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First published online March 25, 2009; 10.1104/pp.108.134098 Plant Physiology 150:348-364 (2009) © 2009 American Society of Plant Biologists OPEN ACCESS ARTICLE
Roles of Morphology, Anatomy, and Aquaporins in Determining Contrasting Hydraulic Behavior of Roots1,[OA]School of Agriculture, Food, and Wine, University of Adelaide (Waite Campus), Plant Research Centre, Glen Osmond, South Australia 5064, Australia (H.B., S.D.T.); School of Plant Biology, Faculty of Natural and Agricultural 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 of organization 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 greater Lax 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 the tip in wheat roots. Despite these contrasting behaviors, the hydraulic conductivity of root cells (Lpc) was similar for all species and increased from the root surface toward the endodermis. Lpc was largely controlled by AQPs, as demonstrated by dramatic reductions in Lpc by the AQP blocker mercury. Modeling the root as a series of concentric, cylindrical membranes, and the inhibition 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 crossed mercury-sensitive AQPs in the endodermis, which significantly influenced Lr. This study demonstrates the importance of examining 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, see Bramley et al., 2007b  ). These membrane integral proteins form water-conducting channels, which are considered responsible for the variable hydraulic conductivity of root systems (Lpr; Javot and Maurel, 2002). Reverse genetics has demonstrated that AQP activity is linked to the hydraulics of some species during abiotic perturbation, as the water relations of antisense or mutant plants tends to be more sensitive to the perturbation (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 expression of other AQPs that were not targeted (Jang et al., 2007) and possibly other transporters involved in osmoregulation. These combined effects may cause changes in the morphology of the root system. For example, antisense Arabidopsis (Arabidopsis thaliana) and tobacco (Nicotiana tabacum) had larger root masses (Kaldenhoff et al., 1998; Martre et al., 2002; Siefritz et al., 2002). The increase in root mass may be a compensatory effect, because a lower expression of plasma membrane AQPs reduced the water permeability of some cells (Martre et al., 2002; Siefritz et al., 2002). However, it is not known if the anatomy of antisense roots was also altered or if water uptake was localized to specific regions of the root. These features would also have an important influence on Lpr.
To fully understand root system hydraulics and the contribution of native AQPs, comprehensive studies at different scales are required (cells, organs, and whole roots). This should be coordinated with examinations of anatomy to identify features that will influence the rate of water flow through the root. Comparative studies on species from different evolutionary lines and with differing root hydraulic properties may also further elucidate AQP behavior. To address these issues, we examined the hydraulic properties of roots of two contrasting species: wheat (Triticum aestivum) and narrow-leafed lupin (Lupinus angustifolius). Wheat develops a root system of several extensively branched individual roots (O'Brien, 1979
To investigate more subtle differences in root hydraulics, we also examined the hydraulic properties of a closely related lupin species: yellow lupin (Lupinus luteus ‘Wodjil’). Water transport in yellow lupin roots has not been measured previously. However, anecdotal evidence indicates that the hydraulic properties differ between the two lupin species. Yellow lupin is more tolerant of waterlogging than narrow-leafed lupin due to characteristics of its roots (Davies et al., 2000a
The aim of this study was to identify the influence of root structure, anatomy, and AQP activity on the rate of water transport in wheat, narrow-leafed lupin, and yellow lupin roots. The rationale and main hypotheses were as follows. (1) Lupin roots have larger and more abundant xylem vessels than wheat (Hamblin and Tennant, 1987
To test these hypotheses, the rate of water flow was measured across whole root systems, individual roots, and root cell membranes of seedlings grown in sand. Anatomical features in different regions of the root were related to the water flow measurements to identify the structural components influencing root water uptake and transport. Cell size, radial path length, and the developmental state of the endodermis and exodermis (if present) were examined. Cross sections of roots were stained for Casparian bands and suberin lamellae (Brundrett et al., 1988
The Total Length of Wheat Roots Was Greater Than That of Lupin Roots, But They Did Not Transport More Water to the Shoot Fourteen days after sowing (DAS), wheat root systems comprised four or five seminal roots (Fig. 1 ). The longest seminal root was 360 to 490 mm long, with the second 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 basal half of the wheat root system also contained many fine lateral roots. The root systems of both lupin species were physically similar at 14 DAS. Lupins had a sturdy taproot with lateral roots in the basal region (Fig. 1) and a few small nitrogen-fixing nodules close to the base of the stem. Wheat root systems were more than twice the total length of lupin root systems (Table I ). However, wheat roots were smaller in diameter than lupin roots, which resulted in no significant difference in surface area of root systems between the three species (P = 0.26; Table I).
