JournalofExperimentalBotany,Vol.51,No.350 WDSpecialIssue,pp.1531–1542,September2000 Water uptake by roots: effects of water deficit ErnstSteudle1 Lehrstuhlfu¨rPflanzeno¨kologie,Universita¨tBayreuth,D-95440Bayreuth,Germany Received22March2000;Accepted24May2000 Abstract switch from the hydraulic to the osmotic mode is purely physical. In the presence of heavily suberized The variable hydraulic conductivity of roots (Lp) is r roots, the apoplastic component of water flow may be explained in terms of a composite transport model. It too small. Under these conditions, a regulation of is shown how the complex, composite anatomical radial water flow by water channels dominates. Since structure of roots results in a composite transport of waterchannelsareundermetaboliccontrol,thiscom- bothwaterandsolutes.Inthemodel,theparallelapo- ponent represents an ‘active’ element of regulation. plastic and cell-to-cell (symplastic and transcellular) Composite transport allows for an optimization of the pathways play an important role as well as the differ- water balance of the shoot in addition to the well- ent tissues and structures arranged in series within known phenomena involved in the regulation of water the root cylinder (epidermis, exodermis, cortex, endo- flow (gas exchange) across stomata. The model is dermis, stelar parenchyma). The roles of Casparian employed to explain the responses of plants to water bands and suberin lamellae in the root’s endo- and deficit and other stresses. During water deficit, the exodermis are discussed. Depending on the develop- cohesion–tension mechanism of the ascent of sap in mental state of these apoplastic barriers, the overall thexylemplaysanimportantrole.Resultsaresummar- hydraulic resistance of roots is either more evenly ized which prove the validity ofthe coehesion/tension distributedacrosstherootcylinder(youngunstressed theory. Effects of the stress hormone abscisic acid roots) or is concentrated in certain layers (exo- and (ABA) are presented. They show that there is an apo- endodermis in older stressed roots). The reason for plasticcomponentoftheflowofABAintherootwhich thevariabilityofrootLp,isthathydraulicforcescause r contributes to the ABA signal in the xylem. On the a dominating apoplastic flow of water around proto- otherhand,(+)-cis-trans-ABAspecificallyaffectsboth plasts, even in the endodermis and exodermis. In the the cell level (water channel activity) and water flow absence of transpiration, water flow is osmotic in driven by gradients in osmotic pressure at the root naturewhichcausesahighresistanceaswaterpasses level which is in agreement with the composite trans- across many membranes on its passage across the port model. Hydraulic water flow in the presence of root cylinder. The model allows for a high capability gradients inhydrostaticpressureremainsunchanged. ofrootstotakeupwaterinthepresenceofhighrates The results agreewiththe composite transportmodel of transpiration (high demands for water from the and resemble earlier findings of high salinity obtained shoot). By contrast, the hydraulic conductance is low, for the cell (Lp) and root (Lp) level. They are in line whentranspirationisswitchedoff.Overall,thisresults r with known effects of nutrient deprivation on root Lp in a non-linear relationship between water flow and r and the diurnal rhythm of root Lp recently found in forces(gradientsofhydrostaticandosmoticpressure) r roots of Lotus. which is otherwise hard to explain. The model allows for special root characteristics such as a high Key words: Composite transport, hydraulic conductivity, hydraulicconductivity(waterpermeability)inthepres- regulation, root, stress, water deficit, water uptake. enceofalow permeabilityofnutrient ionsoncetaken up into the stele by active processes. Low root Introduction reflectioncoefficientsareinlinewiththeideaofsome apoplasticbypassesforwaterwithintherootcylinder. Many efforts have been made in the past to understand According to the composite transport model, the the water balance of plants in terms of a regulation of 1Fax:+49921552564.E-mail:[email protected] © SocietyforExperimentalBiology2000 1532 Steudle transpiration,i.e. ofhowstomatal conductance wouldbe affected by water status, light intensity, nutrition, and other factors and how this would change under stress conditionssuchasduringdeficitsinwatersupply(Cowan, 1977; Schulze, 1986; Kramer and Boyer, 1995; Jones, 1998). Fewer efforts have been made to investigate the input side of the water balance, i.e. the acquisition of water from the soil. It has been known for a long time that the hydraulic conductance of roots is variable (Brewig, 1937; Brouwer, 1954; Weatherley, 1982; Steudle et al., 1987; Steudle, 1994; Steudle and Peterson, 1998; Henzleret al., 1999).It depends onfactors such aswater shortage and salinity of the soil and on the demands for waterfromthetranspiringshoot.Otherimportantfactors are nutrient deficiency, anoxia, temperature, and heavy metals (Munns and Passioura, 1984; Radin and Eidenbock,1984;Azaizehetal.,1992;BirnerandSteudle, 1993; Maggio and Joly, 1995; Carvajal et al., 1996; Peyranoet al.,1997;Schreiberetal.,1999;Henzleretal., 1999; Clarkson et al., 2000). The lack in current know- ledgeofhowthesefactorsinfluencewatersupplyislargely due to technical problems in measuring, in sufficient Fig.1. Simplified model of steady water flow across a plant. Water detail,thehydraulicarchitectureofroots.Basichydraulic uptakebyroots,long-distancetransportinthexylemandthestomatal resistancearedenotedbyvariablehydraulicresistorsarrangedinseries. properties of roots are not yet adequately understoood, Usually, the resistance of the root is smaller than that of the shoot simply because roots in the soil are much less accessible which is dominatedby resistancesof vapour pathways in the leafand than shoots. Comparative measurements of root the boundary layer outside the leaf. The resistance along the xylem is hydraulics(differentspecies,treatmentsetc.)requiremeas- relatively small. Hence, at a given water flow through the system, the root hydraulic resistance in relation to that of stomata will determine urement of water flows, driving forces (pressure and the water status (water potential) of the shoot (arrow). In order to osmotic gradients between root surface and xylem) and improve the water status of the shoot (y ), the plant can either shoot improve water uptake by reducing R or avoid water losses by root surface area. Since growing conditions change the root increasingR . root