roles of morphology, anatomy, and aquaporins in · closely related, the anatomy of yellow lupin...

Roles of Morphology, Anatomy, and Aquaporins inDetermining Contrasting Hydraulic Behavior of Roots1[OA]

Helen Bramley2*, Neil C. Turner, David W. Turner, and Stephen D. Tyerman

School of Agriculture, Food, and Wine, University of Adelaide (Waite Campus), Plant Research Centre, GlenOsmond, South Australia 5064, Australia (H.B., S.D.T.); School of Plant Biology, Faculty of Natural andAgricultural Sciences, University of Western Australia, Crawley, Western Australia 6009, Australia (H.B.,D.W.T.); and Centre for Legumes in Mediterranean Agriculture, Faculty of Natural and Agricultural Sciences,University of Western Australia, Crawley, Western Australia 6009, Australia (N.C.T.)

The contrasting hydraulic properties of wheat (Triticum aestivum), narrow-leafed lupin (Lupinus angustifolius), and yellow lupin(Lupinus luteus) roots were identified by integrating measurements of water flow across different structural levels oforganization with anatomy and modeling. Anatomy played a major role in root hydraulics, influencing axial conductance (Lax)and the distribution of water uptake along the root, with a more localized role for aquaporins (AQPs). Lupin roots had greaterLax than wheat roots, due to greater xylem development. Lax and root hydraulic conductance (Lr) were related to each other,such that both variables increased with distance from the root tip in lupin roots. Lax and Lr were constant with distance from thetip in wheat roots. Despite these contrasting behaviors, the hydraulic conductivity of root cells (Lpc) was similar for all speciesand increased from the root surface toward the endodermis. Lpc was largely controlled by AQPs, as demonstrated by dramaticreductions in Lpc by the AQP blocker mercury. Modeling the root as a series of concentric, cylindrical membranes, and theinhibition of AQP activity at the root level, indicated that water flow in lupin roots occurred primarily through the apoplast,without crossing membranes and without the involvement of AQPs. In contrast, water flow across wheat roots crossedmercury-sensitive AQPs in the endodermis, which significantly influenced Lr. This study demonstrates the importance ofexamining root morphology and anatomy in assessing the role of AQPs in root hydraulics.

The expression of a large number of aquaporins(AQPs) occurs predominantly in roots (for review, seeBramley et al., 2007b). These membrane integral pro-teins form water-conducting channels, which areconsidered responsible for the variable hydraulic con-ductivity of root systems (Lpr; Javot and Maurel, 2002).Reverse genetics has demonstrated that AQP activityis linked to the hydraulics of some species duringabiotic perturbation, as the water relations of antisenseor mutant plants tends to be more sensitive to theperturbation (Martre et al., 2002; Siefritz et al., 2002;Aharon et al., 2003; Yu et al., 2005; Jang et al., 2007).However, reverse genetics can also alter the expressionof other AQPs that were not targeted (Jang et al., 2007)and possibly other transporters involved in osmoreg-ulation. These combined effects may cause changes inthe morphology of the root system. For example,

antisense Arabidopsis (Arabidopsis thaliana) and to-bacco (Nicotiana tabacum) had larger root masses(Kaldenhoff et al., 1998; Martre et al., 2002; Siefritzet al., 2002). The increase in root mass may be a com-pensatory effect, because a lower expression of plasmamembrane AQPs reduced the water permeability ofsome cells (Martre et al., 2002; Siefritz et al., 2002).However, it is not known if the anatomy of antisenseroots was also altered or if water uptake was localizedto specific regions of the root. These features wouldalso have an important influence on Lpr.

To fully understand root system hydraulics and thecontribution of native AQPs, comprehensive studies atdifferent scales are required (cells, organs, and wholeroots). This should be coordinated with examinationsof anatomy to identify features that will influence therate of water flow through the root. Comparativestudies on species from different evolutionary linesand with differing root hydraulic properties may alsofurther elucidate AQP behavior. To address theseissues, we examined the hydraulic properties of rootsof two contrasting species: wheat (Triticum aestivum)and narrow-leafed lupin (Lupinus angustifolius). Wheatdevelops a root system of several extensively branchedindividual roots (O’Brien, 1979), whereas narrow-leafed lupin has a dominant taproot that can developmany primary lateral roots (Clements et al., 1993).Under the same field conditions, wheat has a muchgreater total root length density than narrow-leafedlupin (Hamblin and Tennant, 1987; Gallardo et al.,

1 This work was supported by the Grains Research and Devel-opment Corporation of Australia and the Australian ResearchCouncil.

2 Present address: Department of Renewable Resources, 4-44 ESB,University of Alberta, Edmonton, Canada T6G 2E3.

* Corresponding author; e-mail [email protected] author responsible for distribution of materials integral to the

findings presented in this article in accordance with the policydescribed in the Instructions for Authors (www.plantphysiol.org) is:Helen Bramley ([email protected]).

[OA] Open Access articles can be viewed online without a sub-scription.

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1996; Gregory and Eastham, 1996). However, bothspecies have similar aboveground biomass and ex-tract similar volumes of water from the soil profile(Hamblin and Tennant, 1987). Per unit of root length,narrow-leafed lupin appears to take up and transportwater at a greater rate than wheat (Hamblin andTennant, 1987; Gallardo et al., 1996). The reason forthese differing hydraulics was not previously investi-gated but was speculated to be because wheat andlupin roots have different xylem development (seebelow; Hamblin and Tennant, 1987; Gallardo et al.,1996). Other differences in anatomy and AQP activitycould also be the cause of the disparity.

To investigate more subtle differences in root hy-draulics, we also examined the hydraulic properties ofa closely related lupin species: yellow lupin (Lupinusluteus ‘Wodjil’). Water transport in yellow lupin rootshas not been measured previously. However, anec-dotal evidence indicates that the hydraulic propertiesdiffer between the two lupin species. Yellow lupin ismore tolerant of waterlogging than narrow-leafedlupin due to characteristics of its roots (Davies et al.,2000a, 2000b), which may be related to AQP activity(Bramley et al., 2007b). In addition, comparative mea-surements of leaf water potential in the field (Paltaet al., 2004) suggest that yellow lupin may have greaterconductance to water flow than narrow-leafed lupin.

The aim of this study was to identify the influence ofroot structure, anatomy, and AQP activity on the rateof water transport in wheat, narrow-leafed lupin, andyellow lupin roots. The rationale and main hypotheseswere as follows. (1) Lupin roots have larger and moreabundant xylem vessels than wheat (Hamblin andTennant, 1987). If water flow through xylem vessels isanalogous to flow through pipes, then the axial con-ductance (Lax) of lupin roots should be greater thanthat of wheat. (2) The transport of water through theradial flow path, not the axial path, is considered thegreatest limitation to the rate of water flow throughthe whole root (Steudle and Peterson, 1998). Therefore,the total hydraulic conductance (Lr) of wheat andlupin roots should be less than their Lax. (3) Lupin rootshave abundant root hairs over the whole root system,in comparison with wheat, where root hairs are morelocalized to the apical region (Hamblin and Tennant,1987). This implies that the absorbing region differsbetween the species and that lupin roots may absorbwater more evenly along the length of the root com-pared with wheat. (4) Structural differences and AQPactivity in the radial pathway should be the primaryfactors determining Lpr of wheat and lupin. In theradial path, water crosses a series of tissues from theroot surface to the vascular tissue in the stele. Watercan flow through the apoplast (cell walls and intercel-lular spaces) and/or through cells. The relative con-tribution of the radial pathways depends on theirrelative Lr. Both conductances are proportional to theircross-sectional area and inversely proportional totheir path length (Steudle and Frensch, 1996). Theradial path length was expected to be longer in lupin

than in wheat, because lupin roots are two to threetimes thicker. If the radial path length is longer in lupinroots, then lupin roots should have fewer anatomicalbarriers to water flow in the apoplast and/or greaterAQP activity in the cell-to-cell path, facilitating greateroverall root Lr. AQP activity can only influence radialconductance if water crosses membranes. In the apo-plast, flow can be restricted by the deposition of suberinin cell walls, such as Casparian bands and suberinlamellae in the exodermis and endodermis (Steudle andPeterson, 1998). (5) Because the two lupin species areclosely related, the anatomy of yellow lupin roots wasexpected to be very similar to that of narrow-leafedlupin roots. Therefore, AQPs were expected to mediatedifferences in Lpr between the species.