Midday transpiration of yellow lupin plants, measured gravimetrically, was approximately one-third lower than that of the other species (P = 0.004; Table I). However, transpiration of wheat and narrow-leafed lupin plants was not significantly different (P > 0.05; Table I).
The hydraulic properties of whole root systems were measured with a pressure chamber. Preliminary measurements indicated that the pressure range applied to root systems induced water flux that would have been sufficient to meet the demands of transpiration (Table I). The rate of water transport, per unit pressure gradient, by whole roots was similar for both lupin species, which resulted in a similar mean Lr (P > 0.05; Table I). However, the abscissa intercepts of the linear regression between flow rate and applied pressure ( Root segments (with the tip intact) between 60 and 180 mm long connected to a root pressure probe were used to determine whether Lr varied with root length. Root segments shorter than 60 mm were generally too delicate to connect to the probe, and lateral roots prevented segments longer than 180 mm from sealing to the pressure probe. After connecting the excised end of the detached root segment to the root pressure probe, measurements began when root pressure (Pr) was stable. The Pr of the two lupin species was not significantly different, but Pr of wheat roots was approximately twice that of lupin roots (Table II ).
The Lr of narrow-leafed lupin root segments tended to be greater than that of yellow lupin and wheat root segments (Table II). Lr of lupin root segments increased linearly with length of the segment (P < 0.0001, r2 = 0.61 for narrow-leafed lupin and P < 0.0001, r2 = 0.59 for yellow lupin; Fig. 2, A and B ). In comparison, Lr of wheat root segments was almost constant with segment length (P = 0.937, r2 = 0.0004; Fig. 2C). Because root segments of lupin were larger in diameter than those of wheat (Table II), normalizing Lr by surface area resulted in the average Lpr of lupin being about half that of wheat root segments (Table II). There was no significant difference in Lpr between the two lupin species (Table II). In addition, because Lr was not related to length in wheat, normalizing by surface area resulted in Lpr decreasing linearly with length of the root segment (P = 0.017, r2 = 0.32; Fig. 2F).
Lr of root segments was more than 1 order of magnitude smaller than Lr of whole root systems (Tables I and II). This disparity in Lr for both lupin species was because Lr of whole root systems was related to total root length, with the same regression applying for whole root systems as distal root segments (P > 0.05; Fig. 2, A and B). The increase in water transport with increasing length of lupin roots resulted in an almost constant Lpr with length within each of the two methods of measuring Lr, but whole root systems had on average a higher Lpr than distal root segments (Fig. 2, D and E; Tables I and II). In comparison, although Lr of wheat root systems was greater than for distal root segments (Fig. 2C), this was not related to total root length. Instead, Lpr of distal root segments tended to decrease with length, to a lower constant value in whole roots (Fig. 2F).
Lignified xylem vessels fluoresced bright yellow/white with the aniline blue procedure, which indicated xylem maturity (Brundrett et al., 1988 Xylem vessels developed in a diarch pattern in the roots of both lupin species, initially consisting of two groups of five or six small vessels (Fig. 1K). New vessels developed centripetally, forming a continuous band across the center of the stele between 100 and 120 mm from the root tip (Fig. 1J). Xylem vessel walls became lignified as lupin roots matured (Fig. 1H). The abundance and size of lupin xylem vessels continued to increase with distance from the tip, resulting in a larger and more circular stele (Fig. 1G). Using the fluorescence images, the cross-sectional area of mature xylem vessels (Ax) was measured along the length of wheat seminal roots and the taproot of lupins. For wheat, Ax was constant along the length of the root until at least 100 mm from the root tip, where Ax increased to a larger constant value (Fig. 3A ). Distal to 100 mm from the root tip, Ax of a wheat seminal root was approximately half that of lupin taproots, and as roots matured the difference in Ax increased between the species (Fig. 3A). For lupin, Ax increased with distance from the root tip in the form of logistic growth curves, which were significantly different between the species (P < 0.0001; Fig. 3A). Ax of yellow lupin tended to increase more with distance from the root tip than that of narrow-leafed lupin.
