composite model of roots

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Plant Physiol. (1993) 103: 335-349

Transport of Water and Solutes across Maize RootsModified by Puncturing the Endodermis'Further Evidence for the Composite Transport Model of the Root

Ernst Steudle*, Mart ina Murrmann, and Caro1A. PetersonLehrstuhl fr Pflanzenokologie, Universitat Bayreuth, D-95440 Bayreuth, Cermany (E.S., M.M.); and

Department of Biology, University of Waterloo, Waterloo, Ontario, Canada N2L 3C 1 (C.A.P.)

the intac t root. I f there were such a pathway, ei ther in these areasor across the Casparian band itself, roots woul d have to be treatedas a system composed of two parallel pathways (a cell-to-cell andan apoplasmic path). It i s demonstrated that this composite trans-por t model of the roo t allows integration of severa1 transportproperties of roots that are otherwise difficult to understand,namely (a) the differences between osmotic and hydrostatic waterflow, (b) the dependence of roo t hydraulic resistance on the dr ivingforce or water flow across the root, and (c) low reflection coeffi-C h t S of roots.

The effects of puncturing the endodermis of young maize roots( z e a mays 1.) on their transport properties were measured usingthe root pressure probe. Small boles with a diameter of 18 to 60pm were created 70 to 90 mm from the tips of the roots by pushingfine glass tubes radially i nto them. Such wounds inj ured about 10 2to 10-~% f the total surface ares of the endodermis, which, inthese hydroponically grown roots, had developed a Casparian bandbut no suberin lamellae. The small injury t o the endodermis causedthe original root pressure, which varied from 0.08 to 0.19 MPa, todecrease rapidly (half-time = 10-100 s) and substantially to a newsteady-state value between 0.02 and 0.07 MPa. The radial hydraulicconductivity (Lp, ) of control (uninjured) roots determined usingfactor of 10 than that determined using osmotic gradients (aver-ages: Lp, [hydrostatic] = 2.7 X lO- m s-' MPa-'; Lp, [osmotic] =2.2 X 10-* m s-' MPa-'; osmotic solute: NaCI). Puncturing theconductivi ties measured by either method. Thus, the endodermiswas not rate-li miting root Lp,: apparently the hydraulic resistanceof roots was more evenly distributed over the entire root tissue.However, puncturing the endodermis did substantially change thereflection uJ and permeability ( P d coefficients of roots for NaCI,indicating that the endodermis represented a considerable barrierto the flow of nutrient ions. Values of ur. decreased from 0.64 t o0.41 (average) and P., increased by a factor of 2.6, i.e. from 3.8 x10-' to 10.1 x i r s-' (average). The roots recovered frompuncturing after a time and regained root pressure. Measurableincreases in root pressure became apparent as soon as 0.5 to 1 hafter puncturing, and orig inal or higher roo t pressures were at-tained 1.5 to 20 h after injury. However, after recovery roots oftendid not maintain a stable root pressure, and no further osmoticexperiments could be performed with them. The Casparian bandof the endodermis i s discontinuous at the root tip, where theendodermis has not yet matured, and at sites of developing latera lroots. Measurements of the cross-sectional area of the apoplasmicbypass at the roo t tip yielded an area of 0.031% of the to tal surfacearea of the endodermis. An additionalO.O49% was associated wi thlateral root primordia. These areas are larger than the artificialbypasses created by wounding in this study and may providepathways for a natural bypass flow of water and solutes across

Supported by a grant from EUROSILVA (project No. 39473C) toE.S. and by a Bilateral Exchange Grant jointly funded by theDeutsche Forschungsgemeinschaft and the Natural Sciences andEngineering Research Council of Canada to C.A.P.* Corresponding author; fax 49-921-55-2564.

There is increasing evidence that pathways for the radialsome roots, a cell-to-cell transport of water predominates,whereas in others water flow is mainly around cells, i.e.

of roots with mostlY cell-to-cell transport are young barleyand bean, whereas in Yo'Jng maize and Cott01-1 roots, anapoplasmic transport dominates (Steudle and Jeschke, 1983;Steudle et al., 1987; Radin and Matthews, 1989; Steudle andBrinckmann, 1989). In species exhibiting large differencesbetween osmotic and hydrostatic water flow, the extent towhich apoplasmic transport occurs depends on the nature ofthe force set up between root medium and xylem to drivewater flow, Hydrostatic gradients (which dominate in theintact, transpiring plant) cause a much larger flow due to ahigher L p , than osmotic, and the differences have been foundto be as as an Order Of magnitude or even more (Steudleand Frensch, 1989; Zhu and Steudle, 1991;Cruz et al., 1992;Birner and Steudle, 1993). Differences between species werenot due to different contributions of the longitudinal (axial)

Abbreviations:A,, root surface area m2);,, diffusion coefficient(mZ -I); cx elastic coefficient of root xylem (MPa); ]vr, radial waterflow acrOSS root (m s-~) ; radial solute flow acrOSS root (moi m - ~s-I); active component of radial solute flow (mo1 m-' s-'); k,,(ksr), rate constant of water (solute) exchange across root s-I); Lp,,radial hydraulic conductivity of root (m s - ~MP a- ~ ) ;pE, final rootpressure (MPa);P,, root pressure; P (Pmax),minimum (maximum)root pressure; P,,, steady-state root pressure; P,, permeability coef-ficient of root (m s-'); TI,*/, , alf-time of water exchange across root(s); T V , ~ ,alf-time of solute exchange across root (s); V,, volume ofmature root xylem (m3);APJAVS, elastic coefficient of root pressureprobe (MPa m-3); u reflection coefficient of root (1).

hydrostatic pressure gradients as driving forces was larger by a transport of water acrOSS roots differ for differen t species, In

endodermis did not result in measurable increases in hydraulic apoplasmic (Steudle~ 989~ 992r 1993at 993b). Examples

335


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Composite Transport Model of t h e Root 337

culated for regions lacking a Casparian band, i.e. for root tipsand for lateral root primordia. The total area available at roottips was evaluated from the diameter of the endodermalcylinder 4 mm from the root tip, the point where the Cas-parian band was just maturing. The apoplasmic area wasestimated from cross-sections stained with Cellufluor. Simi-larly, the total area of the endodermis interrupted by a lateralroot primordium was calculated from freehand cross- andlongitudinal sections of the main root, which were medianlongitudinal through the lateral. To estimate the fractionalapoplasmic area of this region, cell diameters of the lateralroot primordia were measured at the level of the main rootendodermis. The type of section that proved most useful forthis purpose was a paradermal, longitudinal section at thelevel of the endodermis of the main root positioned to providea cross-section of the lateral. For these preparations it wasnecessary to use laterals that had emerged from the parentroots to orient them for sectioning. Data for the cell wallthickness were taken from electron micrographs reported byKaras and McCully (1973). The total number of unemergedlateral root primordia, plus those emerged by 2 mm, werecounted on five roots that had been cleared and stained usingthe method of Hackett and Stewart (1969), i.e. by stainingthe nuclear material (which is very dense in the mitoticallyactive primordia) with acetocarmine and clearing most of theremaining cytoplasmic substances with methyl benzoate. Thisgave a conservative estimate of the numbers of laterals with-out Casparian bands because, according to Karas and Mc-Cully (1973), laterals of maize that had emerged by as muchas 4.5 mm still had no Casparian bands. Sections and stained

roots were photographed with slide film, white-light illumi-nation with Ektachrome 64T (ISO 64), and UV/violet illu-mination (excitation wavelengths: 390-420 nm) with Fuji-chrome ISO 100).