To test these hypotheses, the rate of water flow wasmeasured across whole root systems, individual roots,and root cell membranes of seedlings grown in sand.Anatomical features in different regions of the rootwere related to the water flow measurements to iden-tify the structural components influencing root wateruptake and transport. Cell size, radial path length, andthe developmental state of the endodermis and exo-dermis (if present) were examined. Cross sections ofroots were stained for Casparian bands and suberinlamellae (Brundrett et al., 1988, 1991). To determinewhether water uptake is localized to a particularregion of the root, the Lr of root segments and rootsystems was compared against root length. Assumingthat water can enter the root anywhere along the rootsurface, Lr should be proportional to root length. TheLax of small root segments was measured and relatedto changes in xylem vessel development as well astotal root Lr. To estimate the contribution of the radialpathways, a radial profile of the hydraulic conductiv-ity of cells in each cell layer of the root was created. Theprofile was applied to a model, modified from Joneset al. (1988), describing the root as a series of concentriccylindrical membranes. Water flow across the cell-to-cell pathway was inhibited with an AQP blocker to testthe model and the contribution of AQP activity. Here,we assumed that blocking the cell-to-cell pathwaywould not increase the hydraulic conductivity of theapoplastic pathway. By adopting this integrated ap-proach, we identified the contributions of root mor-phology, anatomy, and AQPs to root water flow in thethree contrasting species.

RESULTS

The Total Length of Wheat Roots Was Greater ThanThat of Lupin Roots, But They Did Not Transport MoreWater to the Shoot

Fourteen days after sowing (DAS), wheat root sys-tems comprised four or five seminal roots (Fig. 1). Thelongest seminal root was 360 to 490 mm long, with thesecond and third seminal roots 20 to 50 mm shorter.The remaining roots grew to lengths of up to 200 mm.No nodal roots developed in these young plants. The

Contrasting Hydraulic Properties of Wheat and Lupin Roots

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basal half of the wheat root system also containedmany fine lateral roots. The root systems of both lupinspecies were physically similar at 14 DAS. Lupins hada sturdy taproot with lateral roots in the basal region(Fig. 1) and a few small nitrogen-fixing nodules closeto the base of the stem. Wheat root systems were morethan twice the total length of lupin root systems (TableI). However, wheat roots were smaller in diameterthan lupin roots, which resulted in no significantdifference in surface area of root systems betweenthe three species (P 5 0.26; Table I).

Midday transpiration of yellow lupin plants, mea-sured gravimetrically, was approximately one-thirdlower than that of the other species (P 5 0.004; Table I).However, transpiration of wheat and narrow-leafedlupin plants was not significantly different (P . 0.05;Table I).

Lr Increased with Root Length in Lupin But Notin Wheat

The hydraulic properties of whole root systemswere measured with a pressure chamber. Preliminary

Figure 1. The root systems of wheat and narrow-leafed lupin at 14 DAS. The root systems werestained for 1 h with 0.5% methylene blue toenhance the image contrast. The insets showfreehand, transverse sections of the root (A andF) or stele (B–E and G–K). Sections A and F werestained with 0.05% toluidine blue. Sectionsstained with Sudan Red and viewed under whitelight (B and D) or phase contrast (G and J) showsuberin lamellae in the walls of the endodermis(en). Fluorescence in sections stained with theberberine-aniline blue procedure and viewed un-der UV light (C, E, H, and K) show Casparianbands (E and K; indicated by white arrows) in theendodermis and lignified cell walls, phloem, andxylem vessels (C, E, H, and K). Bars 5 50 mm.

Table I. Dimensions and mean water transport parameters of whole roots used in the pressurechamber measurements

At 14 d after sowing, DP of 0.3, 0.5, and 0.7 MPa was applied to detopped root systems to induce a waterflux. The flow rate at each pressure was plotted against DP, and the slope of the linear regression gave Lr.Normalizing flow rate by surface area gave Lpr. Required DP is the pressure that was needed to induce aflow rate through detopped root systems that was equivalent to midday transpiration. Values are means 6 SE

(n 5 6–8). Asterisks denote significant differences by Tukey’s test (P , 0.05).

Parameter Narrow-Leafed Lupin Yellow Lupin Wheat

Total length (m) 2.3 6 0.1 1.9 6 0.2 5.2 6 0.5*Surface area (m2 3 1023) 8.2 6 0.6 7.1 6 0.6 7.0 6 0.5Transpiration (g h21) 0.6 6 0.05 0.4 6 0.04* 0.6 6 0.01Required DP (MPa) 0.31 6 0.045* 0.25 6 0.05* 0.41 6 0.09*Lr (m3 s21 MPa21 3 10210) 10.61 6 1.02 8.24 6 0.88 3.96 6 0.58*DP intercept (kPa) 180* 100* 23*Lpr (m s21 MPa21 3 1028) 13.12 6 1.7 11.66 6 1.1 5.74 6 0.8*

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measurements indicated that the pressure range ap-plied to root systems induced water flux that wouldhave been sufficient to meet the demands of transpi-ration (Table I). The rate of water transport, per unitpressure gradient, by whole roots was similar for bothlupin species, which resulted in a similar mean Lr (P .0.05; Table I). However, the abscissa intercepts of thelinear regression between flow rate and applied pres-sure (DP) differed significantly between the lupinspecies and were greater than zero (P , 0.05; TableI). The DP intercept of narrow-leafed lupin was greaterthan that of yellow lupin, indicating that a greaterdriving force was required in narrow-leafed lupinroots to induce the same flux as in yellow lupin (P 50.812; Table I). The average Lr of lupin root systemswas more than 2-fold greater than that of wheat (P ,0.001; Table I). Because the roots of all species had asimilar surface area, Lpr of lupin roots was approxi-mately twice that of wheat roots (P , 0.001; Table I),but if flow rate was expressed on a per unit root lengthbasis, Lpr of lupin roots was approximately 6-foldgreater than that of wheat roots (data not shown).

Root segments (with the tip intact) between 60 and180 mm long connected to a root pressure probe wereused to determine whether Lr varied with root length.Root segments shorter than 60 mm were generally toodelicate to connect to the probe, and lateral rootsprevented segments longer than 180 mm from sealingto the pressure probe. After connecting the excised endof the detached root segment to the root pressureprobe, measurements began when root pressure (Pr)was stable. The Pr of the two lupin species was notsignificantly different, but Pr of wheat roots was ap-proximately twice that of lupin roots (Table II).

The Lr of narrow-leafed lupin root segments tendedto be greater than that of yellow lupin and wheat rootsegments (Table II). Lr of lupin root segments increased

linearly with length of the segment (P , 0.0001, r2 50.61 for narrow-leafed lupin and P , 0.0001, r2 5 0.59for yellow lupin; Fig. 2, A and B). In comparison, Lr ofwheat root segments was almost constant with seg-ment length (P 5 0.937, r2 5 0.0004; Fig. 2C). Becauseroot segments of lupin were larger in diameter thanthose of wheat (Table II), normalizing Lr by surfacearea resulted in the average Lpr of lupin being abouthalf that of wheat root segments (Table II). There wasno significant difference in Lpr between the two lupinspecies (Table II). In addition, because Lr was notrelated to length in wheat, normalizing by surface arearesulted in Lpr decreasing linearly with length of theroot segment (P 5 0.017, r2 5 0.32; Fig. 2F).

Lr of root segments was more than 1 order ofmagnitude smaller than Lr of whole root systems(Tables I and II). This disparity in Lr for both lupinspecies was because Lr of whole root systems wasrelated to total root length, with the same regressionapplying for whole root systems as distal root seg-ments (P . 0.05; Fig. 2, A and B). The increase in watertransport with increasing length of lupin roots resultedin an almost constant Lpr with length within each ofthe two methods of measuring Lr, but whole rootsystems had on average a higher Lpr than distal rootsegments (Fig. 2, D and E; Tables I and II). In compar-ison, although Lr of wheat root systems was greaterthan for distal root segments (Fig. 2C), this was notrelated to total root length. Instead, Lpr of distal rootsegments tended to decrease with length, to a lowerconstant value in whole roots (Fig. 2F).

Xylem Development and Lax

Lignified xylem vessels fluoresced bright yellow/white with the aniline blue procedure, which indicatedxylem maturity (Brundrett et al., 1988). The develop-

Table II. The dimensions and hydraulic properties of detached root segments at 14 d after sowing

Root segments, excised from the taproot of lupin or the longest seminal root of wheat, were connected toa root pressure probe. When Pr was stable, the Lr was measured. Lr was normalized by dividing by thesurface area of the root to give Lpr. Because of the dependence of some hydraulic parameters on root length(see text for details), ranges are given with means 6 SE in parentheses. Asterisks denote significantdifferences between species (P , 0.001).