structure and anatomy, this has to be characterized shoot as well. In a schematic way, it is shown in Fig.1 how the ant under conditions of stress, when high tensions in the variableroothydraulicresistancewouldcontributetothe xylemcausecavitationandinterruptwaterflowinvessels water potential (water status) of the shoot. Usually, the between root and shoot (Tyree and Sperry, 1988). vapourpathwaysinmesophyllairspaces,stomatalpores, It is generally accepted that the contribution of active and the boundary layer outside the leaves represent the water transport in plants, i.e. water flow directly coupled highest hydraulic resistance in the soil–plant–air con- to metabolic reaction(s), should be negligible. This is tinuum (SPAC), and most of the water potential differ- because the water permeability of plant cell membranes ence between soil and atmosphere will drop here (Nobel, is larger by several orders of magnitude than that of 1991; Yang and Tyree, 1994). At a given water potential soluteswhichwouldcausearapidshortcircuitofprimary difference (y –y ) and water flow across the soil– activewaterflow(Steudle,1989,1992).Insteadofactively soil atm plant–air continuum, the water potential of the shoot taking up water, roots just allow it to pass through them may be dominated by the drop in water potential across in response to water potential gradients usually set upby the root hydraulic resistance which is usually the highest transpiration. The complex anatomical structure of roots within the liquid part of the SPAC. In other words, to results in a complex pattern of water flow. Transport improve the water status of the shoot, the plant could across various tissues (epidermis, cortex, stele) has to be either increase stomatal or decrease roothydraulic resist- considered during the radial passage across the root ance; the latter does happen (besides the regulation of cylinder as well as a longitudinal flow component (axial transpiration). It is not clear whether or not this is a transport in xylem vessels). During stress, the anatomy purelyphysicaladjustmentorinvolvessomeactiveregula- of root tissue will change, largely, because stress (such as tion of root hydraulic properties (besides the stress- water deficit) induces the development of apoplastic bar- induced changes in anatomy which are obvious). As riers for water and ion flow (Stasovski and Peterson, indicated in the figure, the hydraulic resistance of the 1991; Taleisnik et al., 1999). Their formation represents xylemisrelativelysmall.Itmay,however,becomeimport- a fundamental adaptive strategy of plants to survive in Wateruptakebyroots 1533 anadverseenvironment(NorthandNobel,1995;Peyrano Roleofrootanatomy:theapoplast et al., 1997; Zimmermann and Steudle, 1998; Schreiber The interpretation of root hydraulic data requires suffi- et al., 1999; Zimmermann et al., 2000). In addition, the cient knowledge of their structure. Namely, this refers to hydraulic conductivity of root cell membranes may be thedevelopmentofsuberinlamellaeandCasparianbands reducedduetoaclosureofwaterchannelsoraquaporins intheapoplast.Therearemajordifferencesamongspecies in root cell membranes. These transport proteins have and growing conditions. In the past few years there has received much attentionin the past years (Chrispeelsand beenimportantprogress incharacterizingapoplastic bar- Maurel, 1994; Steudle and Henzler, 1995; Maurel, 1997; riers caused bydrought and other environmental factors. Steudle and Peterson, 1998; Tyerman et al., 1999; In maize, the absorption of water and nutrient ions may Kjellbom et al., 1999). So, besides transport across the be significantly reduced during drought stress (Stasovsky apoplast,thereissomewaterflowacrosscellmembranes. Different from the irreversible chemical modifications of andPeterson,1991).Stressedrootsdevelopedasuberized interface between living tissue and rhizosphere to minim- the apoplast (Casparian bands, suberin lamellae), the ize water losses. It appeared that, upon rehydration, root intensity of the cellular component may be either revers- systems recovered their hydraulic conductance (which, ibly regulated by an opening or closing of existing water however, was not measured) by elongation growth thus channels or the density or aquaporins may be increased forming new roots of high hydraulic conductivity. by recruitment of internalized protein into the plasma Taleisnik et al. grew plants under conditions of water membrane. During root development the relative contri- shortage and controls and compared the ability of roots bution of components of overall water flow may change, toretainwaterfromplantswhichhaveanexodermiswith when the endodermis and exodermis develop (Frensch those which do not (Taleisnik et al., 1999). Rates of etal.,1996).Waterdeficitsreducerootgrowthandcause water loss were smaller in exodermal than in non- a pronounced suberization of the apoplast and, perhaps, also affect the cellular passage (water channels). This exodermal roots. Older parts of roots released less water to the dry environment than younger. Rieger and Litvin influences the water balance by reducing the capacity of concluded from hydraulic measurements on root systems roots to take up water. (drought-stressed and controls) of intact plants of differ- The radial flow of water across plant roots along the ent herbaceous and woody species that the existence of a apoplastwithitsbarriersand theparallelcell-to-cellpath suberized exodermis had some influence on the overall (aquaporins) is best described by a composite transport model which allows for differences in movement through root Lpr (Rieger and Litvin, 1999). However, the resist- anceofthecortexappearedtobeofequalorevengreater individual cell layerssuch as the exo-and endodermis, as importance than that of the suberized layers. Recent wellasthroughvarioustissues(Steudleetal.,1993,1999; Steudle, 1994, 1997; Steudle and Frensch, 1996; Steudle measurements on excised roots of young maize seedlings andPeterson,1998).The modelallowsforaninteraction have shownthatthehydraulicconductivitywaslargerby between parallel flows. The axial component of water afactorof3.6(onaverage)whentherewasnoexodermis flow may be incorporated (Frensch and Steudle, 1989; (roots grown in hydroponic culture) as compared with Steudle and Peterson, 1998). roots having an exodermis (roots grown in aeroponics) In this review, some basic properties of root water (Zimmermann and Steudle, 1998). In these experiments, transport and how they would change during