P, and Lp,Roots were excised under water near the kernel to obtainan unbranched segment of primary root 89 to 174 mm long(diameter: 0.78-1.28 mm). To prepare for an experiment, aroot was inserted into a silicone seal (8-10 mm long) thatwas then placed in the root pressure probe (Fig. 1).The sealwas carefully tightened stepwise until the root pressure beganto increase after tightening. This procedure ensured that theseal around the root was sufficiently closed to prevent leakageof liquid through the root cortex. Whether or not the xylemvessels in the sealed area remained open or were crushedwas tested at the conclusion of each experiment as describedbelow (Steudle and Frensch, 1989; Peterson and Steudle,1993). Steady-state root pressures between 0.08 and 0.19MPa (0.8-1.9 bars) were obtained after 1 to 2 h, and experi-ments commenced. The L p , was evaluated from root pressurerelaxations.

Water flows were induced by either changing the rootpressure with the aid of a movable metal rod in the equipment(Fig. 1; hydrostatic experiments) or by altering the osmoticpressure of the medium. The latter was accomplished bychanging the bathing solution around the root from thenormal culture solution to one amended with a specificamount of NaCl as an osmoticum (exosmotic experiment). In

medium medium t NaClmedium medium t NaCl glass microcapillarylass microcapillary

VsupportsFigure 1. Diagram of a root pressure probe with a root fixed to it. To induce water flows (root pressure relaxations), thestationary root pressure could be changed either by moving the metal rod (hydrostatic experiments) or by exchangingthe root-bathing medium for one with a different osmotic concentration (osmotic experiments). Root pressure wasmeasured continuously with a pressure transducer. From the rate constants (half-times)of the root pressure relaxationsmeasured, the Lp, usr nd P,, were calculated. T o puncture the root endodermis, the tip of a th in microcapillary tube wasinser ted radially into t h e root with the aid of a micromanipulator (not shown). For further explanation, see text.


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338 Steudle et al. Plant Physiol. Vol. 103, 1993

endosmotic experiments, the change was made back to theoriginal culture solution. The changes of root pressure, re-corded after step changes in water potential, were exponentialwith time. From the rate constant (kWJ of the processes, theL p , was evaluated (Steudle et al., 1987; Steudle, 1989, 1992,1993a, 1993b):

TI,^/, is the half-time of the process; APr/AVs (in MPa m-3) isthe elastic coefficient of the measuring system (the inverse ofits water capacity; Vs = water volume of system), and A , isthe effective surface area of the root. M,/AVs was determinedby inducing step changes of volume (AV,) with the aid of themovable rod and measuring the resulting changes of rootpressure (AP,; Steudle and Jeschke, 1983). The effective sur-face area of the root was calculated from its diameter andlength, subtracting the apical part where tracheary elementsof the early and late metaxylem were immature, i.e. theterminal 21.5 mm (Peterson and Steudle, 1993).

In osmotic experiments with permeating osmotica (as inthis paper), root pressure relaxations were biphasic, and thephases were separated by minima or maxima of root pressure,i.e. water flow across the root changed direction during theexperiment. The first rapid phase ( water phase ) was causedby the change of the osmotic pressure of the medium. Thesecond, slower phase was due to the passive permeation ofthe solute across the root cylinder, which changed the osmoticgradient across the root causing water to enter ( solutephase ). Both phases could be fitted by a single exponentialcurve ( r 22 0.98).

In hydrostatic experiments, pressure relaxations caused bya change of root pressure exhibited only one phase. This waseither an uptake of water by the root (endosmotic water flow)or an extrusion of water from the root (exosmoticwater flow),depending on whether the pressure in the head of the probewas decreased or increased. Hydrostatic relaxation curveswere composed of three distinct regions brought about bydifferent rates of changes of root pressure with time, i.e. threedifferent phases could be distinguished. There was an initialvery rapid phase immediately following the change in P,(half-time: 0.7-0.9 s), then a central body of the curve (half-time: 1.9-4.9 s), and finally a very slow component (half-time: 52-66 s).As discussed elsewhere (Peterson and Steudle,1993), the second phase is related to the radial exchange ofwater across the root cylinder. The two other componentscould easily be separated from this phase because of the largedifferences in their rate constants. To calculate the rates ofradial water exchange across the root, chart recorder stripswere digitized on a graphics tablet, and the curves were thenanalyzed using fit programs. The programs also assisted infinding the final root pressure (PE;see Fig. 3) of pressurerelaxations. The use of a proper value of PEhad some impacton the evaluation of k (TsI2 ')Birner and Steudle, 1993;Melchior and Steudle, 1993).

In a typical experiment in which the hydraulic conductivityof a root was measured, the M,/AVs was determined initially,followed by six replicate hydrostatic relaxations and oneosmotic pressure relaxation. The latter biphasic relaxations

also gave P,, and a,, (see below) of the root to NaC1. AFterthese measurements the root endodermis was punctured withthe aid of a glass tube that had a tip diameter of 18 to 60 pm(Fig. 1). t was prepared by drawing a molten capillary tiibeon a vertical puller used to prepare microelectrodes. Themicrocapillary tube was advanced slowly, radially into theroot, and then withdrawn. In this way, a single hole or a fewholes were produced in the cortex, endodermis, and sorne-times in part of the stele; in a11 cases, mature xylem vesljelswere not damaged. Holes were made at distances between70 and 90 mm from the root tip. Puncturing led to a reducedsteady root pressure (Pro).After puncturing a11 procedureswere repeated to measure the transport coefficients Lp,, usr,and Psr.At the conclusion of each experiment, roots were cutwith a razor blade right at the seal, which resulted in animmediate decrease of pressure to zero (atmospheric). Acldi-tional hydrostatic pressure relaxations were performed, imdthese were usually very fast, indicating that, within thesealing area, the vessels remained open and that the hydra ulicresistance of the xylem within the seal was very low com-pared with that of the intact root during the experiments(which lasted for 4-10 h for each root). Experiments weredisregarded when the half-times of pressure relaxations aftersevering the root were larger than one-fifth to one-tenth ofthat obtained for the intact system.Measurement of P, , and usrCoeff ic ients

Using a permeating solute (NaCI), we calculated P,, fromthe rate constant k S J of the second phase (solute phase) ofbiphasic osmotic pressure relaxations (Steudle, 1989, 14192,1993a), i.e.:

where indicates half-time of the solute phase, and V x sthe volume of mature root xylem. For the roots used, V, was3% of the total root volume as estimated from root cross-sections obtained at different distances from the root tip(Steudle and Frensch, 1989). Thus, after measuring k,, (7 ,;),V,, and A , (see above), we calculated Psrfrom Equation 2

usrfor the given solute was obtained from the maximlumchange of root pressure M,)caused by a change of theosmotic pressure of the medium. During hypertonic charigesAP, = P,, - P and during hypotonic changes AP, = P , -P [P,, = steady-state root pressure; Pmun(max)minimlum(maximum) root pressure]. Reflection coefficients were cal-culated from Steudle et al. (1987) and Steudle (1989, 1992,1993a, 1993b):

(3)where Asso is the change of the osmotic pressure in themedium of permeating solute ys* (NaCl), t x is the elasticmodulus of the root xylem, and ax s the osmotic pressure inthe root xylem. Because the elastic modulus of the root xyllemwas large, t x >> a, was valid, and the second term on theright side of Equation 3 was equal to unity to a good approx-


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Composite Transport Model of the Root 339

imation. The third factor on the right side of the equation(exp[k,,. tmin]) corrected for the solute flow into or out of thexylem during the water phase, i.e. during the time taken toreach the minimum or maximum ( As for the osmoticand hydrostatic Lp , , the coefficientsPSr nd u, were measuredbefore and after puncturing the endodermis.