Property Narrow-Leafed Lupin Yellow Lupin Wheat

Diameter (mm)a 1.1–1.9 0.7–1.3 0.5–0.9(1.5 6 0.05*) (1.0 6 0.03*) (0.7 6 0.03*)

Length (mm) 75–181 82–170 69–179(129 6 6) (120 6 5) (120 6 8)

No. of cortical cell layers(including endodermis)

9–11 8–10 5–7

Pr (kPa) 20–119 26–103 77–172(48.5 6 5.1) (52.8 6 3.4) (114.9 6 7.3*)

Lr (m3 s21 MPa21 3 10211) 2.0–7.0 1.4–3.6 1.2–4.1(3.8 6 0.3*) (2.3 6 0.1) (2.5 6 0.2)

Lpr (m s21 MPa21 3 1028) 3.7–8.6 3.8–8.9 5.3–18.4(6.0 6 0.4) (5.9 6 0.3) (10.6 6 1.0*)

No. of measured roots 20 22 17

aDiameter was measured at the excised end of the root.





ment of primary xylem vessels began within 5 mm ofthe root tip in all species. The seminal roots of wheatdeveloped a single large central xylem vessel (59–82mm diameter) surrounded by five to eight smaller ves-sels (16–23 mm diameter; Fig. 1). Walls of the smallvessels became lignified within 10 mm of the root tip(Fig. 1E). In some roots, the central vessel and/or severalsmaller vessels divided into two. Secondary thickeningof the central vessel wall occurred 80 to 100 mm from theroot tip, but lignification of the wall was not observeduntil more than 100 mm from the tip (Fig. 1C).

Xylem vessels developed in a diarch pattern in theroots of both lupin species, initially consisting of twogroups of five or six small vessels (Fig. 1K). Newvessels developed centripetally, forming a continuousband across the center of the stele between 100 and 120mm from the root tip (Fig. 1J). Xylem vessel wallsbecame lignified as lupin roots matured (Fig. 1H). Theabundance and size of lupin xylem vessels continuedto increase with distance from the tip, resulting in alarger and more circular stele (Fig. 1G).

Using the fluorescence images, the cross-sectionalarea of mature xylem vessels (Ax) was measured alongthe length of wheat seminal roots and the taproot oflupins. For wheat, Ax was constant along the length of

the root until at least 100 mm from the root tip, whereAx increased to a larger constant value (Fig. 3A). Distalto 100 mm from the root tip, Ax of a wheat seminal rootwas approximately half that of lupin taproots, and asroots matured the difference in Ax increased betweenthe species (Fig. 3A). For lupin, Ax increased withdistance from the root tip in the form of logistic growthcurves, which were significantly different between thespecies (P , 0.0001; Fig. 3A). Ax of yellow lupin tendedto increase more with distance from the root tip thanthat of narrow-leafed lupin.

After the measurement of Lr, the root in the pressureprobe was excised at the seal and the conductance ofthe segment remaining in the seal (10 mm long) wasmeasured. Here, radial flow would be negligible be-cause the segment was surrounded by the seal; hence,the measurement is defined as the Lax. Lax varied withdistance from the root tip (Fig. 3B) and followed asimilar pattern to the development of Ax in all species(Fig. 3A). Lax increased nonlinearly with distance fromthe tip in lupin roots, with different regressions fittingthe two lupins (P 5 0.011; Fig. 3B). In comparison, Laxincreased only marginally with distance from the tip ofwheat roots (Fig. 3B). Lax of wheat for segments excisedless than 100 mm from the tip was approximately one-

Figure 2. The relationship between root lengthand Lr (A–C) or Lpr (D–F) of detached root seg-ments (black symbols) and whole root systems(white symbols) of narrow-leafed lupin (A and D),yellow lupin (B and E), and wheat (C and F). Notethe log scale of the root length and Lr axes.Comparison of linear regression for root segmentsand whole root systems resulted in the samelinear fit (P . 0.05) between Lr and root lengthfor narrow-leafed lupin (A) and yellow lupin (B).

Bramley et al.




third that of lupins, but values of Lax diverged furtherbetween the species as the root matured (Fig. 3B).

Lax was up to 2 orders of magnitude greater than Lr,particularly for lupin root segments (Fig. 4). Lax washighly correlated with Lr in lupin root segments, withlinear regressions that were significantly differentbetween the two species (P 5 0.004). The regressioncoefficient for narrow-leafed lupin (9.9 6 1.4 3 1023)was more than twice that of yellow lupin (4.1 6 1.0 31023). Therefore, narrow-leafed lupin has a greaterradial conductance than yellow lupin for the sameaxial Lr. There was no correlation between the twohydraulic parameters in wheat (Fig. 4).

Radial Anatomy

Root diameter was related to the number of celllayers and cell sizes in the cortex (Tables I and III; Fig.1). The number of cell layers across the cortex wasconstant in all species along the length of the root for atleast 250 mm. Suberin was not detected in the outercortex of any species, indicating that none of thespecies developed an exodermis when grown insand. The Casparian strip in the endodermis haddeveloped at 5 mm from the tip in all species, but

suberin lamellae developed in the endodermis muchcloser to the root tip in wheat than in lupin (Fig. 1).Around 40 mm from the root tip, endodermal cells ofwheat contained suberin lamellae and the inner tan-gential walls of the endodermis and walls of stellarcells became thickened (Fig. 1D). In the endodermis oflupins, only a few cells contained suberin lamellae at100 to 120 mm from the tip (Fig. 1J). At 200 mm fromthe root tip, all lupin endodermal cell walls containedsuberin lamellae, except cells adjacent to xylem poles(Fig. 1G). No secondary thickening of cell walls oc-curred in roots of either lupin species.

Hydraulic Properties of Root Cells

Since Lr� Lax, radial flow was the limiting factor inroot water transport for all of the species. Therefore,the hydraulic conductivity of cells (Lpc) in each radialcell layer was measured with the cell pressure probe inorder to identify whether the disparity in root watertransport between the species was due to differenthydraulic properties at the cell level. Due to their smallsize and possibly different composition of cell sap,epidermal cells were the most challenging to measure,particularly in lupin roots. Although turgor pressurecould be measured, constant blockages of the micro-capillary or sudden losses of turgor pressure meantthat Lpc was not determined in sufficient lupin epi-dermal cells for statistical analysis.

There was no significant difference in turgor pres-sure of root cells between the lupin species (P . 0.05),but the cells of the epidermis had a lower turgorpressure (0.26 6 0.03 MPa) than all other cell layers(0.37 6 0.01 MPa). The average turgor pressure ofwheat root cells (0.61 6 0.01 MPa) was greater thanthat of lupin but was independent of the radial loca-tion in the root (P 5 0.119).

Figure 3. The relationship between distance from the root tip and thecross-sectional area of lignified xylem as indicated by fluorescence (Ax;A) or Lax (B) for narrow-leafed lupin (black circles), yellow lupin (whitecircles), and wheat (inverted black triangles). Lax was measured on 10-mm-long root segments after measuring Lr with the root pressure probe.The curves represent best statistical fits. The error bars in A represent SE

(n 5 9), and each point in B represents a segment from an individualroot.

Figure 4. The correlation between the Lr of a detached root segmentand its Lax. Lax values are shown in Figure 3B, where distance from theroot tip denotes the length of the original root that Lr was measured on.The correlation is significant for narrow-leafed lupin (black circles; P ,

0.0001, r2 5 0.81) and yellow lupin (white circles; P 5 0.002, r2 5

0.56) but not for wheat (inverted black triangles; P 5 0.712). Each pointrepresents an individual root segment.





Lpc of root cells ranged between 0.3 3 1026 and 3.5 31026 m s21 MPa21, and the average Lpc was notsignificantly different between the three species (P 50.12). The location of the cell within the cortex influ-enced Lpc such that Lpc tended to increase toward theroot axis (Fig. 5A), although due to the variabilitybetween roots and cells, this trend was not statisticallysignificant in the lupin species (P 5 0.072 for narrow-leafed lupin and P 5 0.081 for yellow lupin). For wheatroots, the trend was more pronounced and the innercell layers of the cortex (cell layers 5 and 6) weresignificantly different from those of the epidermis (celllayer 1; P , 0.05).

The increased Lpc near the axis could be compen-sating for the decreasing area for water flow towardthe axis. When Lpc was adjusted by a factor for thedecrease in area (C) for each jth cell layer (Eq. 1), Lpcwas almost constant across the cortex for all species(Fig. 5B).

Lpcadjusted5 Lpc

Cj

Cj 21

ð1Þ

Estimation of the Contribution of the Radial Pathwayswith the Concentric Membrane Model

The radial profile of Lpc (Fig. 5A) was used to predictLr and Lpr by applying a model that describes the rootas a series of concentric cylindrical membranes (see‘‘Materials and Methods’’ for the parameters). Themodel assumes that radial water flow is primarily viathe cell-to-cell flow path and that, after entering theroot, water crosses each cell layer in series to reach thestele. The root diameters and number of cell layersused in the predictions are shown in Table IV. Each celllayer has two concentric membranes, which can beincluded independently of each other in the model.Where Lpc was not measured in lupin, the value of Lpcused was the same as the closest measured cell layer.