conditions apossibleeffectofachangeofthehydraulicconductivity of water deficit are discussed. The focus will be on the ofrootcellmembranes(waterchannels)hasbeenexcluded relative roles of apoplastic barriers and aquaporins, but bymeasuringtherootcellLp(cellpressureprobe)besides changes in the driving forces according to the cohesion– the overall root Lp (root pressure probe; Zimmermann r tension mechanism of the ascent of sap will also be et al., 2000). Hence, there was a substantial effect of considered.Somerecentresults of theeffects ofthestress suberization on the overall hydraulic properties of roots. hormone abscisic acid (ABA) will be included and meas- In the past few years there has been considerable urements in which changes in the chemical composition progress in identifying the chemical composition of apo- ofapoplastic barriershavebeencorrelatedwithmeasure- plastic barriers. After digesting away the rest of the root, ments of the hydraulic properties of roots both at the it has been possible to separate Casparian bands from cell and whole root levels (Freundl et al., 1998, 2000; the exo- and endodermis and to analyse these structures Hose et al., 2000; Zimmermann and Steudle, 1998; chemically and to refer the results to root hydraulic Zimmermann et al., 2000). This review will also refer to properties (Schreiber, 1996; Zeier and Schreiber, 1998; olderdataontheeffectsofsalinityandmorerecentwork Schreiber et al., 1999; Zimmermann et al., 2000). The on nutrient stresses and diurnal aquaporin expression results show that Casparian bands contain both aliphatic which may be important with respect to water deficit and aromatic suberin. The former is thought to be more (Azaizehand Steudle,1991;Azaizeh etal., 1992;Henzler hydrophobic and less permeable to water, the latter is a et al., 1999; Clarkson et al., 2000). lignin-like phenolic polymer esterified with hydroxycin- 1534 Steudle namic acids. In addition, Casparian bands contain con- Pathwaysforwaterandsolutesandcomposite siderable amounts of lignin. Since lignin is a relatively transport hydrophilic compound, the analyses from Schreiber’s When water and solutes (nutrients) move into a root, group suggest that Casparian bands would be fairly there will be a cellular (cell-to-cell) and an apoplastic impermeable to ions and to rather big polar solutes, but component of flow (Steudle, 1989, 1992). The passive may allow some passage of water and small solutes. This cell-to-cell component of solute flow should be small is in agreement with transport measurements. because of the low permeability of solutes to membranes (nutrient ions). For water this is different, because water Natureofwatermovementinroots movesbyseveralordersofmagnitudemorerapidlyacross membranes than do ions. In principle, the cell-to-cell In textbooks, the movement of water in roots is often (‘protoplastic’) component would have to be split into a describedasanosmoticprocessinwhichrootmembranes symplastic and a transcellular component (Fig.2A). The play the important role (Kramer and Boyer, 1995). The former is the component due to transport across plas- rootislookedatasanearlyperfectosmometer.Hydraulic modesmata within the symplast. The latter refers to the resistances involved in the radial movement of water are passage across plasmalemmata (two membranes per cell thought to be constant. However, evidence collected layer). The vacuolar component is not included, the during the past decade indicates that this view has to be contribution of which should be small (Steudle and modified. Along the apoplastic path, water movement Peterson,1998).Experimentally,thesymplasticandtrans- will be hydraulic in nature, i.e. driven by gradients in hydrostatic pressure. Different from cell membranes, cell cellular components can not be separated to date. Therefore, theyare summarized for pragmatic reasonsas walls have no selective properties, i.e. they do not select a cell-to-cell component. During the radial passage of between water and solutes as they pass through. Hence, water across the root, there could be combinations of gradients in solute concentration or osmotic pressure pathways in that water may travel within the symplast alongtheapoplast(Dp,wherethesubscript‘s’standsfor s for some distance and may then cross the plasma mem- solute) do not result in a significant water flow. This is brane and move within the cell wall. These combinations so because, in the apoplast, the osmotic component of water potential has to be modified by a reflection coeffi- can be included in models for water transport as long as allowance ismade fora rapidexchange ofwater between cient (scw, where the additional superscript ‘cw’ stands s pathways (‘local equilibrium’; Molz and Ferrier, 1982; for‘cellwall’),whichisameasureofthe‘passiveselectiv- ity’ of the apoplast. The reflection coefficient of the Westgate and Steudle, 1985; Steudle, 1989, 1992; Steudle apoplastwillusuallybeclosetozero,sothatthe‘effective’ and Frensch, 1996). According to these models, the osmotic component of water potential which drives the contribution of the cell-to-cell component can be derived flow across the apoplast, i.e. scwDp, will be also small. from measurements with the cell pressure probe. The s s Along the cell-to-cell path, the situation is different apoplastic component (which is, to date, not accessible because of the presence of membranes which exhibit a to direct measurement in intact tissue) is obtained from s#1.Theoverallroots willbebetweenzeroandunity the difference between the hydraulic conductivity of the s sr asfoundinexperimentswiththerootpressureprobeand whole root and that of the cell-to-cell component (Zhu other techniques (Miller,1985; Steudle et al., 1987, 1993; and Steudle, 1991). When apoplastic barriers are formed Steudle and Frensch, 1989; Steudle and Peterson, 1998). which interrupt the flow around root protoplasts in the As a consequence of their composite structure, roots do endo-andexodermisalongadevelopingroot,therelative not behave like ideal osmometers which would exhibit a contributions of hydraulic and osmotic flow change reflection coefficient of unity. In addition to the low (Frensch et al., 1996). reflection coefficient of roots, the hydraulic conductivity The composite structure of roots allows a fairly high of roots should depend on the force (osmotic and hydro- and adjustable water permeability in the presence of a staticpressuregradients)usedtodrivewateracrossroots. pronouncedabilitytoretainnutrientsaltstransferredinto