RESULTSAnatomy

The endodermis of a typical maize root used in this workcan be considered a cylinder with a diameter of 0.50 mm anda length of 85 mm, i.e. the total length of the root less thehydraulically isolated tip region of 21.5 mm. The total surfacearea of such an endodermis is 134 mm2. The average lengthand tangential width of its component cells were 280 and 18pm, respectively. Assuming a wall thickness of 1 pm (seeMaterials and Methods ), the per cell area of the apoplast in

a tangential longitudinal view, sectioning midway throughthe endodermis, would be 596 pm2. Thus, the total area ofthe apoplast in this plane of the entire endodermis would be16 mm2 or 12% of the total surface area. Endodermal cellspossessed Casparian bands but not suberin lamellae (Fig.2A). The exodermis was immature, i.e. with neither Casparianbands nor suberin lamellae (Fig. 2A).A discontinuity in the Casparian band exists at the root tipwhere the structure is immature. The diameter of the endo-dermis 4 mm from the root tip was 2R = 350 pm. The cellsof the stele were small in diameter (2r = 9.5 pm) and closelypacked (Fig. 2B). Assuming a wall thickness (Ar) of 1pm, theproportion of one cell occupied by wall would be 2ar. A r / d= 2Ar/r = 2.1/4.75 = 0.42. Therefore, the wall area in thewhole apical bypass would be aR2.0.42 = a.175*.0.42 =40, 400 pm2, i.e. 0.030% of the endodermal surface area.Because the endodermis matures earlier than the trachearyelements, water and solutes following an apoplasmic pathwould have to cross the epidermis and cortex and then movebasipetally across the young stelar tissue before reaching amature vessel. Although this is not a long path, the permea-bility of these walls is low compared to older walls, at leastfor the apoplasmic dye berberine (Enstone and Peterson,1992). This has to be taken into account when consideringboth the hydraulic and diffusional resistances of the tip routeand its possible contribution to the overall apoplasmic bypass.

Discontinuities in the Casparian band are associated withlateral root primordia, which are very numerous in maize(Fig.2C). On average, the roots possessed 170 young laterals,which were within the main root or had emerged 2 mm fromits surface. Their average diameters at the level of the mainroot endodermis were determined from measurements ofcross-sections and longitudinal sections of the main root,which were median longitudinal sections of the primordia(Fig. 2D). The discontinuous area of the endodermis wasusually less than the maximum diameters of the laterals,because primordia were narrower at the level of the endo-dermis than in the nearby cortex (Fig. 2D). The total circulararea in which the Casparian band of the main root wasabsent at the base of a primordium was 30,000 pm2. Anestimate of the potential apoplasmic bypass was made from

paradermal, longitudinal sections taken at the level of theendodermis (Fig. 2E). It was possible to recognize an appro-priate section because the cells of the main root endodermispossessed large primary pit fields that were not present inpericycle or neighboring cortical cells (Fig. 2E). The meandiameter of the cells in the initials was 2r = 11 pm and themean cell wall thickness Ar = 0.035 pm (0.025-0.050 pm;Karas and McCully, 1973). This resulted in a fraction avail-able for apoplasmic transport of 2ar. Ar/r? = 2Ar/r = 2.0.035/5.5 = 0.013 or 1.3% of the total cross-section. Becausethe cross-section of a single root primordium was, on average,30,000 pm2 (mean diameter: 195 pm), the absolute value ofapoplast available in a lateral was 390 pm2. There were 170initials in a root, giving a total apoplasmic cross-section of66,300 pm2. This is only a small fraction, i.e. 0.049% of thetotal surface area of the endodermis. By contrast, the cross-section of the apoplasmic route across the Casparian band ofthe endodermis was as large as 12% of the total surface areaof the endodermis (see above), i.e. it was larger by a factorof 245 than that available in the apoplast of the initials.Root Pressure and Hydraulic Measurements

In a typical example, root pressure, osmotic and hydrostatichydraulic conductivities, and reflection coefficients beforeand after puncturing the endodermis were a11 measured onone root (Fig. 3). In Figure 3A the initial four pressure spikesrepresent measurements of the elastic coefficient of the sys-tem (AP,/AV, = 2.9 X 109 MPa m-3 or 29 bar pL- for thisexperiment). These measurements of elasticity were followedby hydrostatic (Fig. 3, B and D) and osmotic (Fig. 3, C and E)pressure relaxations, including the solute phases for NaCl.From the half-times of hydrostatic pressure relaxations (TableI) and from the water phases of the osmotic experiments, Lp,was calculated (Table 111). From the solute phases of osmoticexperiments, permeability and reflection coefficients weredetermined (Table 11, see below). The hydrostatic hydraulicconductivity was considerably larger, and the Ta valueswere considerably smaller than the osmotic (factor of 10, onaverage; Table 111).

At the time indicated by an arrow in Figure 3F, a thinmicrocapillary tube (tip diameter: 18 pm) was slowly driveninto the root through the cortex, endodermis, and part of thestelar tissue and was then withdrawn. This resulted in adirect injury to 2 to 10 endodermal cells that represent 10-'to 10-3 of the total surface area of the endodermis (Petersonet al., 1993). Injuring the endodermis brought about a rapiddecrease in root pressure (half-time = 100 s) due to anoutflow of solution from the stele across the leak. A new,steady value of root pressure of about 0.04 MPa (0.4 bar)was obtained when the rate of active uptake of solutes(nutrients) compensated for their loss. The osmotic concen-tration of the solution in the apoplast of the stele apparentlyremained somewhat higher than that in the medium, causinga root pressure larger than atmospheric. It should be notedthat the absolute value of the decrease in pressure wasfairly variable in different experiments (see Tables I, 111,and IV), which could have reflected variable damage to theendodermis.

When root pressure attained a new steady value after the


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Composi te Transport Model of the Root 341

endodermis was punctured, the a and P,, were remeasuredfor the modified root (Fig. 3, G-J; Tables 1-111). It is evidentfrom the data given in Figure 3 and in Tables I to 111 thathydraulic parameters (hydrostatic and osmotic and Lp,)were not a ffected by puncturing. For example, the ratio ofhydrostatic TI/,Walues measured before and after puncturingwas 1.2 f 0.3 (mean f SD; Table I). Similarly, hydraulicconductivity did not change in osmotic experiments (Table111). The lack of an effect of puncturing is remarkable becauseof the common view that the endodermis should represent amain hydraulic barrier in the root. If this view were correct,puncturing this barrier should have resulted in a considerableshortening of half-times and in an increase of hydraulicconductivities.

In contrast to the hydraulic parameters, solute relationsparameters (Psr nd usr)did change upon puncturing the roots(Tables I1 and 111). Control values of usrand Psrwere similarto data already published (Azaizeh and Steudle, 1991; Steu-dle, 1992, 1993a, 1993b), that is, on average, usr= 0.64 andP,, = 3.8 X 10-9 m s-. This means that intact roots did notbehave like ideal osmometers (usr= 1 and P,, = O). Injuringonly 10- to 10-3 of the total surface area of the endodermisresulted in a substantial (36%) reduction of usr(on average,usr was reduced from 0.64 to 0.41) and in an increase of P,,by a factor of 2.7 on average (Tables I1 and 111).