The Lr of each concentric cylindrical membrane(Lmembrane) when water uptake is distributed evenlyalong the length of 120-mm-long roots (excluding 5mm at the apex) is shown in Figure 6. The modelpredicted that Lr of narrow-leafed lupin would begreater than that of yellow lupin, which increased withroot length (Fig. 7). The predicted Lr for both lupinspecies was close to the measured Lr for a root segment120 mm long (Table IV). The disparity between pre-dicted and measured values for narrow-leafed lupinwill increase with longer root lengths, because themeasured Lr increased more rapidly with root lengththan predicted (compare regression coefficients inTable IV). Extrapolating the regression for the mea-sured Lr and root length to the abscissa indicated thatthe first 40 mm of narrow-leafed lupin roots was notinvolved in water uptake, but the intercept for yellowlupin was close to that set in the model (Table IV).Normalizing Lr by the surface area of lupin rootsresulted in a predicted Lpr that was not significantlydifferent from the measured Lpr (Table IV). The close-ness of the predicted and measured Lpr values forlupins indicated that the majority of radial water flowcould occur across each cell layer through the cell-to-cell pathway.

For wheat, the concentric membrane model predicteda smaller Lr than for lupin for the same root length(Fig. 7). The predicted Lr for a 120-mm-long wheat rootwas close to the measured value (Table IV). However,unlike the measured Lr, predicted Lr increased withlength (Fig. 7), which implied that a specific region ofwheat roots determines Lr. To explore this observation,the model was adjusted for anatomical structures inthe cell-to-cell path that could influence Lr. To incor-porate the effect of suberization of the endodermis, themodel was adjusted to allow for water uptake onlybetween 5 and 40 mm from the tip. Limiting the lengthof the absorbing region resulted in a predicted Lrindependent of root length (Fig. 7). However, Lmembrane

Table III. Dimensions of root cells in each cell layer

Cell layers are numbered from the epidermis (1) toward the root axis. Values are means 6 SE (n 5 22–59)from 10 roots. The data were combined for cell layers 4 to 6 of yellow lupin and cell layers 2 to 4 of wheatbecause no significant differences could be found between these cell layers. Volumes were onlydetermined for those cell layers punctured by the cell pressure probe.

Cell LayerNarrow-Leafed Lupin Yellow Lupin Wheat

Diameter Volume Diameter Volume Diameter Volume

mm pL mm pL mm pL

1 14 6 1 15 6 1 16 6 0 18 6 1 27 6 1 51 6 22 23 6 1 50 6 3 26 6 1 63 6 4 51 6 1 279 6 153 37 6 2 151 6 22 40 6 1 242 6 19 51 6 1 279 6 154 46 6 3 269 6 30 54 6 1 626 6 37 51 6 1 279 6 155 52 6 2 404 6 33 54 6 1 626 6 37 34 6 2 215 6 76 63 6 3 640 6 60 54 6 1 626 6 37 30 6 4 226 6 107 60 6 4 492 6 51 36 6 2 316 6 348 52 6 3 421 6 46 22 6 1 94 6 89 41 6 1 13 6 0

10 22 6 111 17 6 0

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(Fig. 6), and consequently predicted Lr, was reduced toa value that was only one-quarter of the measured Lr.Therefore, not all cylindrical membranes in the radialpathway contribute equally to water flow. Changingthe model to allow for cell-to-cell water flow acrossonly two cylindrical membranes resulted in a pre-dicted Lr close to the measured Lr. For example, usingLmembrane for the epidermis (both inward- andoutward-facing membranes) predicted Lr of 2.95 310211 m3 s21 MPa21 (Lpr of 10.7 3 1028 m s21 MPa21),and Lmembrane for the endodermis predicted Lr of2.5 3 10211 m3 s21 MPa21 (Lpr of 10.4 3 1028 m s21

MPa21), which were very similar to the measuredvalues (Table IV).

Test of the Model by Inhibiting Water Flow throughAQPs with HgCl2

Mercury had no significant effect on Lpr of wholeroot systems or root segments of either lupin species

(Fig. 8, A and B). However, Lpr of wheat root systemsand individual roots was reduced by up to half aftermercury treatment (P 5 0.0002; Fig. 8, A and B).

On average, mercury reduced Lpc of cells fromtreated roots by 33% in narrow-leafed lupin, by 86%in yellow lupin, and by 77% in wheat (Fig. 8C). Theouter cell layers of narrow-leafed lupin roots wereparticularly sensitive to mercury treatment, and mea-surements of Lpc were only obtained from cells deeperthan the third cell layer. Mercury treatment did notaffect the volumetric elastic modulus of any species(P 5 0.147); therefore, changes in the rate of waterexchange across the cell were due to the effect ofmercury on Lpc.

If water flow occurs entirely through cells by cross-ing membranes, the inhibition in Lpc should also bereflected in Lpr. However, the concentric membranemodel predicted a greater reduction in Lpr than themeasured value, particularly for lupin (Table IV).The values in Table IV are based on the assumptionsthat the entire root length and all cell layers areinvolved. Adjusting the model for wheat, using thesame parameters above (length of absorbing regionof 35 mm and cellular flow across two membranes)resulted in predicted values lower than measuredvalues. A 77% reduction in both Lmembrane of theendodermis resulted in Lpr of 2.44 3 1028 m s21

MPa21. However, if only the exterior membrane ofthe endodermis was inhibited, Lpr was reduced byjust over half (4.4 3 1028 m s21 MPa21) compared withthe 43% reduction in Lpr actually measured.

DISCUSSION

The main hydraulic properties of wheat and lupinroots were identified by integrating measurements ofwater flow, across different structural levels of orga-nization, with anatomy and modeling. An importantobservation from this study was the major role of rootstructure and anatomy on root water transport and thelocalized influence of AQPs. AQP activity was ubiq-uitous in the cortex and epidermal cells of all species,based on the high values of Lpc and the strong inhi-bition by Hg21. The similar magnitudes and profiles ofLpc across the root for each species pointed toward amajor role for AQPs in root water transport. However,the contrasting behaviors and the weaker inhibition ofwater flow by Hg21 in root tissue and root systemsdemonstrated that AQPs have localized influences onroot hydraulics. At the cell level, AQPs considerablyinfluence Lpc of all three species. At the level of the rootsystem, the control of bulk water flow by AQPs waslimited to a small region of the endodermis in wheat.For lupins, bulk water flow occurred predominantlythrough the apoplast without the influence of AQPs.Without manipulation of the rhizosphere, the benefitof these contrasting behaviors can only be speculatedupon, but they may indicate a tradeoff between theoverall rate and the potential to rapidly regulate therate of water flow.

Figure 5. Radial profiles of the mean Lpc (A) and adjusted Lpc (Lpca; B)for each cortical cell layer, 30 to 50 mm from the tips of narrow-leafedlupin (black circles, solid lines), yellow lupin (white circles, dottedlines), and wheat (inverted black triangles, dashed lines) roots. Lpca wascalculated from Equation 1 to account for the decrease in area of eachcell layer toward the root axis. The cell layers are numbered from theoutside of the root (epidermis 5 1) toward the root axis. Error barsrepresent SE (n 5 4–16 cells per layer, except for the epidermis of bothlupin species, where Lpc was not measured).





The Total Length of Wheat Roots Was Greater ThanThat of Lupin, But Wheat Roots Did Not TransportMore Water to the Shoot

The features described here for wheat and lupinroots grown in sand-filled pots are consistent withthose previously described for roots grown in the field(Greacen et al., 1976; Passioura, 1980; Hamblin andTennant, 1987). Even at the relatively early stage ofgrowth used in this study (14 DAS), the total length ofwheat roots was greater than that in lupin roots. Wheatroot systems appear to have more rapid early growthand greater proliferation of roots than lupins (Gregoryand Eastham, 1996). Whole plant transpiration con-firmed that the shorter root systems of lupin suppliedthe shoots with equivalent volumes of water as wheat.Eudicots commonly use the same amount of water asmonocots, despite having smaller root length density(Mason et al., 1983). Therefore, the contrasting hy-draulic properties of wheat and lupin roots may bemore extensive among herbaceous species.