This is also a consequence of composite transport. In the the xylem (low solute permeability). This would be hard presenceofdifferentforces,thetwopathways(apoplastic to achieve with a homogeneous membrane or osmotic andcell-to-cell)willbeusedwithdifferentintensity.Both barrier. The composite transport model explains experi- pathways will be used in the presence of a hydraulic mentalfindingssuchas(i)lowrootreflectioncoefficients, (hydrostatic pressure) gradient such as during transpir- (ii) differencesbetweenosmotic and hydraulicwater flow ation. In the presence of an osmotic gradient, however, and the variability of root Lp, and (iii) differences r cell-to-cell transport will dominate. The hydraulic com- between roots of woody and herbaceous plants. ponent will be small if any. As a consequence, the Low reflection coefficients can be simply explained by hydraulic conductivity of roots should differ depending the existence of apoplastic bypasses in arrays where on the conditions as found experimentally (see below). Casparianbandshavenotyetdevelopedorevenbysome Wateruptakebyroots 1535 Fig.2. (A) Routes of radial flow of water across plant roots. The apoplastic path is around protoplasts. The symplastic path is mediated by plasmodesmatawhichbridgethecellwallsbetweenadjacentcellssothatacytoplasmiccontinuumisformed.Duringthepassagealongtheapoplast and symplast, no membranes have to be crossed. On the transcellular path, two plasma membranes have to be crossed per cell layer. The transcellularpathissomethingspecialforwaterwhichhasahighmembranepermeability(cellhydraulicconductivity).Usually,thiscomponentis negligible for solutes. Note that the symplastic and transcellular flow components can not be separated experimentally and are summarized as a cell-to-cellcomponentofwaterflow(seetext). Duetotherapidwaterexchangebetweenprotoplastsandadjacentapoplast,thereshouldbelocal waterflowequilibriumbetweenthetwocompartmentsatanytime.Intheendodermisandexodermisofroots,theapoplasticflowcomponentcan bemodifiedbytheexistenceofapoplasticbarriers(Casparianbands,suberinlamellae).Theseareusuallythoughttobeimpermeableforwaterand solutes(nutrientions).(B)Thecomplexcompositestructureofrootscauses‘compositetransport’.Thisisduetothefactthatalongtheapoplastic paththerearenomembranesand,hence,thereisnoosmoticwaterflowbecauseofawallreflectioncoefficientofnearlyzero.Ontheotherhand, there is a considerable hydraulic flow across the apoplast. Across the cell-to-cell path, both flow components (hydraulic and osmotic flow) are present,butthispassagehasarelativelyhighhydraulicresistancebecausetherearealotofmembraneswhichhavetobecrossed.Therewillbeno hydraulicwaterflowacrosstheapoplastintheabsenceofhydrostaticpressuregradients(atzerotranspiration).Osmoticflowwillbesmall,because ofthehighresistanceundertheseconditions.Dependingontheefficiencyofapoplasticbarriers,thisresultsinlargedifferencesbetweenhydraulic andosmoticflowwhichcausesvariabilityofrootLp.Tensionsinthexylemplayanimportantrole.Atzeroorlowwaterflows,acirculationflow of water can be set up within the root as indicatedr. Note that, different from (A), only one Casparian band is shown in (B) for the sake of simplicity.Theatmosphericpressureoftherootmediumistakenasareferenceofpressure,i.e.P =0. m bypass flow of water across the Casparian band itself tionsinwhichoverallflows(water,solutes)andreflection (Fig.2B).Theyaresupportedbycombinedmeasurements coefficients have beencalculatedusing measuredrootcell of cell Lp and root Lp and by puncturing experiments data (W Melchior and E Steudle, unpublished results). r (Zhu and Steudle, 1991; Azaizeh et al., 1992; Steudle Differencesbetweenhydraulicandosmoticwaterfloware et al., 1993; Peterson and Steudle, 1993). The existence considerable and more pronounced in roots of woody of parallel elements of low (apoplast) and high (cell-to- than herbaceous plants. In woody species, they can be as cell) reflection coefficients should result in a circulation large as two to three orders of magnitude (Ru¨dinger flow of water in the root at zero overall water flow et al., 1994; Steudle and Meshcheryakov, 1996; Steudle (Fig.2B),whichcanbedemonstratedincomputersimula- and Heydt, 1997). This is in line with the theory. The 1536 Steudle variable root Lp, is largely explained by a change in the show that this is true (North and Nobel, 1991, 1995; r driving force (see below), but other reasons have been Melchior and Steudle, 1993). discussed as well (Fiscus, 1975; Passioura, 1988). During water stress, the formation of suberin lamellae in the endo- and exodermis could result in localized high resistances for water and ion flow in the root apoplast. Physiologicalconsequencesofcomposite The exodermis could, thus, contribute to the regulation transport of water uptake into roots as suggested by experiments The most obvious consequence of composite transport is in which roots were grown in soil with water deficits or that water supply of the shoot by the root may change nutrient solution with high salinity (Stasovski and according to the demand of the shoot, simply by a Peterson, 1991; Cruz et al., 1992; Azaizeh and Steudle, ‘physical adjustment’ of root Lp. In a transpiring plant, 1991; Azaizeh et al., 1992; Peyrano et al., 1997; Taleisnik r the hydrostatic pressure gradient between soil solution et al., 1999). However, recent measurements on young and xylem will be high (tensions in the xylem), and the onion roots grown in vermiculite have shown that root hydraulicresistanceoftherootlow.Thisfacilitateswater Lp increased as the exodermisdeveloped atconstant cell r uptake with increasing demand. It appears that there is a Lp (Barrowclough et al., 2000). These results indicate ‘switching’ between the hydraulic and osmotic modes of that, different from maize, the hydraulic resistance may action of roots. On the other hand, the hydraulic resist- be more evenly distributed across the root cylinder in ance willbe high at low rates oftranspiration, i.e. during onion even during stages where the exodermis is already the night or during periods of water deficit. Under these developed. To date, there are, for technical reasons, no conditions,plants may be protected from excessive water quantitative values of the permeability of the exo- and loss to the soil by the high hydraulic resistance of its endodermisofintactrootstowater.Thesedetailedmeas- roots.Thus,themodeltendstooptimizethewaterbalance urements are necessary to work out the functions of the by adjusting root hydraulics according to