At the end of each experiment, the root was excised nextto the silicone seal (arrow, Fig. 3K), and the root pressureimmediately decreased to zero. Subsequent hydrostatic relax-ations had a very short half-time, confirming that the vesselsof the xylem had been open during the experiment (Fig. 3K).Previous work in which a Cellufluor solution was forcedthrough the root while it was still in the seal also confirmedthat the solution was flowing through the vessels in experi-ments of this type (Peterson and Steudle, 1993).

In one set of experiments, more than one hole was madein the root cylinder. The root pressure decreased stepwiseafter each endodermal puncture, reaching steady values eachtime (Fig. 4). Such experiments were a challenge to performbecause they required more than one puncture of the endod-ermis. It was essential not to injure the root too deeply andthereby damage a mature vessel because this resulted in anirreversible decline of root pressure to zero. During prolongedexperiments (longer than a few hours) with injured roots,root pressures tended to become unstable. Because of thelength of time required for the multiple punctures and sub-sequent stabilizations of root pressure, it was possible toperform only the rapid hydrostatic measurements thatyielded values for Tz,2w nd Lp,. The more lengthy osmotic

experiments that would have also yielded values of P,, andusrcould not be done.Recovery of Roots from Puncturing

Evidently, punctured roots were capable of regaining theirroot pressure (Fig.5 , Table IV). The time required for recoverywas quite variable, ranging from 1.5 to 20 h to reach theoriginal root pressure. In some cases, root pressures evenhigher than the original were achieved (see root numbers 13-15 and 18-20 in Table IV). As in the multiple puncturingexperiments described above, only hydrostatic experiments(TlITw,p,) could be made. The results indicated that therewas no change in hydrostatic Lp , after recovery (Table IV).For some roots, cross-sections were taken through the punc-tured zones at the end of the experiments. Conspicuousbrowning of the walls of the endodermis and nearby cells ofthe stele was evident in unstained preparations (Fig. 2F).

DISCUSSIONPuncturing young maize roots affected transport of water

and solutes quite differently. Steady-state root pressures wereconsiderably reduced by the small injuries inflicted on theendodermis, but the and the Lp, remained unchanged.The solute permeability, measured for NaCl as the test solute,increased by a factor of 3 and osrdecreased by one-third. Theareas of artificial apoplasmic bypasses created by puncturingwere only 10- to 10-3% of the total surface area of theendodermis and of an order of magnitude or even smallerthan those naturally present in intact roots in secondary rootinitials (3 x 10-%) or across the tip route ( 5 x 10-%). Thus,modified roots represent model systems to study the role ofapoplasmic components of water and solute transport acrossroots and their effect on root osmotic properties. They shouldbe valuable systems for testing root transport models.Steady State Root Pressure of Modified Roots

Puncturing the endodermis caused a rapid decrease of rootpressure with a half-time of 10 to 100 s. This half-time wassubstantially larger than that for water exchange in hydro-static experiments but was smaller by 1 to 2 orders of mag-nitude than that measured for the diffusional flow of solutesafter the root was punctured. Therefore, it is safe to concludethat the half-time was not rate limited by the diffusional flowof solutes (nutrients) from the xylem into the medium acrossthe artificial pore or injury. Rather, it was caused by aconvective flow of xylem sap across the hole. The efflux of

(Continued from facing page) are present in the endodermis b u t not in the exodermis (ex).B, Transverse section 4 m m from the t ip stainedwith Cellufluor. The diameters of the immature endodermis (en)and those of the enclosed cells were used to estimate the area of theapoplasmic bypass at the root tip. C, Whole mount of a cleared root stained with acetocarmine. Unemerged lateral roots were evident asdark red conical structures. D, Longitudinal section of a main root with a near-median longitudinal section of a lateral root, stained withCellufluor. The diameter of the lateral root at its widest point is greater t h a n t h e discontinuity in the main root endodermis (en). , Longitudinal(paradermal)section of a main root with a cross-section of the base of a lateral at the leve1 of the endodermis of the main root, stained withCellufluor. The endodermis (arrows) s distinguished by the presence of large primary pit fields in its walls. Such sections were used toestimate t h e apoplasmic bypass area associated with lateral root primordia. F, Transverse section of a root that had been punctured 24 hearlier. The outer walls of severa1 cells of the endodermis (en)were ruptured. Cells of the endodermis and nearby stele underwent a wo u n dreaction characterized by the deposition of brown substances in their walls.


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o, + 50 n0r-l NaCl

Time, t Os)Figure 3. Time course of root pressure during a typical puncturingexperiment. A, Measurements of the elasticity of the system (seeMaterials and Methods ). B to E, Examples of hydrostatic a ndosmotic root pressure relaxations of the intact root. In 6, endosmoticand exosmotic hydrostatic relaxations are given in the absence,andin D n the presence, of the osmotic solute (50mOsmol kg- NaCI).C and E Biphasic osmotic pressure relaxations from which usr ndP,, were calculated in addition to the osmotic Lp,. At the conclusionof E, the root endodermis was punctured (arrow) nd root pressuredecreased rapidly from 0.1 3 to 0.04 MPa F). From a comparison ofB and D to G a nd I it is evident that the hydrostatic T remainedthe same after puncturing, i.e. Lp, remained constant. The same istrue for osmotic half-times, seen by comparingC with H. However,uSr n d JV2'decreased a nd P,, increased. A t t h e conclusion of t h eexperiment, the root was cut right at the seal whereupon the rootpressure decreased to zero (K). Half-times decreased by a n orderof magnitude after the cut, proving that the root xylem in the sealingarea remained open dur ing time of the experiment (10h). Note thedifferent scales on the time axes.

sap would lower the concentration of solutes in the xylemuntil a new steady state and an osmotic equilibrium wereattained. This would occur when the loss of solutes was justbalanced by the active uptake of solutes (nutrients) and theirdelivery to the apoplast of the stele. In such a system, theabsolute value of P,, is a measure of the rate of active uptakeof nutrients into the xylem.

To quantify the effect of changes in root transport param-

eters on the absolute value of the P,,, the root may bedescribed in terms of a pump/leak model, assuming a certainrate of active pumping of solutes (nutrients; Jsr*) and someleakage caused by the permeability of the root cylinder (en-dodermis). The P,, is a measure of the latter. Using the linearforce/flow relations of irreversible thermodynamics, the ra-dial water (Ivrn m3 m-' s-') and solute (Isr n mo1 m-2 s- )flows across the root are (according to Steudle, 1989, 1992,1993a, 1993b):

andIsr = Psr (C,l - C,O) + (1 - usr c vr + Isr*, ( 5 )

where C, and C are the solute concentrations in the mediumand root xylem, respectively, cs s the mean of both concen-trations, R is gas constant, and T is temperature. By conl'en-tion, Jsr, Jvr < O denotes an uptake and ISr,vr > O indicaks aloss of solutes and water from the root. For the steady state,it is valid that IS vr= O. From Equations 4 and 5 (Steudle,1993a, 1993b; Birner and Steudle, 1993), this yields a P,, of

