gas chromatography/mass spectrometry. Root Lpr was measured using the root pressure probe, and the hydraulic conductivity of cortical cells (Lp) using the cell pressure probe. Roots from the two cultivation methods differed significantly in (i) the Lpr evaluated from hydrostatic relaxations (factor of 1.5), and (ii) the amounts of lignin and aliphatic suberin in the hypodermal layer of the apical root zone. Aliphatic suberin is thought to be the major reason for the hydrophobic properties of apoplastic barriers and for their relatively low permeability to water. No differences were found in the amounts of suberin in the hypodermal layers of basal root zones and in the endodermal layer. In order to verify that changes in root Lpr were not caused by changes in hydraulic conductivity at the membrane level, cell Lp was measured as well. No differences were found in the Lp values of cells from roots cultivated by the two different methods. It was concluded that changes in the hydraulic conductivity of the apoplastic rather than of the cell-to-cell path were causing the observed changes in root Lpr.

Transport of Water and Solutes across Maize Roots Modified by Puncturing the Endodermis (Further Evidence for the Composite Transport Model of the Root)

The effects of puncturing the endodermis of young maize roots (Zea mays L.) on their transport properties were measured using the root pressure probe. Small holes with a diameter of 18 to 60 [mu]m were created 70 to 90 mm from the tips of the roots by pushing fine glass tubes radially into them. Such wounds injured about 10–2 to 10–3% of the total surface area of the endodermis, which, in these hydroponically grown roots, had developed a Casparian band but no suberin lamellae. The small injury to the endodermis caused the original root pressure, which varied from 0.08 to 0.19 MPa, to decrease rapidly (half-time = 10–100 s) and substantially to a new steady-state value between 0.02 and 0.07 MPa. The radial hydraulic conductivity (Lpr) of control (uninjured) roots determined using hydrostatic pressure gradients as driving forces was larger by a factor of 10 than that determined using osmotic gradients (averages: Lpr [hydrostatic] = 2.7 x 10–7 m s-1 MPa-1; Lpr [osmotic] = 2.2 x 10–8 m s-1 MPa-1; osmotic solute: NaCl). Puncturing the endodermis did not result in measurable increases in hydraulic conductivities measured by either method. Thus, the endodermis was not rate-limiting root Lpr: apparently the hydraulic resistance of roots was more evenly distributed over the entire root tissue. However, puncturing the endodermis did substantially change the reflection ([sigma]sr) and permeability (Psr) coefficients of roots for NaCl, indicating that the endodermis represented a considerable barrier to the flow of nutrient ions. Values of [sigma]sr decreased from 0.64 to 0.41 (average) and Psr increased by a factor of 2.6, i.e. from 3.8 x 10–9 to 10.1 x 10.-9 m s-1(average). The roots recovered from puncturing after a time and regained root pressure. Measurable increases in root pressure became apparent as soon as 0.5 to 1 h after puncturing, and original or higher root pressures were attained 1.5 to 20 h after injury. However, after recovery roots often did not maintain a stable root pressure, and no further osmotic experiments could be performed with them. The Casparian band of the endodermis is discontinuous at the root tip, where the endodermis has not yet matured, and at sites of developing lateral roots. Measurements of the cross-sectional area of the apoplasmic bypass at the root tip yielded an area of 0.031% of the total surface area of the endodermis. An additional 0.049% was associated with lateral root primordia. These areas are larger than the artificial bypasses created by wounding in this study and may provide pathways for a “natural bypass flow” of water and solutes across the intact root. If there were such a pathway, either in these areas or across the Casparian band itself, roots would have to be treated as a system composed of two parallel pathways (a cell-to-cell and an apoplasmic path). It is demonstrated that this “composite transport model of the root” allows integration of several transport properties of roots that are otherwise difficult to understand, namely (a) the differences between osmotic and hydrostatic water flow, (b) the dependence of root hydraulic resistance on the driving force or water flow across the root, and (c) low reflection coefficients of roots.