the demand two structures during water deficit and other conditions fromtheshoot(Fig.1).Theleakrateforsolutes(nutrient of stress. Although limited in application, the approach salts) is kept low throughout. Typically, permeability of Zimmermann and Steudle already mentioned may be coefficients of roots to nutrient salts are similar to those usedinwhichavariationofgrowingconditionsproduced of cell membranes (Steudle and Brinckmann, 1989). roots with or without an exodermis (Zimmermann and Steudle, 1998). Another approach would imply measure- ments of changes in cell Lp and of the propagation of Roleoftheendodermisandexodermis changes inwaterpotential(osmotic andhydrostaticpres- Measurementswithexcisedrootsofyoungmaizeseedlings sure) across the rootcylinder (cell pressure probe). These grown in hydroponic culture have shown that the experiments are not easy to perform, but the techniques hydraulicresistanceisnotconcentratedintheendodermis for making them are, in principle, developed (Zhu and (which would be the conventional view). When grown in Steudle,1991).Theyshouldbeusedtoseehowhydraulic hydroponics, roots developed no exodermis (Peterson resistances of the two layers would changein response to and Steudle, 1993; Peterson et al., 1993; Steudle et al., water deficits. Suberized cellsof theexo-and endodermis 1993).Ratherthanbeingconcentratedintheendodermis, shouldhaveasmallercellLp.Hence,ratesofthepropaga- the resistance was more evenly distributed in the living tion of changes in water potential of the root medium roottissue. Vessel walls which were already thickend and across the root (as measured with the cell pressure probe contained lignin, contributed to only 10–30% of the in cortex cells) should be smaller in the presence of a overall resistance to water movement. The endodermis suberized exodermis. was modified by Casparian bands, but there were no suberin lamellae. So, the conclusion that the endodermis Solutetransport (with Casparian bands only) was not a major barrier to water movement could only be drawn for these young Water is much more mobile along both the apoplastic roots grown in hydroponics. In roots grown under ‘real’ and cell-to-cell path than solutes. Therefore, the move- conditions,extrapolationfromthemeasurementsonroots ment of water in the apoplast can not be quantified by growninaeroponicsandfromotherresultsindicatedthat followingthe movementofapoplastic dyes.For example, this is not usually the case. Definitely, the exodermis in the experiments of Zimmermann and Steudle, the represents a barrier for water movement for maize roots external media contained the ionic apoplastic tracer PTS grown under real conditions. It is expected that suberin (3-hydroxy-5,8,10-pyrene-trisulphonate; Peterson et al., lamellaelaiddowninthetangential,radialandtransverse 1981; Hanson et al., 1985; Skinner and Radin, 1994) to walls reduce the hydraulic conductivity. The same may test the permeability of the roots of this ionic compound occur after the thickening of both layers which may be besidesthewater (Zimmermannand Steudle,1998).PTS suberized and/or lignified. There are experiments which passed across the roots and, different fromthe water, the Wateruptakebyroots 1537 permeability of PTS was the same regardless of whether losses to the dry soil when transpiration is switched off. or not the root had a mature exodermis. During its It appears that, to some extent, the composite transport passage across the root, PTS was diluted to a concentra- model solves the dilemma. According tothe model, there tion as low as 0.2% of that in the medium. If PTS were is a variable root resistance which is low in the presence a good tracer for water, this would mean that the apo- of transpiration, when the tension (negative pressure) in plastic component of water flow was only 0.2%. This, the xylem provides a water potential gradient which is however, was not true, because the comparison between sufficient to take up water even from a dry soil. In the root Lp and root cell Lp showed that there must have absence of transpiration (e.g. at night), suberization of r been a considerable flow of water around protoplasts. roots (at water deficits) provides a minimization of water Thisisalsoindicatedfrommeasurementsonspeciesother losses to the dry soil, when the water potential gradient than maize (Radin and Matthews, 1989). It was con- is in the wrong direction. It should be noted that matric cluded that, at least for roots of maize plants grown in forcesareequivalenttohydrostaticforces(asareosmotic aeroponics up to an age of 3 weeks, the endodermis forces), but they are physically different. This may cause rather than the exodermis was the main barrier for the differences in their role as a driving force. Matric forces PTS flow so that the development of the exodermis did originate from the component of water potential which not result in a measurable decrease of the PTS flow. It derives from surface tension effects rather than from appears that charged solutes are effctively retained in the volume work which is the origin of the hydrostatic presence of Casparian bands. pressure component. With respect to the model, it is The picture of the root depicted so far is that of a important howchanges in thematric potentialofthe soil structureinwhichtheapoplaststillhassomepermeability are translated into changes of components of water towater,evenintheexo-andendodermis.Thisconclusion potential in the root apoplast (as shown in detail by was drawn from both the comparison between root Lp Steudle, 1992). When changes cause a change in the r and cell Lp and from the chemical analysis of apoplastic osmotic component, the resulting flow would be largely barriers (see above). It does not refer to nutrient ions. In from cell to cell (high resistance). On the other hand, young maize roots, there was a rapid decrease in root when they cause a gradient in hydrostatic pressure or pressure, even when only a few cells of the endodermis matric potentials, the flow will be largely apoplastic (low were injured (Steudle et al., 1993). As for PTS, the resistance). At a substantial water shortage in the soil, diffusionofionsoutofthestelewas stronglyimpededby the hydraulic resistance of the root may be too high to theendodermis.Therapiddeclineofrootpressurefollow- allow for a sufficient acquisition of water, even when ing an injury suggested that there was a bulk flow of waterpotentialgradientsareintherightdirection.Under xylem solution across the leak which constituted