s:P,, = - usr RT -.P S ,

According to Equation 6, P, will decrease with decreasingusr (decreasing passive root selectivity ) and will decreasewith increasing solute permeability P,, (solute leakage). 'Theequation can be used to (a) calculate the rate of activepumping (Jsr*), if usr, P,,, and P,, are measured (as in thispaper), and to (b) calculate changes in P, in modified rootsfrom changes in usrand P,, and compare the calculated valueswith measured values, assuming that Jsr* remained constmt.In this paper, typical values for intact roots were P,, = 0.15MPa, P,, = 3.8 X 10-9 m s-', and usr= 0.64. According toEquation 6, this results in an active Jsr* = 360 X 10-9 mo1 in-'s-', which is similar to uptake rates measured for excisedmaize roots for main nutrients (e.g. 30-200 10-9 mo1 of K+m-2 s-'; Anderson, 1976). On the other hand, changes of usrfrom 0.64 to 0.41 and of P,, from 3.8 to 10.1 X 10-9 m s-'upon puncturing (Table 11) result in a calculated reductioii ofP,, by 76% of the original or from P,, = 0.15 MPa (on averiige)to 0.04 MPa. This is similar to the measured value (0.04 h4Paon average; Table I), although the effect of puncturing on theabsolute value of P,, was rather variable. The variability couldhave been due to changes in Jsr* and/or to variations in thearea of the endodermis injured. For example, when using thesame capillary tube, hitting the endodermis at right anglesshould have resulted in a smaller hole than a glancing blowbecause of the tendency of the tissue to tear for a distancealong the endodermal cells in the latter case. On the otherhand, pores produced by puncturing could partially close byclogging with debris or by an enlargement of the surroundlingturgescent tissue. After multiple puncturing, P,, declinedstepwise as expected from Equation 6, because of a furtherdecrease in usrand an increase in P,, (Fig. 4). Thus, the simplepump/leak model applied to the root explained the measuredchanges in root pressure reasonably well. It has been dem-onstrated that it may be used to calculate rates of activeuptake of nutrients into roots.


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Table 1. Effect of puncturing the endodermis of young maize roots with a glass cannula tip diameter:18 t 60 pm) on stationary root pressure (P,J and hydrostatic l,, s a measure of Lp,

Root pressures decreased considerably, but TIA Lp,) remained unchanged, i.e. the ratio of T,Avalues was not significantly different from unity t test; P = 0.05). Means f SD a nd ranges are givenfor n = 10 roots. The data for T,A for individual roots are mean values a nd SD for n = 6 experimentson a given root.Pr0

Numher Before After P,, afterpuncturing puncturingMPa

Root P hefore/

1 0.15 0.02 7.52 0.1 5 0.04 3.73 0.10 0.04 2.54 0.16 0.07 2.35 0.14 0.05 2.86 0.08 0.05 1.67 0.15 0.05 3.08 0.19 0.06 3.19 0.15 0.03 5.0

10 0.19 0.06 3.1

17hBefore After

puncturing puncturing

5.0 f 0.64.1 1.23.9 f 0.45.8 f 0.72.7 f 0.43.1 0.34.3 f 0.74.3 0.93.8 f 1.33.5 f 0.2

s3.3 0.42.8 0.33.8 + 0.55.0 f 0.33.4 f 0.83.1 f 0.54.6 f 0.94.2 f 0.32.6 k 0.42.8 f 0.6

1% before/17,2"fter

51.51.51 o1.20.81 o0.91 .o1.51.3

Mean 0.15 f 0 . 0 3 0.05 f 0.01 3.5 1.7 4.1 0.9 3.6 f 0.8 1.2 k 0.3Range 0.08-0.19 0.02-0.07 1.6-7.5 2.7-5.8 2.6-5.0 0.8-1.5

Effects of Endod ermal lnjury on Root Hydraulic Properties(Tv, and Lp,)

In both intact and modified roots, there were large differ-ences between root hydraulic conductivities obtained fromosmotic and hydrostatic experiments. This is a well-knownphenomenon found for maize and other roots (Steudle et al.,1987; Cruz et al., 1992; Steudle, 1992, 1993a). It indicatessubstantial differences in the pathway of water movementdepending on the nature of the driving force. In the case ofhydrostatic pressure gradients, radial water flow is predomi-nantly apoplasmic (i.e. circumventing protoplasts) in the root

cylinder. By contrast, there is a considerable cell-to-cell com-ponent of radial water flow in the presence of osmoticgradients. The reason for this difference, which has beenobserved to date in severa1(but not all) species, is that osmoticgradients across the apoplast constitute only 'a very smalldriving force for water, because the us of the wall materialfor solutes is close to zero. Thus, the variable root hydraulicconductivity is a consequence of the composite nature of roottransport, i.e. it is due to the fact that there are two differentparallel pathways in the root contributing to the overall watertransport.

Despite the large changes in the P, caused by puncturing,

Table 11 Effects of endodermal puncture on parameters determined from osmotic pressure relaxationsP,, upon punctur ing were significant t test; P = 0.05). Individual roots (1-10) were the same as in Table IT uSr, nd P,, were measured using NaCl as the osmoticum (osmotic concentration: 50 mOsmol kg- of NaCI). Changes in TKs ,urr,and

TKIRootNumber Before After

puncturing puncturings

1 1500 19102 1720 3853 1670 7404 1060 5205 985 16706 3900 24607 890 7808 1630 2259 1520 350

10 1030 285

17hseiore/17h'fter

0.84.52.32.00.61.61.17.24.33.6

os os.beforelBefore After o afterpuncturing puncturing

0.730.770.590.760.740.410.920.370.700.40

0.380.590.330.440.270.220.450.280.670.43

1.91.31.81.72.71.92.01.31 o0.9

Before Afterpuncturing puncturing

m s- ) i 093.2 2.52.7 12.33.9 8.94.8 9.95.8 3.41 . 2 1.94.5 5.23.3 24.13.1 13.55.2 19.0

Mean 1590 870 930 f 790 2 . 8 f 2 . 1 0.6 4f 0.1 9 0.41 k0.14 1. 7f 0 . 5 3 .8+ 1.4 10.1 7.4Range 890-3900 225-2460 0.6-7.2 0.37-0.92 0.22-0.67 0.9-2.7 1.2-5.8 1.9-24.1


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Table 111. Summary of the effects ofpuncturing maize roots on Pro,Lp, a,,, and P,,

Note that the average hydrostatic Lp, was about 10-fold largerthan the osmotic. The values are means D for the same 10 rootsas in Tables I and II. Changes in P us,,and Psrwere significant (seeTables I and II),but there were no changes in either the hydrostaticor osmotic Lo, after Duncturinz.

Before AfterPuncturina Puncturina

P (MPa)Mean 0.15 0.03Range 0.08-0.1 9Mean 2.7 f 1.3Range 0.8-6.3

Osmotic Lp, X108 (m s-l MPa-')Mean 2.2 f 2.1Range 0.3-6.4

Hydrostatic Lp, x107 (m s-' MPa-')

U S CMean 0.64 f 0.19Range 0.37-0.92

P,, x109 (m s-')Mean 3.8 1.4Range 1.2-5.8

0.05 f 0.010.02-0.072.8 f 1.60.7-6.7

2.5 f 2.50.7-7.5

0.41 0.140.22-0.67

10.1 7.41.9-24.1

hydraulic conductivity measured either by hydrostatic orosmotic experiments was not affected. This was so, eventhough, according to Poiseuille's law, a hole with a diameterof 20 pm and a length of 400 pm (i.e. across the entire rootcylinder up to the xylem) would exhibit the very high hy-draulic conductance of 0.98 X 10-8m3 s-l MPa-' in hydro-static experiments (viscosity of water at 20C: 10-3 Pa s).Expressing this conductance in relation to the total A , resultsin an L p , as large as 3.8 X 10-5 m s-l MPa-', i.e. a value thatis larger by 2 orders of magnitude than that found for bothintact and punctured roots (hydrostatic L p , = 2.7 X 10-7 ms-' MPa-'). The reason for this discrepancy may be that theholes (pores) made were not entirely across the stelar tissueup to the vessels and across the vessel walls. Damaging thevessels resulted in irreversible damage to the root, probablybecause the outflow of solutes from the xylem could nolonger be compensated for by active pumping. Thus, thesituation in the experiments could be different from that inthe calculation given above.