After the measurement of Lr, the root in the pressure probe was excised at the seal and the conductance of the segment remaining in the seal (10 mm long) was measured. Here, radial flow would be negligible because the segment was surrounded by the seal; hence, the measurement is defined as the Lax. Lax varied with distance from the root tip (Fig. 3B) and followed a similar pattern to the development of Ax in all species (Fig. 3A). Lax increased nonlinearly with distance from the tip in lupin roots, with different regressions fitting the two lupins (P = 0.011; Fig. 3B). In comparison, Lax increased only marginally with distance from the tip of wheat roots (Fig. 3B). Lax of wheat for segments excised less than 100 mm from the tip was approximately one-third that of lupins, but values of Lax diverged further between 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 was highly correlated with Lr in lupin root segments, with linear regressions that were significantly different between the two species (P = 0.004). The regression coefficient for narrow-leafed lupin (9.9 ± 1.4 x 10–3) was more than twice that of yellow lupin (4.1 ± 1.0 x 10–3). Therefore, narrow-leafed lupin has a greater radial conductance than yellow lupin for the same axial Lr. There was no correlation between the two hydraulic parameters in wheat (Fig. 4).
Radial Anatomy Root diameter was related to the number of cell layers and cell sizes in the cortex (Tables I and III ; Fig. 1). The number of cell layers across the cortex was constant in all species along the length of the root for at least 250 mm. Suberin was not detected in the outer cortex of any species, indicating that none of the species developed an exodermis when grown in sand. The Casparian strip in the endodermis had developed at 5 mm from the tip in all species, but suberin lamellae developed in the endodermis much closer to the root tip in wheat than in lupin (Fig. 1). Around 40 mm from the root tip, endodermal cells of wheat contained suberin lamellae and the inner tangential walls of the endodermis and walls of stellar cells became thickened (Fig. 1D). In the endodermis of lupins, only a few cells contained suberin lamellae at 100 to 120 mm from the tip (Fig. 1J). At 200 mm from the root tip, all lupin endodermal cell walls contained suberin lamellae, except cells adjacent to xylem poles (Fig. 1G). No secondary thickening of cell walls occurred in roots of either lupin species.
Hydraulic Properties of Root Cells Since Lr << Lax, radial flow was the limiting factor in root water transport for all of the species. Therefore, the hydraulic conductivity of cells (Lpc) in each radial cell layer was measured with the cell pressure probe in order to identify whether the disparity in root water transport between the species was due to different hydraulic properties at the cell level. Due to their small size and possibly different composition of cell sap, epidermal cells were the most challenging to measure, particularly in lupin roots. Although turgor pressure could be measured, constant blockages of the microcapillary or sudden losses of turgor pressure meant that Lpc was not determined in sufficient lupin epidermal cells for statistical analysis. There was no significant difference in turgor pressure of root cells between the lupin species (P > 0.05), but the cells of the epidermis had a lower turgor pressure (0.26 ± 0.03 MPa) than all other cell layers (0.37 ± 0.01 MPa). The average turgor pressure of wheat root cells (0.61 ± 0.01 MPa) was greater than that of lupin but was independent of the radial location in the root (P = 0.119). Lpc of root cells ranged between 0.3 x 10–6 and 3.5 x 10–6 m s–1 MPa–1, and the average Lpc was not significantly different between the three species (P = 0.12). The location of the cell within the cortex influenced Lpc such that Lpc tended to increase toward the root axis (Fig. 5A ), although due to the variability between roots and cells, this trend was not statistically significant in the lupin species (P = 0.072 for narrow-leafed lupin and P = 0.081 for yellow lupin). For wheat roots, the trend was more pronounced and the inner cell layers of the cortex (cell layers 5 and 6) were significantly different from those of the epidermis (cell layer 1; P < 0.05).
The increased Lpc near the axis could be compensating for the decreasing area for water flow toward the axis. When Lpc was adjusted by a factor for the decrease in area (C) for each jth cell layer (Eq. 1), Lpc was almost constant across the cortex for all species (Fig. 5B).