a low- these conditions, water channels may act as valves to resistance pathway between a compartment of higher reversibly increase the hydraulic conductivity and allow pressure (stelar apoplast) and one of lower pressure for a water uptake under adverse conditions. It has been (medium).Theresultsconfirmedthattheendodermisacts shown that water channels can be activated by phos- asanefficientbarriertotheback-diffusionofionsreleased phorylation which, in turn, is affected by factors such as into the apoplast of the stele, which allows root pressure water potential, turgor, or Ca2+ concentration in the tobuildupunderconditionsoflowtranspiration.Onthe apoplast (Johansson et al., 1996; Kjellbom et al., 1999). other hand, the hydraulic resistance of the endodermis is To date, current understanding of how this regulation rather low, at least for cells with just Casparian bands. takes place in the intact root is fairly limited. Thus, the transport properties of roots are optimized for For roots of Lotus japonicus it has been demonstrated both ions and water. The former are taken up selectively that the concentration of the mRNA encoding for putat- and are held safely in the stele, while the resistance to ive water channels displayed a distinct diurnal rhythm waterflowislow,allowingasufficientsupplytotheshoot. with a maximum at noon. This coincided with the max- imum in the root hydraulic conductivity measured with either osmotic or pressure gradients. However, maxima Arewaterchannelsthealternativepassage? in root Lp were not correlated with appropriate changes r Intheshoot,waterflowislargelycontrolledbystomates. inthehydraulicconductivityofrootcortexcellsasshown Stomatarepresentan‘active’elementofcontrolorregula- by measurements with the cell pressure probe. The result tion which is tuned by the water status of the shoot and was interpreted in that regulation of cell Lp may be signals from the root (ABA). The cuticle of the shoot confined to endodermal and stelar cellsonly assuggested can be regarded as ‘water-tight’ or nearly so. In the root, by molecular markers (Scha¨ffner, 1998). In the stele, a the situation is less clear. There are no stomates and regulation would be more efficient, anyhow, because, for waterandnutrientuptakerequireapassageacrossarrays geometric reasons, this part contributes more to overall ofnon-suberized tissue.Onthe other hand,thehydraulic radial resistance than the cortex and epidermis. conductivityhastobereducedtoavoidsubstantialwater For some species such as bean (Phaseolus coccineus) 1538 Steudle and barley, it has been demonstrated that, due to a high the intensification of water flow during periods of a high membraneLp,thecell-to-cellpassagedominates(Steudle demand from the transpiring shoot. At water shortage, and Jeschke, 1983; Steudle and Brinckmann, 1989). For however, the hydraulic resistance would be high, thus others, the effects are variable. A few attempts have been preventing a backflow to the dry soil when transpiration made todemonstrate the actionof water channels(aqua- is switched off. The phenomenon of a variable root porins) in roots such as by closing them with mercurials resistance has been interpreted in different ways. It has (Maggio and Joly, 1995). However, in these experiments beeninterpretedintermsofchangesinthewaterpermeab- effects of the blocking agent on other transporters and ility of root cell membranes (Brewig, 1937). Fiscus side effects have not been completely ruled out. developed a physical model which explained the ‘appar- Experimentsonwheatindicatedthatnutrientdeprivation ent’ decrease of root hydraulic resistances at high flow reduced water channel activity (Carvajal et al., 1996; rates by a change in the driving force (Fiscus, 1975). Clarkson et al., 2000). The conclusion was drawn from According to the ‘dilution model’ of Fiscus, the change the fact that, different from controls, the hydraulic con- was due to a change in the osmotic component of the ductivityofdeprivedrootswasnotaffectedbymercurials. driving force at a constant root Lp. Under conditions of r Recently,ZhangandTyermanhaveusedthecellpressure water shortage, the effect should increase as the signific- probe to measure the reversible inhibition of water chan- ance of the osmotic pressure of the xylem sap increases. nelsbyHgCl incorticalcells ofwheat roots(Zhangand 2 However, detailed measurements with pressure probes Tyerman, 1999). Interestingly, hypoxia caused similar have shown that the dilution effect can not completely effects as HgCl and hypoxia-treated cells could not be 2 explain non-linearity (Zimmermann and Steudle, 1998). further inhibited. Zhang and Tyerman did not measure Tensions in the root xylem provide the major force the effect of closure of water channels on root Lp as did r moving water across the root cylinder into xylem vessels other authors (Azaizeh and Steudle, 1991; Azaizeh et al., and up to the shoot. Only occasionally, should osmotic 1992). The latter authors found that, in maize, salinity forcesdominate.Hence,thecohesion–tensionmechanism (100mM)causedareductionofcellLpbyfactorofthree plays an important role during the acquisition of water to six. However, at the root level, the reduction was only byroots,namely,underconditionsofwaterstress.During 30–60%. The result has been explained in terms of the the past ten years, the validity of the cohesion–tension composite transport model by a substantial apoplastic theory has been questioned by Zimmermann and bypass-flow of water. co-workers on the basis of direct measurements of xylem For intact internodes of Chara, there are, to date, pressure using the cell pressure probe (Balling and detailed data on the function of water channels (Henzler Zimmermann, 1990; Melcher et al, 1998). In the early andSteudle,1995;Schu¨tzandTyerman,1997;Herteland papersfromthisgroup,tensionsinthexylemwererather Steudle,1997).Theworkdemonstratedboththeselectiv- low (pressuresless negative),and werehardly affectedby ity of water channels and its significance for cell Lp. transpiration. Very often sub-atmospheric pressures have Similar to the wheat cortex cells (Zhang and Tyerman, been measured rather than pressures below the absolute 1999), closure of water channels reduced cell Lp by 75%. zero(vacuum).ValuesofP asmeasuredwiththepressure Onthe otherhand,thepermeabilityofthemembranefor x probeweresubstantiallyhigher(lessnegative)thanthose small uncharged solutes such as low molecular weight found with the Scholander bomb (pressure chamber). alcohols, amides, ketones