The lack of any change in the osmotic L p , is to be expected.In contrast to hydrostatic gradients, osmotic gradients acrossthe pore could not have caused water flow because there wasno selective membrane on this path. The lack of change inthe hydrostatic and osmotic L p , to puncturing shows that theradial hydraulic resistance of the root was rather evenlydistributed over a11 of its tissues. This idea differs considerablyfrom the commonly held view of the endodermis being themain hydraulic resistance in roots. It is supported by recentmeasurements of Peterson et al. (1993), who removed someof the root cortex of young maize roots by scraping and alsopunctured roots with thin glass cannulae. In both cases thehydrostatic L p , remained unchanged despite reductions inroot pressure that occurred during puncturing.

Location of Barriers to Solute Flow: Effect of Puncturingon Root Psrand usrThe effect of puncturing on the root P,, and asrwas sub-

stantial, indicating that the endodermis was the main barrierfor solute transport. Thus, the contribution of the apop1as:micsolute transport in regions lacking a Casparian band (rootinitials, tip route) to overall solute transport is potentidlyimportant, although the mobility of solutes in these pathwaysis known to be restricted (Peterson et al., 1981; Enstone iind

' MPii

O= 17 I I

o= -I IO 2 1 I2 I

10 2-3 WTime, t (s)Figure 4. Effect of multiple puncturing of the endodermis on rootpressure. Hydrostatic relaxations; B D, F, and H are as descri bedin Figure 3. The first puncturing ( C ) esulted in a decrease of rootpressure from 0.13 to 0.07 MPa, and the second (E) resulted in adecrease from 0.07 to 0.05 MPa. The third hole (G)caused a fur herpressure decrease from 0.05 to 0.03 MPa, i.e. t o a rather low value.Note that multiple puncturing did not change T,, (and therer'oreLp,) in hydrostatic pressure relaxations.


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Puncturing of endodermisDiameter of microcapillary: 60 p mI

oI

02.0

15 O 6 b i i I ~ lO 15 30 O 15

Time, t (s)Figure 5. Typical experiment demonstrating the recovery of root pressure in punctured roots. The different regions(A to C and E a nd F) are as explained in Figure 3. D, Recovery took about 20 h for this root. During this time the rootregained its ability to accumulate ions i n t h e stele (which includes the xylem), t h u s increasing its root pressure. In thisparticular root, the root pressure after recovery was even larger t h a n the original.

Peterson, 1992). The only apoplasmic pathway from thecortex to the main root stele that is open to molecules thesize of fluorescent dyes exists at the flanks of emerged lateralroots (Peterson et al., 1981).

Solute transport can be analyzed in detail by using theexperimental data (permeability coefficients and areas avail-able for solute transport in different pathways). Of interestis the contribution of different pathways to the overall solutetransport in the roots and the solute mobility in these path-ways. For this evaluation the solute transport across theCasparian band itself has to be taken into account as well,because this structure occupied an area as large as 12% ofthe total surface area of the endodermis. Two extreme casesare of interest: (a) the case in which the Casparian band iscompletely impermeable to solutes (the commonly accepted

view) and (b) the case in which the transport across theCasparian band and apoplasmic bypasses (tip and lateralroots combined) contribute equally to the overall solutepermeability. In the latter case, the Casparian band would beextremely leaky. Transmembrane solute transport across en-dodermal cells may be considered negligible in this contextto a first approximation (but see below).

(a) If the Casparian band were completely impermeable tosolutes, in this case NaCl, a permeability coefficient for thetransport across secondary root initials and the main root tipmay be estimated by first. referring the root P,,of 3.8 X 10-9m s-' (Table 11) to the surface area of the endodermis (as thelimiting barrier) instead of that of the root surface. Becausethe surface area of the endodermis was about half that of theroot, this yields a P,,= 7.6 X 10-9 m s-l for the endodermis.

Table IV. Effects of recovery on P,, and ThW n punctured rootsoriginal was attained. Hydrostatic J,, Lp,)did not change d u r in g recovery.Recovery took from 1.5 to 20 h, after which a root pressure equal to or even higher t h a n the

P,, TV?Root Ti me RequiredNumber for Recovery Before After After Before Afterpuncturing puncturing recovery puncturing recovery

h MPa 511 7.5 0.1 7 0.05 0.1 7 4.7 f 0.7 9.6 f 0.21 2 1 2 0.17 0.07 0.1 5 4.0 rt 0.5 5.2 rt 0.613 17 0.11 0.04 0.20 7.7 -C 0.5 5.9 f 1.414 13 0.17 0.05 0.22 3.6 0.3 2.7 +. 0.515 14 0.16 0.12 0.27 5.3 f 0.4 4.6 .616 10 0.09 0.02 0.05 4.5 f 0.7 6.2 1.217 16 0.08 0.03 0.02 3.2 f 0.1 7.2 3.118 1 1 0.12 0.04 0.19 3.6 f 0.7 2.9 .519 1.5 0.07 0.04 0.08 5.7 rt 1.1 6.8 rt 1.020 20 0.10 0.06 0.17 2.2 f 0.6 1.6 rt 0.2

Mean 12.2 f 5.2 0.12 f 0.04 0.05 0.03 0. 15 f 0.08 4 . 5 f 1.5 5.3 f 2.4Range 1.5-20 0.07-0.17 0.02-0.12 0.02-0.27 2.2-7.7 1.6-9.6


12/15


If the total flow is assumed to go through the walls in areaslacking a Casparian band (8 X 10-2 of the surface area ofthe endodermis), we get a P,, = 7.6/0.08 X 10-7= 9.5 X 10-6m s-l for that apoplasmic path. Assuming a homogeneouspath of length of 1 2 pm (the thickness of the endodermis),an apoplasmic diffusion coefficient(Os)or NaCl of D, = 9.5X 1.2 X 10- = 1.1X 10-l' m2s-' is estimated. This value iswithin an order of magnitude of those given in the literaturefor Na+ and C1- ions in walls of barley root tissue (3 X 10-to 10 X 10- m2s-'; for refs., see Walker and Pitman, 1976).It is considerably smaller than that of NaCl in bulk aqueoussolution (1.5 X 10-9 m2 s-' at 25OC).