The radial profile of Lpc (Fig. 5A) was used to predict Lr and Lpr by applying a model that describes the root as a series of concentric cylindrical membranes (see "Materials and Methods" for the parameters). The model assumes that radial water flow is primarily via the cell-to-cell flow path and that, after entering the root, water crosses each cell layer in series to reach the stele. The root diameters and number of cell layers used in the predictions are shown in Table IV . Each cell layer has two concentric membranes, which can be included independently of each other in the model. Where Lpc was not measured in lupin, the value of Lpc used was the same as the closest measured cell layer.
The Lr of each concentric cylindrical membrane (Lmembrane) when water uptake is distributed evenly along the length of 120-mm-long roots (excluding 5 mm at the apex) is shown in Figure 6 . The model predicted that Lr of narrow-leafed lupin would be greater than that of yellow lupin, which increased with root length (Fig. 7 ). The predicted Lr for both lupin species was close to the measured Lr for a root segment 120 mm long (Table IV). The disparity between predicted and measured values for narrow-leafed lupin will increase with longer root lengths, because the measured Lr increased more rapidly with root length than predicted (compare regression coefficients in Table IV). Extrapolating the regression for the measured Lr and root length to the abscissa indicated that the first 40 mm of narrow-leafed lupin roots was not involved in water uptake, but the intercept for yellow lupin was close to that set in the model (Table IV). Normalizing Lr by the surface area of lupin roots resulted in a predicted Lpr that was not significantly different from the measured Lpr (Table IV). The closeness of the predicted and measured Lpr values for lupins indicated that the majority of radial water flow could occur across each cell layer through the cell-to-cell pathway.
For wheat, the concentric membrane model predicted a smaller Lr than for lupin for the same root length (Fig. 7). The predicted Lr for a 120-mm-long wheat root was close to the measured value (Table IV). However, unlike the measured Lr, predicted Lr increased with length (Fig. 7), which implied that a specific region of wheat roots determines Lr. To explore this observation, the model was adjusted for anatomical structures in the cell-to-cell path that could influence Lr. To incorporate the effect of suberization of the endodermis, the model was adjusted to allow for water uptake only between 5 and 40 mm from the tip. Limiting the length of the absorbing region resulted in a predicted Lr independent of root length (Fig. 7). However, Lmembrane (Fig. 6), and consequently predicted Lr, was reduced to a value that was only one-quarter of the measured Lr. Therefore, not all cylindrical membranes in the radial pathway contribute equally to water flow. Changing the model to allow for cell-to-cell water flow across only two cylindrical membranes resulted in a predicted Lr close to the measured Lr. For example, using Lmembrane for the epidermis (both inward- and outward-facing membranes) predicted Lr of 2.95 x 10–11 m3 s–1 MPa–1 (Lpr of 10.7 x 10–8 m s–1 MPa–1), and Lmembrane for the endodermis predicted Lr of 2.5 x 10–11 m3 s–1 MPa–1 (Lpr of 10.4 x 10–8 m s–1 MPa–1), which were very similar to the measured values (Table IV).
Mercury had no significant effect on Lpr of whole root systems or root segments of either lupin species (Fig. 8, A and B ). However, Lpr of wheat root systems and individual roots was reduced by up to half after mercury treatment (P = 0.0002; Fig. 8, A and B).
On average, mercury reduced Lpc of cells from treated roots by 33% in narrow-leafed lupin, by 86% in yellow lupin, and by 77% in wheat (Fig. 8C). The outer cell layers of narrow-leafed lupin roots were particularly sensitive to mercury treatment, and measurements of Lpc were only obtained from cells deeper than the third cell layer. Mercury treatment did not affect the volumetric elastic modulus of any species (P = 0.147); therefore, changes in the rate of water exchange across the cell were due to the effect of mercury on Lpc. If water flow occurs entirely through cells by crossing membranes, the inhibition in Lpc should also be reflected in Lpr. However, the concentric membrane model predicted a greater reduction in Lpr than the measured value, particularly for lupin (Table IV). The values in Table IV are based on the assumptions that the entire root length and all cell layers are involved. Adjusting the model for wheat, using the same parameters above (length of absorbing region of 35 mm and cellular flow across two membranes) resulted in predicted values lower than measured values. A 77% reduction in both Lmembrane of the endodermis resulted in Lpr of 2.44 x 10–8 m s–1 MPa–1. However, if only the exterior membrane of the endodermis was inhibited, Lpr was reduced by just over half (4.4 x 10–8 m s–1 MPa–1) compared with the 43% reduction in Lpr actually measured.