Wheat Roots Predominantly Absorb Water in a Region

Close to the Root Tip, But Lupin Roots Absorb WaterMore Evenly along Their Whole Length

If the root is analogous to a leaky pipe, where watercan enter anywhere along the length of this pipe, thenLr should increase with root length. Hence, Lpr shouldbe constant with length. In this study, wheat rootsystems consisted of up to five seminal roots. Lr of anindividual, unbranched, seminal root did not varybetween 70 and 200 mm from the tip, resulting in adecrease in Lpr with root length. This indicated thatwater was not uniformly absorbed along the entireroot but instead occurred predominantly in a distal

region. The region coincided with a less mature endo-dermis. Farther from the tip, the endodermis was inthe tertiary state of development, containing suberinlamellae and thickened cell walls. Water uptake by aregion close to the root tip appears to be a commonfeature of cereal roots (Greacen et al., 1976). Lr of wholeroot systems was more than 10-fold greater than thatof seminal root, indicating that more than one rootcontributes to water uptake in whole root systems.Using the 95% confidence interval of Lr from rootsegments (1.95 3 10211 to 2.88 3 10211 m3 s21) andassuming that water uptake was confined to apicalregions, 10 to 26 root tips contributed to flow in wheatroot systems. At 14 DAS, each of the three longest

Table IV. Comparison of the estimated values for various root water flow parameters from the concentric membrane model and the valuesmeasured with the root pressure probe

The Lr of roots with an average length of 120 mm was calculated from Equation 2, assuming that all of the root length, except 5 mm at the apex, wasabsorbing water and that all radial water flow occurred through the cell-to-cell flow path. The Lpr was calculated from Equation 3 using the sameassumptions. To compare the linear regressions between Lr and root length, the length of the concentric membranes was varied to coincide with therange of root length segments measured with the root pressure probe. n/a, Not applicable.

Parameter Type of Value Narrow-Leafed Lupin Yellow Lupin Wheat

Root diameter (mm) 1,537 1,050 660No. of cell layers including endodermis 11 9 6Lr for 120-mm-long root (m3 s21 MPa21 3 10211) Predicted 3.0 2.1 1.9

Measured 3.4 2.3 2.5Regression coefficient for Lr versus root length (m2 s21 MPa21 3 10211) Predicted 26.0 18.4 16.2

Measured 43.3 19.2 0.68a

Root length when Lr 5 0 (mm) Predicted 5 5 5Measured 40.5 2.1 n/a

Lpr (m s21 MPa21 3 1028) Predicted 5.4 5.6 7.8Measured 6.0 5.9 10.6

P (predicted versus measured Lpr) 0.106 0.271 0.015Reduction in Lpr after 50 mM HgCl2 (%) Predicted 33 85 77

Measured 10 14 41

aRegression does not significantly deviate from zero (P 5 0.937).

Figure 6. Lmembrane of each cell layer predicted by the concentricmembrane model, for a 120-mm-long root of narrow-leafed lupin(black circles, solid line), yellow lupin (white circles, dotted line), andwheat (inverted triangles, dashed lines). For wheat, the predictions areshown for all of the root length involved in water uptake (inverted blacktriangles) or if water uptake predominantly occurs between 5 and 40mm from the root tip (inverted white triangles).

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seminal roots had more than 10 lateral roots greaterthan 10 mm long (Fig. 1). Therefore, there may bevariability between root tips in their contribution toflow. This variability may depend on maturity andheterogeneity between local root environments.

For both lupin species, Lr of unbranched taprootsegments and whole root systems increased propor-tionally with root length. This implies that water wasabsorbed more evenly along the entire root length,including the lateral roots. Unlike wheat roots, theendodermis of lupins did not become as heavilysuberized and there was no secondary thickening.The presence of suberin lamellae in the endodermisdid not appear to restrict water flow, as there was nocorresponding change in Lr with the development ofthis structure. However, a large region of the endo-dermis, adjacent to xylem poles, did not becomesuberized. Lateral roots may also prevent the endo-dermis from forming a tight barrier in more matureroots as they emerge from this unsuberized region.Eudicots undergo secondary growth, so more maturelupin roots may develop a periderm. If a peridermdoes develop, then the impermeability of this tissue towater may alter the relationship between Lr and rootlength. Hamblin and Tennant (1987) did not describe aperiderm in field-grown lupin roots but observedabundant root hairs on all parts of roots, suggestingthat water uptake also potentially occurs along thelength of more mature root systems.

Lax Is Greater in Lupin Roots because of GreaterXylem Development

Hamblin and Tennant (1987) speculated that lupinroots have higher specific rates of water uptake thanwheat roots due to increased xylem development. In

this study, Lax of the roots of both lupin species wasgreater than that in wheat, which was related to thearea of lignified xylem. Larger vessels have greaterpotential for water flow, since according to the Hagen-Poiseuille equation, Lr varies to the fourth power of thevessel radius. In lupin roots, the number and diameterof xylem vessels increased nonlinearly with maturity.The logistic growth curves imply that the amountof lignified xylem will increase in the taproot untilaround 450 mm from the tip. However, this extrapo-lation does not take into account secondary xylemdevelopment in mature roots. The increase in xylemmay be beneficial in the region of lateral root devel-opment (Steudle and Peterson, 1998). The strong rela-tionship between Lax and Lr indicates that increasingFigure 7. Lr predicted by the concentric membrane model in relation to

root length for narrow-leafed lupin (black circles, solid line), yellowlupin (white circles, dotted line), and wheat (inverted triangles, dashedlines). For wheat, the predictions are shown for all of the root lengthinvolved in water uptake (inverted black triangles) or if water uptakepredominantly occurs between 5 and 40 mm from the root tip (invertedwhite triangles). The regression coefficients for the linear regressionsthat were significantly greater than zero (P , 0.01) are given in Table IV.

Figure 8. The effect of mercury on Lpr and Lpc of whole root systems(A), individual roots (B), and root cortical cells (C) of narrow-leafedlupin (NL), yellow lupin (YL), and wheat (W). Measurements wereconducted before (black bars) and after (white bars) treatment of rootswith 50 mM HgCl2. Asterisks denote significant differences due totreatment at the 5% (*), 1% (**), and 0.1% (***) levels of significance.Error bars represent SE (n 5 4–7 for roots and n 5 10–14 for cells).





xylem development in lupin roots may also compen-sate for more water arriving at the xylem as water isabsorbed along the length of the taproot.

Because monocots do not undergo secondarygrowth, the development of xylem vessels in wheatroots is determined at an early stage in root growth.Each seminal root contains a large central vesselsurrounded by several smaller vessels. Fluorescenceimagery indicated that the small vessels become lig-nified at least 100 mm closer to the root tip than thecentral vessel. However, the central vessel does notappear to be functional up to 200 mm from the root tip,as there was no abrupt change in Lax that correspondedwith fluorescence of the vessel. According to theHagen-Poiseuille equation, the Lax of a 10-mm-longwheat root segment (average of seven vessels of 21 mmdiameter) is 3.7 3 1029 m3 s21 MPa21, if we assume thatthe central vessel is nonfunctional. This is almost5-fold greater than the average measured Lax of a10-mm root segment. Although the measured Lax inthis study may be artificially high, flow through xylemvessels is not as ideal as the assumptions used incalculating Lax with the Hagen-Poiseuille equation;therefore, measured Lax values are typically less thancalculated (Greacen et al., 1976; Steudle and Peterson,1998). The central vessel may become functional atgreater distances from the root tip, where lateral rootsdevelop. However, the presence of lateral roots pre-vented the sealing of root segments to the root pres-sure probe to test this hypothesis.

Yellow Lupin May Be More Conservative in Its Water

Use Than Narrow-Leafed Lupin

Yellow lupin roots tended to have a greater cross-sectional area of xylem vessels but had similar Lax tonarrow-leafed lupin. This disparity between the twospecies was manifested in the relationship between Laxand Lr. The regressions in Figure 4 imply that yellowlupin has a greater capacity to carry water axiallyrelative to the radial path. For narrow-leafed lupin, theradial conductance increases at a greater rate per Lax,so greater water potential gradients may exist in thexylem. Corresponding to this, yellow lupin tends tomaintain a higher leaf water potential than narrow-leafed lupin, but it also tends to have lower rates ofstomatal conductance and transpiration (Davies et al.,2000a; Palta et al., 2004). In this study, the transpira-tion of yellow lupin plants was also less than thatof narrow-leafed lupin plants. Minimizing the waterpotential gradient driving water flow will reduce therisk of cavitation, particularly because wide vessels aremore vulnerable to cavitation (Tyree et al., 1994).

The Flow of Water Radially Determines Lr

Lax was 2 orders of magnitude greater than Lr,indicating that the rate of water flow through theradial pathway was limiting to Lr for all three species.Similar differences in magnitude between axial and

radial conductance have been reported for a widevariety of species (Steudle and Peterson, 1998). Theradial pathway of all three species has the same funda-mental structure, with the main differences being thelength of the pathway (distance across the cortex) andthe development of the endodermis. None of thespecies developed a subepidermal layer that wasdifferent in structure from the other cortical cells. Anexodermis may develop in older root systems underfield conditions or in adverse environments. However,an exodermis was not induced in lupin by growingthe roots in aeroponic culture, as has been demon-strated for maize (Zea mays; Zimmermann et al.,2000; Hartung et al., 2002). In addition, Hamblin andTennant (1987) did not identify an exodermis in ma-ture root systems of wheat or narrow-leafed lupingrown under the same field conditions, but the cortexof wheat roots deteriorated.