(b) If, on the other hand, the transport across the Casparianband and that across apoplasmic bypasses contributedequally to the overall transport ( leaky Casparian band ), thiswould mean that the permeability of the Casparian bandwould be smaller by a factor of 12/0.080 = 150 than thatalong apoplast lacking a hydrophobic barrier. Using thisvalue and considering a thickness of the Casparian band of2 pm yields a P,, = 3.2 X 10-8 m s-l and a D , = 6.4 X 10-14m2 s-' for the Casparian band. This value is smaller by 3orders of magnitude than the D , for NaCl in the apoplastlacking a hydrophobic barrier (see above), despite the factthat the calculation assumed a leakage of as large as 50%across the Casparian band. It is clear that, for a properfunction of the Casparian band as an effective barrier forsolutes, the D , would have to be considerably smaller. Thus,it could become comparable to D, values of univalent salts incuticles (1 X 10-15 to 3 X l O - I 7 m2 s-' for KCl; Tyree et al.,1992). The calculations show that, if the Casparian bandwere almost impermeable, its D, would have to be even lessthan that of a waxy layer like the cuticle. This is unlikely tobe the case because suberin is less hydrophobic than wax.Therefore, it is reasonable to assume that Casparian bandswould allow some bypass of solutes and water even in amature endodermis. Because the bypass would be small, itwould not really affect the function of the endodermis afilter for solutes. From root pressure probe data (Steudle,

l989,1992,1993a, 1993b) and also from other work (Hansonet al., 1985; Yeo et al., 1987; Radin and Matthews, 1989;Behrmann and Heyser, 1993), there is experimental evidenceof apoplasmic bypasses in the endodermis, bu t more data areundoubtedly required for proof.

Although the membrane permeability of the solute (NaC1)was not directly measured in this paper, we may estimate thepossible contribution of the membrane-bound component(endodermal protoplasts) to solute transport from the overallpermeability and from the morphometric data. For example,if the components across the apoplast lacking a Casparianband (0.08% of total surface area of endodermis), across theCasparian band itself (12%), and the endodermal cells (88%)each contributed equally to the overall transport (i.e. each byone-third), this would result in a permeability coefficient (Ps )of the endodermal cell membrane of P, = 5.8 X 10-9 m s-'.This value is somewhat higher than those given in theliterature for P. values of root cell membranes to Na+ andC1- (1 X 10-9 to 3 x 10-I' m s-'; Pitman, 1976). Hence, thecontribution of the cellular path may be smaller than one-third. The comparison of the data shows that the membrane-

bound path across the endodermis cannot be neglected iii arigorous evaluation, at least for an endodermis lacking sub-erin lamellae.

Recovery from PuncturingRepair of punctured roots appeared to be a process t l iat

usually started 0.5 to 1 h after wounding, but completerecovery took up to 20 h. This indicates that, following aninjury, roots soon improved their selectivity and their abilityto accumulate nutrient ions in the stele. The rapid closure ofinjuries in the endodermis or stele may be favored by thelack of air spaces that would otherwise have to be filled withhydrophobic material (Peterson et al., 1993). However, i:heosmotic experiments attempted on recovered roots showedthat they represented rather unstable osmometers, tending toleak and lose root pressure when subjected to osmotic stress.For this reason, no satisfactory osmotic experiments could beperformed on recovered roots. Perhaps by improving itheconditions during recovery, more stable roots could be pro-duced in the future. This would be a worthwhile achievementbecause punctured roots could be filled with different sol-utes, dyes, inhibitors, hormones, and the like while the poreis open. After the pore closes, different types of modifiedroots would thus be obtained to study transport and otherroot functions.

Composite Transport Mod elAt first glance, it is astonishing that after making a hole as

large as 60 pm in the endodermis the roots maintained apositive root pressure and exhibited some passive selectivity,i.e. usr> O. The solute permeability of the modified roots wasstill rather low, and the osmotic responses after punctur:mgwere basically the same as those of intact roots, i.e. they weretypical for an osmometer in the presence of a permea thgsolute as measured for roots or plant cells and derived fromirreversible thermodynamics. Similarities in osmotic re-sponses of intact and modified roots may be explained by anextended osmometer model of the root, which is shownschematically in Figure 6 . The root can be represented by anosmotic system having a composite osmotic barrier consistingof the cell-to-cell path (superscript b ), which is an almostsemipermeable barrier because of the presence of numerousmembranes in series, and the nonselective (porous) wall paththat should leak solutes (superscript a ). The function of theCasparian band (Fig. 6A) is to minimize this leakage. ,41-though there may be some leakage present in the system clueto (a) a not completely tight Casparian band (Fig. 6A), (b)apoplasmic pathways lacking Casparian bands (Fig. 6B), or(c) an artificial pore or injury (as in this paper; Fig. 6C), theroot will respond as an osmometer because of the existenceof the cell-to-cell path. If the solute concentration in thexylem is larger than that in the medium, water will be suckedinto the xylem along the cell-to-cell path and root pressiirewill build up. However, this will immediately induce a back-flow of water (solution) across the nonselective apoplast or,


13/15


Composite transport modelof root

Cadparian band@ Root tissue lacking lnside

Endo-dermis lnside

A I m tpressurCasparian band

-e

Figure 6. Diagrammatic scheme of composite transport in a plantroot. A a nd B denote transport across the intact system (root tissuewith or without a n endodermis, respectively),and C depicts thesituation in a punctured root. For the sake of simplicity, rhizodermisa nd cortex (including the endodermis) are represented by threecell layers and the stelar tissues by a single layer. In C, puncturedroots vessels were not hit by the microcapillary tube. Differentroutes of radial flow are denoted, i.e. the flow across the apoplastor an artificial hole (superscript a)and the cell-to-cell path (su-perscript b) . The figure indicates t h a t there will be a n osmoticwater flow jvb)irected from the root medium into the xylem (stele)as longas the osmotic concentration in the xylem is larger than tha tin the medium. The uSr long this path should be high ( 0 ; = 1 ) .However, as soon as a root pressure builds u p , there will be abackflow of water (xylem solution) across the nonselective apo-

in the modified root, across the artificial pore. Because thereis some active pumping of nutrients, a steady root pressurewill be established when the rate of the osmotic water uptakejust balances the hydraulic extrusion. In the steady state, theopposing water flows will cancel, and some kind of a circu-lation flow of water across the entire barrier will be estab-lished (Fig. 6). It is clear that, because of the backflow, theroot osmometer will not behave like an ideal (semipermeable)osmotic cell. In osmotic equilibrium, the hydrostatic pressuredifference set up will be smaller than the osmotic pressuredifference between the xylem and medium. In quantitativeterms, this is expressed by usr< 1, and it will hold that P, =uSr.Ar, . The rate of backflow will increase with increasingsize and number of holes (leaks), and P,, and a will decreasefurther as demonstrated in this paper. On the other hand,the backflow will be reduced by a decreasing permeability ofthe Casparian band during root development, and it is clearthat usrwill increase with a decreasing hydraulic conductanceof the endodermal or other apoplasmic path .

Composite systems such as that depicted for the root inFigure 6 have been readily treated in the theory of thethermodynamics of irreversible processes as applied to mem-branes composed of arrays with different transport properties(Kedem and Katchalsky, 1963; Steudle, 1989, 1992, 1993a,1993b). The theory shows that for a system composed of twoparallel pathways we get for the overall usr of the barrier(root):

(7)Here the same notation is used as in Figure 6, i.e. thesuperscript a for transport across the hole or apoplast andthe superscript b for the cell-to-cell path. The ya and ybdenote the fractional cross-sectional areas of the two differentpathways (ya + yb = 1) and Lp and Lpb represent theirhydraulic conductivities. The u, of the two different arraysare denoted by ut, and u It can be seen from Equation 7that the overall usr is a weighted mean of the individual usvalues and that the different components of the barriercontribute to the overall usr according to their hydraulicconductance (ya Lp and yb Lpb, respectively), whereby itholds that L p , = yLp+ yb Lpb. Hence, the contribution ofthe very low us of the apoplasmic or pore path lacking aCasparian band (ut = O) to the overall usr could be highbecause of the high hydraulic conductance of the apoplast orpore, although the area available for apoplasmic transport ismuch smaller than that along the cell-to-cell path.

plasmic paths (usa = O) in B, C, and, perhaps, also in A if the matureCasparian band is somewhat permeable to water. When the op-posing water flows exactly cancel each other, root pressure willhold steady a nd will be smaller t han expected on the basis of thedifference in osmotic pressure between the xylem sap and themedium, i.e. urrwill be smaller t h a n unity. It can be seen from themodel that, according to the composite structure of the entirebarrier, a circulation flow of water will be set u p across the root atzero or low net water flow jVJ.