Water transport in plants: Role of the apoplast

The present state of modelling of water transport across plant tissue is reviewed. A mathematical model is presented which incorporates the cell-to-cell (protoplastic) and the parallel apoplastic path. It is shown that hydraulic and osmotic properties of the apoplast may contribute substantially to the overall hydraulic conductivity of tissues (Lpr) and reflection coefficients (67-1). The model shows how water and solutes interact with each other during their passage across tissues which are considered as a network of hydraulic resistors and capacitances (‘composite transport model’). Emphasis is on the fact that hydraulic properties of tissues depend on the nature of the driving force. Osmotic gradients cause a much smaller tissue Lpr than hydrostatic. Depending on the conditions, this results in variable hydraulic resistances of tissues and plant organs. For the root, the model readily explains the well-known phenomenon of variable hydraulic resistance for the uptake of water and non-linear force/flow relations. Along the cell-to-cell (protoplastic) path, water flow may be regulated by the opening and closing of selective water channels (aquaporins) which have been shown to be affected by different environmental factors. H Lambers Section editor

Determination of permeability coefficients, reflection coefficients, and hydraulic conductivity ofChara corallina using the pressure probe: Effects of solute concentrations

SummaryThe pressure probe technique which has been used for measurement of water relations parameters of plant cells [hydraulic conductivity (Lp), elastic modulus (ε) and half-time of water flow equilibration for individual cells (T12/w)] can be used also for measuring reflection and permeability coefficients (σ,Ps) of permeable solutes. In the presence of a permeable osmoticum the pressure/time curves are biphasic, i.e. after a rapid water flow bringing turgor pressure to a minimum value (Pmin), a second phase occurs in which turgor pressure increases back to the original value (Po). The second phase (“solute phase”) is due to an equilibration of the solute across the cell membrane and can be used to evaluatePs. The responses are strictly reversible, i.e. when the osmoticum is removed a pressure maximum is quickly reached followed by a slower equilibration of solute. The reflection coefficients for the solutes can be calculated from the change in osmotic pressure of the medium (Δψs0) and from the change in turgor at the minimum (Po−Pmin) after correcting for solute flow. For internodal cells ofChara corallina, reflection and permeability coefficients for certain nonelectrolytes (sugars, polyols, monohydroxyalcohols, amides, ketones) are given and compared with data obtained by other methods. ForChara, Ps and osmotic values ofLp depended on external stirring, whereas σ corrected for solute flow did not. As expected hydrostaticLp did not depend on stirring. No polarity of water flow was found for hydrostaticLp (Lpen/Lpex=1.02±0.05, 95% confidence limits) whereas a polarity was observed for osmoticLp which can be explained in terms of a concentration effect. Using permeable solutes, the concentration dependence ofLp, σ andPs could be measured over large concentration ranges (up to 1.4m) at constant cell turgor.Ps was independent of solute concentration for concentrations up to 1.4m while both σ andLp decreased with increasing concentration such that there was a linear relationship between (1−σ) and 1/Lp as predicted by the frictional model for a lipid membrane with pores. The slope of the (1−σ)vs. 1/Lp plot gives a value ofPs and the intercept with the (1−σ) axis gives the degree of frictional interaction between solute and water. The frictional term was found to be significantly greater than zero. The values ofPs evaluated from the solute phase were smaller than those obtained from the (1−σ)vs. 1/Lp plots. However, they were of the same order of magnitude and showed the same sequence for the different solutes. The technique for determiningPs and σ is of importance for obtaining quantitative data for the permeation of water-soluble pollutants into plant cells and tissues and for their ecotoxicological significance.

Osmotic responses of maize roots: water and solute relations

Water and solute relations of excised seminal roots of young maize (Zea mays L) plants, have been measured using the root pressure probe. Upon addition of osmotic solutes to the root medium, biphasic root pressure relaxations were obtained as theoretically expected. The relaxations yielded the hydraulic conductivity Lpr) the permeability coefficient (Psr), and the reflection coefficient (σsr) of the root. Values of Lpr in these experiments were by nearly an order of magnitude smaller than Lpr values obtained from experiments where hydrostatic pressure gradients were used to induce water flows. The value of Psr was determined for nine different osmotica (electrolytes and nonelectrolytes) which resulted in rather variable values (0.1·10-8–1.7·10-8m·s-1). The reflection coefficient σsr of the same solutes ranged between 0.3 and 0.6, i.e. σsr was low even for solutes for which cell membranes exhibit a σs≈1. Deviations from the theoretically expected biphasic responses occured which may have reflected changes of either Psr or of active pumping induced by the osmotic change. The absolute values of Lpr, Psr, and σsr have been critically examined for an underestimation by unstirred layer effecs. The data indicate a considerable apoplasmic component for radial movement of water in the presence of hydrostatic gradients and also some solute flow byppassing root protoplasts. In the presence of osmotic gradients, however, there was a substantial cell-to-cell transport of water. Cutting experiments demonstrated that the hydraulic resistance for the longitudinal movement of water was much smaller than for radial transport except for the apical ends of the segments (length=5 to 20 mm). The differences in Lpr as well as the low σsr values suggest that the simple osmometer model of the root with a single osmotic barrier exhibiting nearly semipermeable properties should be extended for a composite membrane model with hydraulic and osmotic barriers arranged in series and in parallel.