The conductance of the radial pathway is inverselyproportional to the length of the flow path or thenumber of cell layers, since the cells are arranged inseries (Steudle and Frensch, 1996). The length of theradial flow path was constant with distance from theroot tip for all species, even though the developmentof the stele caused lupin roots to increase in diameter.The length of the flow path, from the root surface to thexylem, was greatest in narrow-leafed lupin, followedby yellow lupin and then wheat. However, Lr waslowest in wheat roots; therefore, the width of thecortex was not the only factor determining Lr.

There Was Strong Evidence for PredominantlyCell-to-Cell Water Flow

Water flow from the root surface to the vasculartissue can occur through two parallel pathways: theapoplast and the cell-to-cell pathway. Water passingthrough cells may cross into adjacent cells throughconnecting plasmodesmata and/or across mem-branes. The symplastic connection through plasmo-desmata has been demonstrated with tracer dyes andpredominantly occurs within a few millimeters of theroot tip (Hukin et al., 2002). Plasmodesmata have alsobeen observed in regions farther from the root tip (Maand Peterson, 2001), but their involvement in sym-plastic transport is less documented. Measurements ofLpc with the cell pressure probe are a composite valueof the plasma membrane, tonoplast, and plasmodes-mata. To determine the contribution of the cell-to-celland apoplastic pathways for each species, radial pro-files of Lpc were created. The profiles of Lpc for all of thespecies were similar in magnitude and behavior. Lpctended to increase from the root surface toward theendodermis. As water travels across the root, thesurface area for flow decreases. Lpc increased propor-tionally to the decrease in area, implying a compensa-tory effect. The trend in Lpc also suggested that waterflow might occur predominantly through the cell-to-cell pathway. This possibility was tested by the con-centric membrane model.

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A model describing the root as a series of concentricmembranes has been used previously to predict Lpr ofbarley (Hordeum distichon), wheat, and maize roots(Jones et al., 1983, 1988; Steudle and Jeschke, 1983).However, the length of the absorbing region was nottaken into consideration in these previous models,which has an important influence on Lr. Here, weexpanded the model for a root composed of membranecylinders to calculate Lr and included the radial profileof Lpc (instead of an average value of Lpc). The modelassumes that water flow occurs predominantly throughthe cell-to-cell pathway. If this assumption is correct,then the measured and predicted values of Lr shouldbe similar. Using membrane cylinders, the influenceof the length of the absorbing region, anatomy, and,hence, specific membranes could be investigated.

For both lupin species, the predicted Lr values weresimilar to the measured Lr if water crossed all celllayers through the cell-to-cell pathway. Lr was alsopredicted to increase with root length. For wheat, thepredicted and measured values were also in closeagreement, but only when the length of the absorbingregion was confined to the distal region, where theendodermis was less developed and only two cylin-drical membranes were included in the flow path.Although suberization of the endodermis did not ap-pear to influence Lr of lupin roots, state III developmentof the endodermis (suberin lamellae and tertiary thick-ening) has been correlated with a decline in wateruptake of cereal roots (Sanderson, 1983). It is notapparent from the model alone which cell layer con-trols radial water flow, but the most likely candidate isthe endodermis, because Lr was influenced by itsanatomy and in older wheat roots the cortex shrivelsand deteriorates (Greacen et al., 1976; Hamblin andTennant, 1987). We also considered the epidermis,because the cortex of wheat roots apparently shrinkswhen the water potential of the root decreases(Greacen et al., 1976). This implies that it is notbuffered against large changes in water potential inthe xylem and, therefore, the greatest resistance toradial water flow could occur in the epidermis. Al-though the epidermis had smaller Lpc, Lpc was pro-portional to the surface area for transport; therefore,the conductivity of the epidermis was no less than thatof the endodermis.

Water Flow Occurs Predominantly through theApoplast in Lupin Roots But Is Controlled by AQPs inthe Endodermis in Wheat Roots

Because the model provided strong evidence for alarge component of cell-to-cell flow, we further testedthe predictions by inhibiting flow through the cell-to-cell pathway. Despite the shortcomings with Hg21

being relatively nonspecific in its mechanism of inhi-bition of water transport (Zhang and Tyerman, 1999),the inhibition is evidence for the activity and control ofwater flow by AQPs in root cell membranes. If watertransport occurred across specific cylindrical mem-

branes and/or through all cell layers, then the effect ofmercury at the root level should have been propor-tional to the effect at the cell level. However, Lpc wasreduced more than Lr.

Mercury failed to affect Lr of lupin root segments orroot systems, despite dramatic reductions of Lpc in allcell layers. Therefore, water primarily flowed aroundcells in lupin roots, without crossing membranes. Thisalso implies that water did not cross membranes in theendodermis, despite the Casparian strip and the laterpartial development of suberin lamellae. The Caspar-ian strip in the endodermis is also not a significantbarrier to apoplastic water flow in maize roots (Steudleet al., 1993). It is possible that mercury failed to per-meate lupin roots sufficiently to reach the endodermis.AQPs in the endodermis may also be insensitive tomercury, which has been reported for some plasmamembrane intrinsic proteins (PIPs) and tonoplast in-trinsic proteins (TIPs; Daniels et al., 1994; Biela et al.,1999).

For wheat roots, the model and the inhibition of Lrand Lpc indicated that Lr is regulated by AQPs in asmall region of the endodermis. At the endodermis, itis unlikely that mercury could permeate fully becauseof the Casparian strip. If only the exterior ‘‘membrane’’of the endodermis was inhibited by mercury, a similarinhibition in Lr was predicted by the model to whatwas measured. Some studies have removed cell layersto measure Lpr in conjunction with mercury treatment,because of the presence of Casparian bands and su-berin lamellae (Martre et al., 2001; North et al., 2004).However, the delicate nature of wheat roots preventsthis type of manipulation. AQPs were previouslysuspected to be involved in root hydraulics of wheatbecause of the high water permeability of cells. Lpc ofwheat equates to osmotic water permeability of 35 to491 mm s21, which is within the range of AQP-facilitatedwater transport reported for membrane vesicles ofwheat and membrane vesicles and protoplasts of arange of other species (Niemietz and Tyerman, 1997;Ramahaleo et al., 1999; Suga et al., 2003; Alleva et al.,2006). In addition, Lpr varies diurnally and changesrapidly in response to abiotic perturbation (Carvajalet al., 1996; Clarkson et al., 2000). The first majorcharacterization of AQP genes in wheat has identified24 PIPs and 11 TIPs (Forrest and Bhave, 2008), al-though their expression patterns and regulation oftheir transcripts and proteins are not yet known. How-ever, most PIPs of wheat contain up to four conservedCys residues, which could be involved in the mercuryinhibition (Forrest and Bhave, 2008).

What Are AQPs for in Lupin Roots?

Although AQPs in narrow-leafed lupin or yellowlupin have not yet been characterized, the high valuesof Lpc and the effect of mercury demonstrated highAQP activity in both lupin species. In addition, theincrease in Lpc toward the root axis suggests that AQPactivity increases to compensate for a concentrating





effect of the root geometry. For the measurements inthis study, water flow was induced by hydrostaticpressure, and under these conditions, bulk water flowmay bypass cells. Hence, water flow generated bytranspiration also probably travels predominantlythrough the apoplast. However, water flow inducedby osmotic gradients must cross membranes, so thismay be where AQP activity influences water flowacross lupin roots. Lpr of lupin roots measured usinghydrostatic pressure is at least twice the Lpr measuredusing an osmotic gradient, in comparison with wheat,where Lpr was the same under both types of gradient(Bramley et al., 2007a). This suggests that water flowmay follow different radial pathways in lupin rootswhen influenced by different driving forces. Accord-ing to the composite transport model, water flowshould primarily cross the cell-to-cell pathway wheninduced osmotically (Steudle and Peterson, 1998).Therefore, mercury should have a greater inhibitoryeffect on osmotically induced Lpr of lupin, but thisremains to be tested. Lupin roots are particularlysensitive to mercury treatment, which has adverseeffects when combined with an osmotic experimentcausing Pr to decline without subsequent recovery(data not shown).

One of the hypotheses of this study was that thehydraulics of narrow-leafed lupin and yellow lupindiffered because of AQP activity. Here, AQPs were notfound responsible for Lr under these water-sufficientconditions. However, AQPs may be required for os-moregulatory processes (Tyerman et al., 1999) andduring drought as lupin roots undergo osmotic ad-justment to maintain turgor pressure (Jensen et al.,1989). During water deficit, roots often become moresuberized (Vandeleur et al., 2009), which could changethe radial pathway for water flow and lead to theinvolvement of AQPs.