etc., did not change. A careful Zimmermannandco-workersconcludedthatthecohesion inspection of the transport properties of these cells indi- theory was wrong and that the pressure chamber did not cated that there was some slippage of the solutes across correctly measure water potentials (xylem pressure, P ). the water channels (Steudle and Henzler, 1995; Hertel x However, as the group improved its skills in measuring andSteudle,1997;HeuzlerandSteudle,2000).According totheresultswithChara,waterchannelsinplantsdonot xylempressure,valuesbecamemorenegativeandtensions appeartobeasselectiveashasbeensometimesproposed of up to 0.5–0.6MPa were found which reacted on (Chrispeels and Maurel, 1994; Maurel, 1997; Scha¨ffner, transpiration. It was now concluded that the cohesion– 1998).Thereisalackofcombinedmeasurementsatboth tension mechanism can not be completely excluded. the root and cell level to evaluate the effects of channel Rather, it would serve as an additional mechanism. The blockage on overall transport. These measurements have change in the attitude may indicate that, after all, the toincludecellsoftheexo-andendodermisandstelarcells. groupisonitswaytoacceptthecohesion–tensiontheory. However,this isnottruebecausetheoldfindingsarestill cited and claimed to be correct. They are still referred to Thecohesion–tensionmechanismprovidesthe anddiscussedbyotherswhothinkthattheyhaveasound switchingbetweendifferentmodesoftransportin basis (Tomos and Leigh, 1999). roots Recently, combined measurements with the pressure Thenon-linearforce/flowrelationshipfoundduringwater probe and the pressure chamber on intact transpiring uptakebyplantrootsisfavourablefortheplant.Itallows maize plants have shown that both techniques yield Wateruptakebyroots 1539 similar values, at least for tensions of up to 1MPa (10 roottips.AccordingtothemodelofSlovikandHartung, bar; Wei et al., 1999a, b, 2000). This was in agreement ABA is distributed between the apoplast and symplast withthecohesion–tensiontheory.Differentfromtheearly according to the difference in pH between the two com- resultsoftheZimmermanngroup,theresultsofWeietal. partments (Slovik and Hartung, 1992). A more alkaline showed that xylem pressure strongly and rapidly (less acidic) pH of the apoplast would increase the ABA responded to changes in transpiration which is in agree- concentration in the apoplast and, hence, intensify the mentwiththecohesion–tensionmechanism.Mostinteres- solvent drag of ABA to the shoot with the transpiration tingly, a careful examination of the limits of the pressure stream.Thewaterflowacrosstheplantwouldbedirectly probeto measure tensions showed that the equipment by linked to the signal of ABA which is produced in the itself was able to sustain tensions of up to only root during water stress. 1.6–1.8MPa in short periods (Wei et al. 1999a). When In accordance with these findings was the fact that the the tip of the probe was positioned in the xylem, the reflection coefficient of ABA (calculated from solvent upper limit was further reduced to about 1MPa. So, the drag) was close to unity at high pH (all ABA in anionic techniqueasitstandscannotbeusedtoproveordisprove form) and low at low pH, where a considerable amount theexistenceoftensionsofupto10MPa(100bar)which waspresentasundissociatedacid.Whenmaizerootswere grown in aeroponic culture and developed an exodermis, havebeenindirectlymeasuredusingthepressurechamber. both the transport of ABA and the water flow were Up to tensions of 1MPa, the data were in agreement reducedwhichresultedinaconcentrationofABAsimilar with the cohesion–tension theory. It appears that the to that found in the absence of an exodermis (Freundl early and present claims from the Zimmermann group et al., 2000). Hence, the size of the signal did not change are largely based on artefactual results because of limita- much. It was concluded that the apoplastic passage of tions of the technique used. Until recently, these limita- ABA may be significant during the signalling of water tions have not been thoroughly checked for, although deficits from the root to the shoot. There were big differ- there had been warnings in the literature (Steudle and ences between the two species used in that the apoplastic Heydt, 1988; Heydt and Steudle, 1991; Steudle, 1995). component was much larger in maize as compared with With respect to the uptake of water by roots at low sunflower.Thedifferencesmaybeduetodifferencesinthe soil water potential the results are of crucial importance. fine structure of the roots of the two species, namely, in According to the cohesion–tension theory, transpiring the ultrafiltration properties of the endodermis. At the plants could take up water from a dry soil at a high root endodermis, there should be concentration polarization Lp (as predicted by the composite transport model). On r effects which may contribute to the rather high solvent the other hand, when transpiration is switched off, root drag and the low apparent reflection coefficients of ABA. Lp would switch to low values thus limiting water loss r It is interesting that ABA also increased the hydraulic to the soil under these conditions. conductivity of maize both at the level of individual cells (Lp) and entire roots (Lp), perhaps, by affecting water r TransportofABA:therootsignal channels (Hose et al., 2000). Tests with other hormones and ABA derivates have shown that the effects on Lp TheroleofABAasalong-distancestresssignalproduced and Lp are specific for the undissociated (+)-cis-trans- in the root is well established. Usually, the concentration ABA. Erffects were much stronger at the cell than at the of ABA in the xylem is thought to be the signal which is root level. At the root level, Lp did not change in carried up to the shoot with the transpiration stream. r the presence of a hydraulic water flow, when the apo- However, water uptake into the xylem would modify the plastic component was dominating. Thisis in accordance signal by dilution, unless there is some apoplastic trans- with the composite transport model. The results show portofABAwhichcouldcontributetothesignal.Recent that water flow (transpiration) which is usually thought measurements on excised roots of sunflower and maize to be regulated with the aid of the stress hormone ABA indicate that this is the case (Freundl et al., 1998, 2000). atthelevelofstomata,mayinteract withtheABAsignal In sunflower and maize grown in hydroponic culture coming from the root. On the other hand, ABA may there was