The main hydraulic properties of wheat and lupin roots were identified by integrating measurements of water flow, across different structural levels of organization, with anatomy and modeling. An important observation from this study was the major role of root structure and anatomy on root water transport and the localized influence of AQPs. AQP activity was ubiquitous in the cortex and epidermal cells of all species, based on the high values of Lpc and the strong inhibition by Hg2+. The similar magnitudes and profiles of Lpc across the root for each species pointed toward a major role for AQPs in root water transport. However, the contrasting behaviors and the weaker inhibition of water flow by Hg2+ in root tissue and root systems demonstrated that AQPs have localized influences on root hydraulics. At the cell level, AQPs considerably influence Lpc of all three species. At the level of the root system, the control of bulk water flow by AQPs was limited to a small region of the endodermis in wheat. For lupins, bulk water flow occurred predominantly through the apoplast without the influence of AQPs. Without manipulation of the rhizosphere, the benefit of these contrasting behaviors can only be speculated upon, but they may indicate a tradeoff between the overall rate and the potential to rapidly regulate the rate of water flow.
The features described here for wheat and lupin roots grown in sand-filled pots are consistent with those previously described for roots grown in the field (Greacen et al., 1976
If the root is analogous to a leaky pipe, where water can enter anywhere along the length of this pipe, then Lr should increase with root length. Hence, Lpr should be constant with length. In this study, wheat root systems consisted of up to five seminal roots. Lr of an individual, unbranched, seminal root did not vary between 70 and 200 mm from the tip, resulting in a decrease in Lpr with root length. This indicated that water was not uniformly absorbed along the entire root but instead occurred predominantly in a distal region. The region coincided with a less mature endodermis. Farther from the tip, the endodermis was in the tertiary state of development, containing suberin lamellae and thickened cell walls. Water uptake by a region close to the root tip appears to be a common feature of cereal roots (Greacen et al., 1976
For both lupin species, Lr of unbranched taproot segments and whole root systems increased proportionally with root length. This implies that water was absorbed more evenly along the entire root length, including the lateral roots. Unlike wheat roots, the endodermis of lupins did not become as heavily suberized and there was no secondary thickening. The presence of suberin lamellae in the endodermis did not appear to restrict water flow, as there was no corresponding change in Lr with the development of this structure. However, a large region of the endodermis, adjacent to xylem poles, did not become suberized. Lateral roots may also prevent the endodermis from forming a tight barrier in more mature roots as they emerge from this unsuberized region. Eudicots undergo secondary growth, so more mature lupin roots may develop a periderm. If a periderm does develop, then the impermeability of this tissue to water may alter the relationship between Lr and root length. Hamblin and Tennant (1987)
Hamblin and Tennant (1987)
Because monocots do not undergo secondary growth, the development of xylem vessels in wheat roots is determined at an early stage in root growth. Each seminal root contains a large central vessel surrounded by several smaller vessels. Fluorescence imagery indicated that the small vessels become lignified at least 100 mm closer to the root tip than the central vessel. However, the central vessel does not appear to be functional up to 200 mm from the root tip, as there was no abrupt change in Lax that corresponded with fluorescence of the vessel. According to the Hagen-Poiseuille equation, the Lax of a 10-mm-long wheat root segment (average of seven vessels of 21 µm diameter) is 3.7 x 10–9 m3 s–1 MPa–1, if we assume that the central vessel is nonfunctional. This is almost 5-fold greater than the average measured Lax of a 10-mm root segment. Although the measured Lax in this study may be artificially high, flow through xylem vessels is not as ideal as the assumptions used in calculating Lax with the Hagen-Poiseuille equation; therefore, measured Lax values are typically less than calculated (Greacen et al., 1976
Yellow lupin roots tended to have a greater cross-sectional area of xylem vessels but had similar Lax to narrow-leafed lupin. This disparity between the two species was manifested in the relationship between Lax and Lr. The regressions in Figure 4 imply that yellow lupin has a greater capacity to carry water axially relative to the radial path. For narrow-leafed lupin, the radial conductance increases at a greater rate per Lax, so greater water potential gradients may exist in the xylem. Corresponding to this, yellow lupin tends to maintain a higher leaf water potential than narrow-leafed lupin, but it also tends to have lower rates of stomatal conductance and transpiration (Davies et al., 2000a
