Journal of Experimental Botany

JXB Advance Access originally published online on May 31, 2005
Journal of Experimental Botany 2005 56(417):1853-1865; doi:10.1093/jxb/eri175

This Article Abstract FREE Full Text (PDF) OA All Versions of this Article: 56/417/1853    most recent eri175v1 E-letters: Submit a response Alert me when this article is cited Alert me when E-letters are posted Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in ISI Web of Science Similar articles in PubMed Alert me to new issues of the journal Add to My Personal Archive Download to citation manager Search for citing articles in: ISI Web of Science (21) Disclaimer Google Scholar Articles by Kinraide, T. B. Articles by Zobel, R. W. Search for Related Content PubMed PubMed Citation Articles by Kinraide, T. B. Articles by Zobel, R. W. Agricola Articles by Kinraide, T. B. Articles by Zobel, R. W. Social Bookmarking          What's this?

Published by Oxford University Press [2005] on behalf of the Society for Experimental Biology.

RESEARCH PAPER

Organic acid secretion as a mechanism of aluminium resistance: a model incorporating the root cortex, epidermis, and the external unstirred layer

Thomas B. Kinraide^1,*, David R. Parker² and Richard W. Zobel¹

¹Appalachian Farming Systems Research Centre, Agricultural Research Service, USDA, Beaver, West Virginia 25813-9423, USA
²Department of Environmental Sciences, University of California, Riverside, California 92521, USA

^* To whom correspondence should be addressed. Fax: +1 304 256 2921. E-mail: tom.kinraide{at}ars.usda.gov

Received 13 January 2005; Accepted 29 March 2005

	Abstract

Top Abstract Introduction Materials and methods Results Discussion References

 
The resistance of some plants to Al (aluminium or aluminum) has been attributed to the secretion of Al³⁺-binding organic acid (OA) anions from the Al-sensitive root tips. Evidence for the ‘OA secretion hypothesis’ of resistance is substantial, but the mode of action remains unknown because the OA secretion appears to be too small to reduce adequately the activity of Al³⁺ at the root surface. In this study a mechanism for the reduction of Al³⁺ at the root surface and just beneath the epidermis by complexation with secreted OA^2– is considered. According to our computations, Al³⁺ activity is insufficiently reduced at the surface of the root tips to account for the Al resistance of Triticum aestivum L. cv. Atlas 66, a malate-secreting wheat. Experimental treatments to decrease the thickness of the unstirred layer (increased aeration and removal of root-tip mucilage) failed to enhance sensitivity to Al³⁺. On the basis of additional modelling, the observed spatial distribution of Al in roots, and the anatomical responses to Al, it is proposed that the epidermis is an essential component of the diffusion pathway for both OA and Al. We suggest that Al³⁺ in the cortex must be reduced to small concentrations in order substantially to alleviate the inhibition of root elongation and so that the outer surface of the epidermis can tolerate relatively large concentrations of Al³⁺. If OA secretion is required for reducing Al³⁺ mainly beneath the root surface, rather than in the rhizosphere, then the metabolic cost to plants will be greatly reduced.

Key words: Aluminium, aluminum, diffusion, haematoxylin, malate, organic acid, toxicity, wheat

	Introduction

Top Abstract Introduction Materials and methods Results Discussion References

 
Many investigators appear to have concluded that the resistance of some plant roots to Al results from the secretion, into the rhizosphere, of Al³⁺-binding organic acid (OA) anions from the Al-sensitive regions of the root tips (Ojima et al., 1987; Miyasaka et al., 1991; Ryan et al., 2001; Kochian et al., 2004). The correlative evidence for the OA secretion hypothesis of Al resistance is substantial, and now an Al resistance gene from wheat, ALTM1, has been cloned and expressed in other genotypes (Sasaki et al., 2004; Delhaize et al., 2004). Nevertheless, the hypothesis is problematical, principally because the estimated OA concentrations at the root surface appear to be too small to reduce adequately the root-surface activity of Al³⁺ (a_{Al3+, root surf}) (Ryan et al., 1995b; Parker and Pedler, 1998; Parker et al., 2005).

Current investigations of Al resistance appear to concentrate upon the genetics and controls of OA secretion (Kochian et al., 2004) rather than upon a mechanism by which resistance may occur (but see the recent physiological study by Piñeros et al., 2005). The authors are not aware of any direct experimental evidence for a detailed mechanism, nor of any attempt to model such a mechanism quantitatively. According to our understanding of the literature, OA secretion is considered to confer resistance principally by reducing a_{Al3+, root surf}, but there does not seem to have been any consideration of the possibility that reduction of a_{Al3+, root surf}, by itself, may be inadequate, and that a further significant reduction of a_Al3+ across the epidermis may be a crucial component of the mechanism. That is, reduction of a_Al3+ in the cortex may be more important than reduction at the root surface.