14/15


For the same system composed of two parallel pathways,the overall P,, will be given by (Kedem and Katchalsky, 1963;see also House, 1974):

Lp L p bLPrPsr= yP,+ yb P,b + u: - u,a)2 RT , (8)

Again, the superscripts a and b denot e properties ofindividual pathways as above. The first two terms on theright side of Equation 8 represent the contributions of thediffusional components of the individual pathways to theoverall solute flow. The additional compone nt (third term onthe right side), which tends t o increase P,,, esults from thesolvent drag compone nt of solute flow (see second term onthe right side of Equation 5; for details of the theory, see refs.given). Thus, an increase of P,, caused by puncturing theendodermis was due both to the creation of an additionaldiffusional path for solutes (7 P:) and to a solvent-dragcomponent. For the osmotic experiments with puncturedroots, it is hard to judge what the relative contributions ofthe different components (solvent drag versus diffusional)would be. However, owing to the rapid adjustment of rootpressure to the new stationary value after puncturing (i.e. tothe large water flow) and to the rather long half-times ofsolute flow found before and after puncturing, the effect ofsolvent drag should be considerable. Experimentally, it wouldbe difficult to sepa rate the different compon ents given inEquation 8. The same applies to direct measureme nts of theinwardly a nd outwardly directed wate r flows in th e system.

The composite transport model predicts so me variability inroot hydraulic resistances, i.e. a decrease of the hydraulicresistance with increasing water flow across the root. Thiseffect has been known for a long time, but no satisfactoryexplanation h as been given for it (Fiscus, 1975; Weatherley,1982; Passioura, 1988; Katou a nd Taura, 1989). The compos-ite model could resolve the problem. Th e model predicts thata t low rates of water flow, when the osmotic component inthe driving force would be substantial and water flows in theroot would, in part, cancel, t he resistance shou ld be high Lp ,low). This should be t he case at zero or low rates of transpir-ation. At higher flow rates (higher rates of transpiration),tensions in the root xylem (i.e., t he hydrostatic compo nent)will domi nate th e overall driving force for water. This willresult in a water flow along the two pathways in the samedirection an d in a lower hydraulic resistance of the root L p ,high).The composite transport appro ach integrates quite differentan d important properties of roots in a single model. It has asound theoretical an d anatomical basis. It may be extendedto roots exhibiting zones of various developmental stages bytreating these zones as arrays of different transport propertiesarranged in parallel, an d to entire root systems (Steudle,1989, 1992, 1993a, 1993b).

ACKNOWLEDCMENTSThe authors thank Marcus Rdinger for his help in performing

some of the puncturing experiments and Burkhard Stumpf for his

expert technical assistance. The help of Ms. Daryl Enstone in pro-ducing Figure 2, A, C, and D, is also gratefully acknowledged.Received April 14, 1993; accepted June 25, 1993.Copyright Clearance Center: 0032-0889/93/l03/0335/15

LITERATURE CITEDAnderson WP (1976) Transport through roots. I n U Lttge, IdGPitman, eds, Encyclopedia of Plant Physiology, Vol 2, Part B.Springer-Verlag,Heidelberg, Germany, pp 129-156Azaizeh H, Steudle E (1991) Effects of salinity on water transportof excised maize Zea mays L.) roots. Plant Physiol97: 1136-1145Behrmann P, Heyser W (1993) Apoplasmic transport through thefunga1 sheath of Pinus sylvestris/Suillus bovinus ectomycorrhizae.Bot Acta 105: 427-434Bell JK, McCully ME (1970) A histological study of lateral rootinitiation and development in Zea mays. Protoplasma 70: 179-205Birner TP, Steud le E (1993) Effects of anaerobic conditions on waterand solute relations and active transport in roots of maize .Zeamays L.). Planta 190: 474-483Brundrett MC, Enstone DE, Peterson CA (1988) A berberine-anilineblue fluorescent staining procedure for suberin, lignin and callosein plant tissue. Protoplasma 14 6 133-142Cruz RT, Jordan WR, Drew MC (1992) Structural changes andassociated reduction of hydraulic conductance in roots of Sorghumbicolor L. following exposure to water deficit. Plant Physiol 9 9Enstone DE, Peterson CA (1992) The apoplastic permeability of rootapices. Can J Bot 7 0 1502-1512Fiscus EL (1975) The interaction between osmotic- and pressure-induced water flow in plant roots. Plant Physio l55 917-922Frensch J, Stelzer R, Steudle E (1992) NaCl uptake in roots of .Zeamays seedlings: comparison of root pressure probe and EDX data .Ann Bot 70: 543-550Frensch J, Steudle E (1989) Axial and radial hydraulic resistance toroots of maize Zea mays L,). Plant Physiol 91: 719-726Hack ett C, Stewart HE (1969)A method for determining the positionand size of lateral primordia in the axes of roots without sectioning.Ann Bot 33: 679-682Hanson PJ, Sucoff EI, Markhart AH (1985) Quantifying apop1a:jticflux through red pine root systems using trisodium 3-hydrorcy-5,8,10-pyrenetrisulfonate.lant Physiol 77: 21-24House CR (1974) Water Transport in Cells and Tissues. EdwardArnold, LondonKaras I, McCully ME (1973) Further studies of the histology oflateral root development in Zea mays. Protoplasma 77: 243-2651Katou K, Taura T (1989) Mechanism of pressure-induced water flowacross plant roots. Protoplasma 150 124-130Kedem O , Katchalsky A (1963) Permeability of composite mem-branes, Part 2: Parallel elements. Trans Far Soc 59 1931-1940Melchior W, Steudle E (1993) Water transport in onion Al l ium cepaL.) roots. Changes of axial and radial hydraulic conductivitiesduring root development. Plant Physiol101: 1305-1315Passioura JB (1988) Water transport in and to roots. Annu Rev PlantPhysiol Plant Mo1 Biol 3 9 245-265Peterson CA, Emanuel ME, Humphreys GB (1981) Pathway ofmovement of apoplastic fluorescent tracers through the endoder-mis at the site of secondary root formation in corn Zea mays) andbroad bean Vicia f aba ) . Can J Bot 5 9 618-625Peterson CA, Murrmann M, Steudle E (1993) Location of the majorbamers to water and ion movement in young roots of Zea mays L.Planta 190: 127-136Peterson CA, Steudle E (1993) Lateral hydraulic conductivity,ofearly metaxylem vessels in Zea mays L. roots. Planta 189 288-297Pitman MG (1976) Ion uptake by plant roots. Zn U Lttge, M GPitman, eds, Encyclopedia of Plant Physiology, Vol 2B. Springer-Verlag, Berlin, pp 95-128Radin JW, Matthe ws MA (1989) Water transport properties of c d sin the root cortex of nitrogen- and phosphorus-deficient cotkonseedlings. Plant Physiol89 264-268Steudle E (1989) Water flow in plants and its coupling to otherprocesses: an overview. Methods Enzymol l74 183-225

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composite model of roots

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