a significant solvent drag of ABA offered to regulate water uptake under non-transpiring conditions, the nutrient solution at low concentrations (0–500nM). i.e. under conditions of water shortage. It may be specu- The effect compensated or even overcompensated for the latedthat,undertheseconditions,thecell-to-cellcompon- dilution effect caused by the concomitant water uptake. ent of water flow is increased by an activation of water It was concluded that in water-stressed plants, ABA channel activity. produced in the root could be apoplastically transported across the root, at least to some extent. According to Conclusions Hartung et al., the biosynthesis of ABA takes place in the cytosol of all root cells (Hartung et al., 1999). The From transport and anatomical studies it is concluded strongest capacity is found in the non-vacuolated cells of that the composite structure of roots plays an important 1540 Steudle roleduringtheregulationofwateruptake.Thecomposite correlated with the expression of mRNA encoding for structure of roots results in composite transport of water water channels, but not with a rhythm in the Lp of and solutes. There is a switching between the cell-to-cell corticalcells. Itwas concludedthat regulationtook place (aquaporins) and the apoplastic path which isthought to in the endodermis and/or stele. causemostofthevariabilityofthehydraulicconductance Inthecompositetransportmodel,thecohesion–tension of plant roots. The switching between transport models mechanism of the ascent of sap plays an important role. dependsontheforcesactinginthesysteminthepresence At high rates of transpiration and tensions in the root and absence of transpiration (‘hydraulic’ and ‘osmotic’ xylem, the preferred apoplastic path for water provides a modes of water flow). In the presence of high transpir- sufficient supply of shoots with water even under condi- ation, the force driving water across the root will be the tions of water shortage (hydraulic mode of water flow). hydrostaticpressure differencebetweenroot mediumand The cohesion–tension mechanism has been questioned. xylem. This allows both the apoplastic and cell-to-cell Directmeasurementsofxylempressureinleavesofintact pathways to be used, even in the exo- and endodermis. transpiring maize plants now show that the cohesion– Whentranspirationisswitchedoff,thecell-to-cellpassage tensionmechanismisvalid,atleastforarangeoftensions is left which has a high hydraulic resistance. There is a of up to 1 MPa (10 bar). coarse adjustment of root Lp which is purely physical in Future work should concentrate on a more detailed r nature and has no equivalent in the shoot. Provided that mapping of hydraulic resistances in the root cylinder and thereissufficienttranspiration,theswitchingtohydraulic how they would change in response to water shortage water flow provides access of the plant to soil water at and other stresses. Namely, the hydraulic resistances of low water potential. In the absence of transpiration root the exo- and endodermis and of stelar cells should be Lp becomes small. This minimizes water losses to the known for modelling water flow across roots. This can r drysoil.Forplantsgrowingatwaterdeficits,rootgrowth be done by combined measurements with the cell and is slowed down and there are considerable apoplastic root pressure probe and by measuring the propagation barriers blocking the hydraulic route. Under these condi- of changes in water potential (turgor) across the root tions, root Lp may be low and could be regulated along cylinder. r the cell-to-cell path by an opening or closing of water channels. Water channel activity has been shown to exist inrootcortical cellsincludingthe endodermis. The activ- Acknowledgement ity of water channels may be inhibited by heavy metals, This work was supported by a grant from the Deutsche hypoxia, nutrient deficiency, low temperature, and high Forschungsgemeinschaft (DFG), Schwerpunktprogramm salinity.Measurementsofinhibitionatthecellandoverall ‘Apoplast’. root Lp are in agreement with predictions from the r composite transport model. In youngroots ofmaize, there isa correlationbetween References the chemical composition of apoplastic barriers and root Lp. Aliphatic suberin appears to act as the hydrophobic Azaizeh H, Steudle E. 1991. Effects of salinity on water r transport of excised maize (Zea mays L.) roots. Plant material which blocks the water flow across Casparian Physiology97, 1136–1145. bands. Compared with the water, the solute (nutrient) Azaizeh H, Gunse B, Steudle E. 1992. Effects of NaCl and permeability of roots is low. Nutrient ions once taken up CaCl on water transport across root cells of maize (Zea 2 are retained in the xylem and do not leak back. There is mays L.)seedlings.Plant Physiology99, 886–894. an apoplastic component of ABA transport in roots Balling A, Zimmermann U. 1990. Comparative measurements of the xylem pressure of Nicotiana plants by means of the which contributes to the stress signal in the xylem (apo- pressurebomb and pressureprobe.Planta 182,325–346. plastic flow of ABA). The stress signal in the xylem Barrowclough DE, Peterson CA, Steudle E. 2000. Radial depends on the intensity of the rate of transpiration. The hydraulicconductivityalongdevelopingonionroots.Journal flow of ABA is affected by the presence of apoplastic ofExperimental Botany51, 547–557. barriers (exodermis). At least to some extent, ABA pro- Birner TP, Steudle E. 1993. Effects of anaerobic conditions on water and solute relations, and on active transport in roots duced in the rootduring water stress, will bedelivered to ofmaize(Zea maysL.).Planta 190,474–483. theapoplastandthentakentotheshootwiththetranspir- Brewig A. 1937. Permeabilita¨tsa¨nderungen der Wurzelgewebe, ationstream.Ontheotherhand,ABAincreasesthewater die vom Spross beeinflusst werden. Zeitschrift fu¨r Botanik permeability of root cell membranes and the osmotic 31, 481–540. hydraulic conductivity of whole roots as shown by meas- Brouwer R. 1954. The regulating influence oftranspiration and suction tension on the water and salt uptake by roots of urements with the cell and root pressure probe. These intactViciafabaplants.ActaBotanicaNeerlandica3,264–312. results are in line with the composite transport model. CarvajalM,CookeDT,ClarksonDT.1996.Responsesofwheat Nutrientstressesreducewaterchannelactivityandoverall plantstonutrition deprivationmayinvolvethe regulation of root Lp. In Lotus roots, the diurnal rhythm of root Lp water-channelfunction. Planta199,372–381. r r