Lax was 2 orders of magnitude greater than Lr, indicating that the rate of water flow through the radial pathway was limiting to Lr for all three species. Similar differences in magnitude between axial and radial conductance have been reported for a wide variety of species (Steudle and Peterson, 1998
The conductance of the radial pathway is inversely proportional to the length of the flow path or the number of cell layers, since the cells are arranged in series (Steudle and Frensch, 1996
Water flow from the root surface to the vascular tissue can occur through two parallel pathways: the apoplast and the cell-to-cell pathway. Water passing through cells may cross into adjacent cells through connecting plasmodesmata and/or across membranes. The symplastic connection through plasmodesmata has been demonstrated with tracer dyes and predominantly occurs within a few millimeters of the root tip (Hukin et al., 2002
A model describing the root as a series of concentric membranes has been used previously to predict Lpr of barley (Hordeum distichon), wheat, and maize roots (Jones et al., 1983
For both lupin species, the predicted Lr values were similar to the measured Lr if water crossed all cell layers through the cell-to-cell pathway. Lr was also predicted to increase with root length. For wheat, the predicted and measured values were also in close agreement, but only when the length of the absorbing region was confined to the distal region, where the endodermis was less developed and only two cylindrical membranes were included in the flow path. Although suberization of the endodermis did not appear to influence Lr of lupin roots, state III development of the endodermis (suberin lamellae and tertiary thickening) has been correlated with a decline in water uptake of cereal roots (Sanderson, 1983
Because the model provided strong evidence for a large component of cell-to-cell flow, we further tested the predictions by inhibiting flow through the cell-to-cell pathway. Despite the shortcomings with Hg2+ being relatively nonspecific in its mechanism of inhibition of water transport (Zhang and Tyerman, 1999
Mercury failed to affect Lr of lupin root segments or root systems, despite dramatic reductions of Lpc in all cell layers. Therefore, water primarily flowed around cells in lupin roots, without crossing membranes. This also implies that water did not cross membranes in the endodermis, despite the Casparian strip and the later partial development of suberin lamellae. The Casparian strip in the endodermis is also not a significant barrier to apoplastic water flow in maize roots (Steudle et al., 1993
For wheat roots, the model and the inhibition of Lr and Lpc indicated that Lr is regulated by AQPs in a small region of the endodermis. At the endodermis, it is unlikely that mercury could permeate fully because of the Casparian strip. If only the exterior "membrane" of the endodermis was inhibited by mercury, a similar inhibition in Lr was predicted by the model to what was measured. Some studies have removed cell layers to measure Lpr in conjunction with mercury treatment, because of the presence of Casparian bands and suberin lamellae (Martre et al., 2001
Although AQPs in narrow-leafed lupin or yellow lupin have not yet been characterized, the high values of Lpc and the effect of mercury demonstrated high AQP activity in both lupin species. In addition, the increase in Lpc toward the root axis suggests that AQP activity increases to compensate for a concentrating effect of the root geometry. For the measurements in this study, water flow was induced by hydrostatic pressure, and under these conditions, bulk water flow may bypass cells. Hence, water flow generated by transpiration also probably travels predominantly through the apoplast. However, water flow induced by osmotic gradients must cross membranes, so this may be where AQP activity influences water flow across lupin roots. Lpr of lupin roots measured using hydrostatic pressure is at least twice the Lpr measured using an osmotic gradient, in comparison with wheat, where Lpr was the same under both types of gradient (Bramley et al., 2007a
One of the hypotheses of this study was that the hydraulics of narrow-leafed lupin and yellow lupin differed because of AQP activity. Here, AQPs were not found responsible for Lr under these water-sufficient conditions. However, AQPs may be required for osmoregulatory processes (Tyerman et al., 1999
The apoplast should have a higher conductivity to water flow than the cell-to-cell pathway because of the absence of membranes. Therefore, by having a greater proportion of water flow via the apoplast, lupins are able to achieve the same or superior radial conductance as the thinner wheat roots. However, the main consequence of predominantly apoplastic water flow is that Lr is invariable in the short term. Lupins may rely on anatomical and growth-related changes to regulate water flow at the root level and/or manipulate the driving force through closure of stomata. Stomatal conductance of lupins decreases under drought, as more of the root system is exposed to the drying soil and root water uptake decreases (Jensen et al., 1989
In comparison with lupins, wheat root systems are not dependent on growth-related changes in anatomy to vary Lpr. Wheat can rapidly adjust Lpr because flow through a seminal root is predominantly controlled by AQPs. Under the growing conditions of this study, the control occurs in the endodermis in the apical region. It is not known which specific AQPs are expressed in the endodermis, but the expression of different AQPs can be localized to specific tissues (for review, see Bramley et al., 2007b