Contrasting Root Hydraulics

The apoplast should have a higher conductivity towater flow than the cell-to-cell pathway because of theabsence of membranes. Therefore, by having a greaterproportion of water flow via the apoplast, lupins areable to achieve the same or superior radial conduc-tance as the thinner wheat roots. However, the mainconsequence of predominantly apoplastic water flowis that Lr is invariable in the short term. Lupins mayrely on anatomical and growth-related changes toregulate water flow at the root level and/or manipu-late the driving force through closure of stomata.Stomatal conductance of lupins decreases underdrought, as more of the root system is exposed to thedrying soil and root water uptake decreases (Jensenet al., 1989). In addition, without increasing the gradi-ent to drive water flow, the apical part of lupin rootsmay become hydraulically isolated when surface soilsbecome dry, unless the radial permeability in the olderparts of the root is reduced (e.g. through suberization).

In comparison with lupins, wheat root systems arenot dependent on growth-related changes in anatomyto vary Lpr. Wheat can rapidly adjust Lpr because flowthrough a seminal root is predominantly controlled byAQPs. Under the growing conditions of this study, thecontrol occurs in the endodermis in the apical region.It is not known which specific AQPs are expressed inthe endodermis, but the expression of different AQPscan be localized to specific tissues (for review, seeBramley et al., 2007b) and even oriented within thetissue (Hachez et al., 2006). Because the wheat rootsystem consists of several individual roots, it is pos-sible that Lpr of each of these roots can be adjustedindependently. For example, after excising four of theseminal roots in durum wheat (Triticum durum), waterflow in the remaining root increased, maintaining thewater supply to the shoot (Vysotskaya et al., 2004). Afeature like this may provide an advantage in hetero-geneous or fluctuating environments.

CONCLUSION

The hydraulic properties of wheat and lupin rootsare highly contrasting, which suggests a balance be-tween root diameter, Lr, and the ability to rapidlyadjust to prevailing conditions. By absorbing watermore evenly along the root length, which is related toincreasing xylem development, lupin roots have ahigh capacity to transport water, although yellowlupin may be more conservative than narrow-leafedlupin in its water use strategy. In addition, predomi-

Figure 9. Diagram of the concentric membrane model used to calcu-late Lr and Lpr from radial profiles of Lpc. The diagram shows acylindrical root segment consisting of five cell layers, from the epider-mis (Ep) to the endodermis (En). Each cell layer consists of twomembranes, inner and outer, the radii of which were determinedfrom anatomical studies and measurements of cell diameters. Watercrosses each cell layer in series to reach the vascular tissue in the stele.The model assumes that radial water flow occurs by the cell-to-cell flowpath, except in the stele. The cell layers where cellular flow occurs andthe length of the root segment can be varied to examine the effect on Lr.

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nantly apoplastic flow endows lupin roots with thesame or superior radial conductance as thinner wheatroots, but it provides little ability to rapidly adjust Lr inthe short term. However, osmotically induced waterflow must cross membranes, which could be facilitatedby AQPs. In comparison, preferentially absorbingwater in the distal region and low Lax diminish thepotential for high water flow in wheat. However,because a significant component of water flow crossesat least two membranes, which is controlled by AQPs,wheat has the ability to rapidly adjust Lr.

The strong evidence of AQP activity in all cell layersof the cortex of all species implies that their role ismore extensive than solely transporting water fromthe surface of the root to the vascular tissue. Thesimilarity in root cortex profile of Lpc yet contrastinghydraulics of whole roots demonstrate the importanceof examination of root structure and anatomy inassessing the role of AQPs in whole root transport.

A cautionary note should be included about the useof Lpr. Because of problems of scale, measurements ofLpr are generally normalized by root dimensions, suchas length, surface area, or mass. Water uptake is limitedby the absorbing area; therefore, normalization by theentire root size may be inappropriate for some species.The form of the normalization is particularly importantwhen comparing the effects of treatments that alter thedimensions or structure of the root system, especiallyif the absorbing area is unaltered. In these cases, it maybe more meaningful to normalize hydraulic conduc-tivity by another parameter such as leaf area.

MATERIALS AND METHODS

Plant Material

The details of growing wheat and lupin seedlings were described previ-

ously by Bramley et al. (2007a). For all experiments, seedlings were used at 14

DAS. Seedlings were used because the growth of root systems was not

constrained by pot size and roots could be removed without damage. Briefly,

seeds of narrow-leafed lupin (Lupinus angustifolius ‘Merrit’), yellow lupin

(Lupinus luteus ‘Wodjil’), and wheat (Triticum aestivum ‘Kulin’) were germi-

nated and planted into individual pots (75 mm diameter, 350 mm depth)

containing medium to fine river sand. Lupin seeds were inoculated with

Bradyrhizobium (Nitrogerm 100; Adelaide Seed Company) at the time of

planting. Plants were watered daily with nutrient solution, pH 6.0 (Davies

et al., 2000a).

For measurements on whole root systems, seeds were planted individually

into specially designed pots that fitted into a pressure chamber, as described

by Gallardo et al. (1996). To force the cotyledons to grow above the top of the

pot surface and enable the lupin stem and wheat mesocotyl to protrude

through the lid of the pressure chamber, the seeds were planted beneath a

small collar of sand constructed in the lid of the pot. The collar was removed

when the seedling had emerged. Plants were grown in a growth cabinet with a

12-h-light/12-h-dark photoperiod (photosynthetically active radiation of 450–

500 mmol m22 s21), temperature of 21�C/17�C 6 1�C, and 68% relative

humidity.

Whole Plant Transpiration

The volume of water transpired per plant was measured gravimetrically.

Plants were grown as described above. At the start of the experiment, the pots

were watered early in the morning and allowed to freely drain for 1 h. The

pots were then covered to prevent further drainage or soil evaporation. Pots

were weighed hourly between 10:00 AM and 2:00 PM, with the average volume

transpired recorded from five individual plants of each species.

Water Flow through Whole Root Systems

The Lr of whole root systems was measured by the pressure chamber

technique. Before a measurement, the pot was watered with nutrient solution

and drained on absorbent paper for 0.5 h. Shoots were excised below the

cotyledon of lupin and just above the crown of wheat. The pot was sealed (Lab

Putty; Halas Dental Supplies) in the pressure chamber with the cut lupin stem

or wheat mesocotyl protruding through the lid of the chamber.

Pressurizing the chamber induced water flow through the root system, and

the water flux was determined from the rate of exuding sap. Preliminary

measurements indicated that the flux-pressure relationship was linear be-

tween 0.2 and 0.7 MPa. In addition, upon initial pressurization at 0.7 MPa, the

exudation rate of sap gradually increased until becoming steady after 2 to 3 h.

The water flux at each subsequent pressure was stable within 0.3 h. Thereafter,

all root systems were initially pressurized at 0.7 MPa for 3 h, before collecting

exudates, to ensure steady-state conditions (Markhart and Smit, 1990). Ex-

periments commenced at the same time each day to eliminate possible

confounding diurnal effects (Passioura and Munns, 1984).

After the initial 3-h equilibration period, the pressurization protocol

followed that of Gallardo et al. (1996), collecting exudate at 0.7, 0.5, and 0.3

MPa. Lr (in m3 s21 MPa21) of the root system was calculated from the linear

regression of water flux against applied pressure. Lr was converted into Lpr (in

m s21 MPa21) by dividing by the unit surface area of the root system.

The total lengths and surface areas of the root systems were measured after

washing roots from pots and staining them for 1 h in 0.5% methylene blue.

Roots were scanned using a flatbed scanner (600 pixels mm21; ScanJet;

Hewlett-Packard Australia) covered with a thin film of water. Images were

analyzed using Rootedge version 2.3b software (National Soil Tilth Labora-

tory, U.S. Department of Agriculture). Roots were assumed cylindrical for

converting image areas to surface areas.

Radial and Axial Lr of Individual Roots

Roots were carefully washed from the sand, and the longest seminal root of

wheat or the taproot of lupin was excised at various distances from the root

tip, below emerging lateral roots. The root was connected to a root pressure

probe (Steudle, 1993) and supported inside a glass tube (5 mm diameter 3 140

mm length) in which aerated nutrient solution was continuously circulated.

When Pr was constant (2–12 h), the rate of water flow through the root was

measured with the pressure-clamp technique (Bramley et al., 2007a). Lr was

calculated from the linear regression of flow rate against applied pressure and

normalized to Lpr by dividing by the surface area of the root segment.

After measurements of Lr, the root was cut from the probe near the seal. Lax

was measured on the segment of root remaining within the seal (10 mm),

where radial flow was negligible. The rate of decline in Pr when the root was

cut from the probe and applied hydrostatic pressure pulses were used to

determine Lax according to Frensch and Steudle (1989) and Melchior and

Steudle (1993). It should be noted that the measurement of Lax on this small

segment of root could potentially overestimate the real Lax of the root, because

xylem vessels will be less developed near the tip than where Lax was measured

and vessel lengths may exceed 10 mm.

Radial Profile of the Lpc

The cell pressure probe (Steudle, 1993) was used to measure the water

transport parameters of cells in each of the radial cell layers of the root

(epidermis and cortical). Microcapillaries for the cell pressure probe were

made from borosilicate glass with 1 mm o.d. 3 0.58 mm i.d. (GC 100-15

Harvard Apparatus; SDR Clinical Technology). Tips were pulled to external

diameters of 5 to 10 mm using one pull on a capillary puller (Narishige PP-83).

The microcapillary was filled with silicon oil and connected to the probe using

nitrile rubber seals. The cell pressure probe was mounted on a micromanip-

ulator (Narishige MX1) with 1-mm increments so that the depth of the

microcapillary tip, within the root, could be measured.

Roots were excised 80 mm from the root tip and secured inside a small

Perspex chamber (Zhang and Tyerman, 1991). The tip of the microcapillary

was introduced into the root perpendicular to the root axis using the

micromanipulator. When the microcapillary punctured a cell, turgor pressure

forced cell sap into the tip of the microcapillary and a meniscus formed





between the sap and oil. The meniscus in the capillary was observed at 1443

magnification with a stereo microscope (SZX12; Olympus Australia) with

illumination from a fiber-optic light source (LG-PS2; Olympus Australia).

The root was punctured in the root hair zone, 30 to 50 mm behind the root tip.

The location of the cell in the cortex was estimated from the depth of the

microcapillary tip from the root surface and the pulses of the meniscus within

the microcapillary when the tip impaled the cells. Since the angle of the

microcapillary was determined visually to be perpendicular to the root axis,

the error of selecting a specific cell layer increased toward the center of the

root. The tip of the microcapillary was assumed located in the correct cell layer

by allowing for a 5� error and a radius of 25 mm, which gave a distance into the

root from the outside surface of 285 mm. Therefore, cells were measured in

each cell layer from the epidermis to the endodermis of wheat roots but only to

the seventh or eighth cell layer of lupin roots because of their larger diameter.

The methodology for measuring Lpc was described previously by Zhang

and Tyerman (1991). Changes in pressure were limited to approximately 0.05

MPa to prevent the inhibition of water permeability caused by large pressure

pulses (Wan et al., 2004).

Estimation of the Contribution of the Cell-to-Cell FlowPath to Radial Flow

The contribution to radial flow of the cell-to-cell flow path was estimated

by modifying the model of Kedem and Katchalsky (1963) that considers the

permeability of concentric membranes in series. In this study, the root is

described as a cylinder composed of concentric, cylindrical ‘‘membranes.’’

Water crosses each cylindrical membrane in series before reaching the

vascular tissue (Fig. 9). This simplified model assumes that radial water

flow only occurs via the cell-to-cell pathway, and if this assumption is correct,

then Lr and Lpr calculated from the model should be similar to the measured

values.

Using the radial profile of Lpc for the hydraulic conductivity of the

concentric membranes, Lr was calculated from

1

Lr

5 +n

j51

1

Lmembranej

5 +n

j51

1

LpjAj

ð2Þ

where Lmembranej is the conductance, Lpj is the hydraulic conductivity, and Aj is

the surface area of the jth membrane. To calculate A of each concentric

membrane, the inner and outer radii of each cell layer were determined from

root and cell diameters (Table III), measured 30 to 50 mm from the root tip, and

the length of the concentric cylinders was varied according to the length of the

root segment or absorbing region. Each individual membrane could be

included/excluded in the model to determine how specific membranes

influence Lr.

To normalize Lr by the surface area of the root, Lpr was calculated from

1

Lpr

5 rr +n

j51

1

Lpjrj

ð3Þ

where rr and rj are the radius of the root and the jth membrane, respectively.

The following conditions were included in the model. (1) Lpc is a composite

measurement for the tonoplast, plasma membrane, and plasmodesmata;

therefore, the same value of Lpc was used for the two membranes, inner and

outer, that enclosed each cell layer. (2) Water flow through the stele was

considered apoplastic, because the cells in this tissue are very small and the

apoplast constitutes a larger cross-sectional area than in the cortex (Steudle

and Jeschke, 1983). (3) Where Lpc could not be measured (e.g. epidermis and

endodermis of lupins), the average Lpc of the nearest measured cell layer was

used. (4) The first 5 mm of the root apex was assumed not to be involved in the

uptake of water, because this region typically has very low axial and radial

conductances to water flow (Steudle, 2001) and the development of lignified

(assumed to be functional) xylem occurred beyond this region.

Mercury Treatment

Lpr was measured on whole root systems before and after mercury

treatment using the pressure chamber technique (Gallardo et al., 1996). After

initial measurements (untreated), the pot was removed from the pressure

chamber and the root system was rewatered with nutrient solution containing

50 mM HgCl2. So that the seal in the lid of the plant pot was not disturbed, 100

mL of mercury solution was injected into a small hole (5 mm diameter) located

in the side of the pot, just below the top of the pot. A total of 100 mL was

sufficient volume to wet the soil and drain from the bottom of the pot. The pot

was drained for 0.5 h on absorbent paper before repressurizing the root

system. The pressurization protocol described above was then repeated,

collecting exudate from the cut stem when flow rate was constant. Repres-

surizing root systems after watering the root system without mercury resulted

in linear regressions identical to initial regressions (data not shown). There-

fore, any changes in Lpr were due to mercury treatment only.

Lpr of root segments was measured with the root pressure probe before and

after mercury treatment. After initial measurements, the nutrient solution

bathing the root was changed to include 50 mM HgCl2, and after 0.5 h, the

measurements for Lpr were repeated.

Preliminary measurements with the cell pressure probe indicated that

mercury had an effect on the half-time of the rate of water exchange across

root cell membranes within 600 to 1,200 s of application, but treatments for

more than 0.5 h could cause turgor pressure to decrease. Therefore, measure-

ments to determine Lpc were conducted on roots pretreated for 600 s, with or

without 50 mM HgCl2.

Anatomy

Freehand cross sections of roots were taken at 5, 10, and 20 mm from the

tip, then every 20 mm up to 120 mm from the tip, and every 40 mm thereafter.

Due to their small diameters, wheat roots were sectioned using the Parafilm

method of freehand sectioning (Frohlich, 1984). Fluorescence under UV

illumination, after staining root cross sections with the berberine/aniline

blue procedure, indicated the presence of Casparian bands (Brundrett et al.,

1988). Suberin lamellae were identified as red/brown cell walls after staining

cross sections with Sudan Red 7B (Brundrett et al., 1991). Sections were

observed with a Zeiss Axiophot microscope (Carl Zeiss) with video capture

(Nikon DXM1200F digital camera and Nikon ACT-1 version 2.62 software;

Coherent Life Sciences) using bright-field, phase-contrast, or epifluorescence

optics. Ultraviolet illumination with filter number 1 (excitation BP365,

chromatic beamsplitter FT396, emission LP397) was used for fluorescence

microscopy.

Since the berberine-aniline blue procedure also stains lignified cell walls

(Brundrett et al., 1988), the sections stained with berberine-aniline blue were

also used to measure the cross-sectional area of functional xylem along the

length of the root. Mature vessels were identified as those with lignified walls

and hence fluorescing bright yellow-white under UV illumination (Brundrett

et al., 1988). The cross-sectional area of fluorescing vessels was measured

using Global Lab Image/2 version 3.0 (Data Translation, Total Turnkey Solu-

tions). The same software was used to measure the dimensions of root cells

(Table III) to calculate Lpc. In some instances, particularly for wheat roots, the

dimensions of the cells actually punctured with the microcapillary could be

delineated by using the probe to fill the cell with oil (Zhang and Tyerman, 1991).

Statistical Analyses

Statistical analyses were performed using SPSS version 11.0 and GraphPad

Prism version 3.02. Data were log or inverse transformed, where necessary, to

satisfy the requirements of normal distribution and homogeneity of variance

for statistical analysis. For linear regression, plots of flow rate against applied

pressure that were not significantly linear (P , 0.05) were eliminated from the

analysis. Differences between species for regression coefficients and intercepts

were tested by ANOVA with Tukey’s posthoc test. t tests were used to examine

whether the intercepts of the regression for whole root systems were signif-

icantly different from zero. Linear and nonlinear regression analyses were

performed to examine the relationship between root length and root water

transport, selecting the best fits that minimized the absolute sum of squares.

To determine whether the axial and radial components of root water transport

were related, correlation analysis was performed. To compare the water

relations of cells from different cell layers, ANOVA with Tukey’s posthoc test

was used. Lpr of whole root systems or root segments, before and after

mercury treatment, was compared in a paired t test. The effects of mercury on

Lpc were tested with independent t tests.

ACKNOWLEDGMENTS

Technical support was provided by Christiane Ludwig at CSIRO Plant

Industry, Perth, and Wendy Sullivan at the University of Adelaide.

Bramley et al.




Received December 10, 2008; accepted March 19, 2009; published March 25,

2009.

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