The hydraulic properties of wheat and lupin roots are highly contrasting, which suggests a balance between root diameter, Lr, and the ability to rapidly adjust to prevailing conditions. By absorbing water more evenly along the root length, which is related to increasing xylem development, lupin roots have a high capacity to transport water, although yellow lupin may be more conservative than narrow-leafed lupin in its water use strategy. In addition, predominantly apoplastic flow endows lupin roots with the same or superior radial conductance as thinner wheat roots, but it provides little ability to rapidly adjust Lr in the short term. However, osmotically induced water flow must cross membranes, which could be facilitated by AQPs. In comparison, preferentially absorbing water in the distal region and low Lax diminish the potential for high water flow in wheat. However, because a significant component of water flow crosses at 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 layers of the cortex of all species implies that their role is more extensive than solely transporting water from the surface of the root to the vascular tissue. The similarity in root cortex profile of Lpc yet contrasting hydraulics of whole roots demonstrate the importance of examination of root structure and anatomy in assessing the role of AQPs in whole root transport. A cautionary note should be included about the use of Lpr. Because of problems of scale, measurements of Lpr are generally normalized by root dimensions, such as length, surface area, or mass. Water uptake is limited by the absorbing area; therefore, normalization by the entire root size may be inappropriate for some species. The form of the normalization is particularly important when comparing the effects of treatments that alter the dimensions or structure of the root system, especially if the absorbing area is unaltered. In these cases, it may be more meaningful to normalize hydraulic conductivity by another parameter such as leaf area.
Plant Material
The details of growing wheat and lupin seedlings were described previously by Bramley et al. (2007a)
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)
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.
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 between 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
After the initial 3-h equilibration period, the pressurization protocol followed that of Gallardo et al. (1996) 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 mm–1; ScanJet; Hewlett-Packard Australia) covered with a thin film of water. Images were analyzed using Rootedge version 2.3b software (National Soil Tilth Laboratory, U.S. Department of Agriculture). Roots were assumed cylindrical for converting image areas to surface areas.
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
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)
The cell pressure probe (Steudle, 1993
Roots were excised 80 mm from the root tip and secured inside a small Perspex chamber (Zhang and Tyerman, 1991
The methodology for measuring Lpc was described previously by Zhang and Tyerman (1991)
The contribution to radial flow of the cell-to-cell flow path was estimated by modifying the model of Kedem and Katchalsky (1963)
Using the radial profile of Lpc for the hydraulic conductivity of the concentric membranes, Lr was calculated from
To normalize Lr by the surface area of the root, Lpr was calculated from
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
Lpr was measured on whole root systems before and after mercury treatment using the pressure chamber technique (Gallardo et al., 1996 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 µM 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, measurements to determine Lpc were conducted on roots pretreated for 600 s, with or without 50 µM HgCl2.
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
Since the berberine-aniline blue procedure also stains lignified cell walls (Brundrett et al., 1988
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 significantly 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.
Technical support was provided by Christiane Ludwig at CSIRO Plant Industry, Perth, and Wendy Sullivan at the University of Adelaide. Received December 10, 2008; accepted March 19, 2009; published March 25, 2009.
1 This work was supported by the Grains Research and Development Corporation of Australia and the Australian Research Council. 
2 Present address: Department of Renewable Resources, 4-44 ESB, University of Alberta, Edmonton, Canada T6G 2E3. The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Helen Bramley (helen.bramley{at}afhe.ualberta.ca).
[OA] Open Access articles can be viewed online without a subscription. www.plantphysiol.org/cgi/doi/10.1104/pp.108.134098 * Corresponding author; e-mail helen.bramley{at}afhe.ualberta.ca.
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ABSTRACT
RESULTS
P of 0.3, 0.5, and 0.7 MPa was applied to detopped root systems to induce a water flux. The flow rate at each pressure was plotted against 