The mechanism to be evaluated is embodied in the ‘biphasic diffusion hypothesis’. It states that as OA diffuses through the epidermis (the first phase) and then away from the root surface through an unstirred layer (the second phase) it encounters Al. In each phase the divalent anion (OA^2–) binds Al³⁺ according to the equilibrium a_{OAAl+ =} a_Al3+a_OA2–K_OAAl+, which relates chemical activities to an equilibrium constant, K_OAAl+ (Table 1). Binding to OA^2– will deplete a_Al3+ at the root surface and in the cortical free space, reduce toxicity, and establish a concentration gradient resulting in a flux of Al³⁺ toward the root surface and into the root. The influx of Al³⁺ (and some other species) will be countered by an efflux of OAAl⁺ (and some other species).

View this table: [in this window] [in a new window]   Table 1. Some parameter values for the standard diffusion model (OA = malate)

 
This study has two objectives. The first is to compute a spatial profile of species concentrations, activities, and fluxes across the diffusion pathway (epidermis and unstirred layer) of seedlings cultured in solution in order to evaluate the plausibility of the ‘biphasic diffusion hypothesis’. The second is to test components of the hypothesis experimentally.

	Materials and methods

Top Abstract Introduction Materials and methods Results Discussion References

 
Growth experiments
The biphasic diffusion hypothesis, for which a quantitative model will be presented, inspired four lines of experimentation: the first to determine whether increasing the agitation of the culture medium (thereby reducing the thickness of the unstirred layer at the root surface) increases the sensitivity to Al³⁺ (i.e. to a given a_{Al3+, medium}), the second to determine whether removing the root-tip mucilage increases the sensitivity, the third to determine whether increasing the Al³⁺ buffering capacity increases the sensitivity, and the fourth to determine the distribution of Al in seedlings of Al-resistant Atlas 66 and Al-sensitive Scout 66 wheats (Triticum aestivum L.).

To do these experiments, seeds were soaked in 1% sodium hypochlorite for 15 min then rinsed for about 1 h. The seeds were then germinated on paper in contact with 1 mM CaCl₂ and incubated at 25 °C for 2 d. The seedlings were inserted into floats and placed on the surfaces of 500 ml test solutions and incubated for 2 d at 25 °C in the dark, usually with aeration. After incubation, the two longest roots of each of the five seedlings per solution were measured for length. Thus each datum point in the figures that follow refers to a single solution and the mean length of ten roots.

To remove mucilage without injury, the root tip was touched to a tissue paper to remove adhering water. The water was drawn off by capillarity in about 1 s, but the mucilage remained. Next, the root tip was touched to a dry section of the tissue paper for a few seconds to allow the mucilage to adhere. Then the root was drawn along the surface of the paper in an axial direction so that the strand of mucilage pulled away from the root and remained attached to the paper. This operation was repeated on different sides of the tip until all visible mucilage was removed.

Root elongation was sometimes expressed as relative root length computed as

(1)

where RL_Al is the root length in an Al-containing solution, RL_S is the root length in a solution containing enough Al to minimize elongation, and RL_C is the root length in an Al-free solution. Growth in response to a toxicant, such as Al³⁺, may be expressed as

(2)

which generates a downward exponential curve when q=1, a sigmoidal curve when q >1, and a curve with a steep initial decline when 0 <q <1. p is a strength coefficient. When a_{Al3+, medium} = 1/p, RRL = 36.8%, irrespective of the value of q. Equation 2 may be expanded to incorporate other toxicants or ameliorants (Kinraide, 2003b).

Haematoxylin staining for Al
Scout and Atlas wheat seedlings were cultured in 1 mM CaCl₂ at pH 4.5 with variable concentrations of AlCl₃. Seedlings were withdrawn from solutions in which they had been cultured for 1 or 2 d, rinsed by dipping a few times in one then another large volume of distilled water, placed in stain for periods of 5–60 min, then rinsed again as just described. The roots were excised, mounted, and photographed; the stain consisted of 2 g haematoxylin and 0.2 g NaIO₃ l^–1.

A model for the solute fluxes and concentrations
Anatomical considerations and diffusion exterior to the root surface:
Readers may consult Fig. 3 in Degenhardt et al. (1998) for a depiction of an Arabidopsis root, its shape, and its principal region of H⁺ influx. Readers may also consult Fig. 1 in Sivaguru and Horst (1998) for a depiction of a maize root, its shape, and its principal region of Al sensitivity. These roots are smaller and larger, respectively, than the Atlas wheat roots considered here. Photographs of the latter are presented in Fig. 1A in Puthota et al. (1991), and a scale drawing from the photographs is presented in this paper. It illustrates that the root is about 0.5 mm in diameter 2 mm from the tip and that the apical 2 mm is mostly enclosed in a drop of mucilage. The greatest diameter of root plus mucilage is about 1.36 mm. The region 1–2 mm from the tip is considered to be the region of principal Al sensitivity, alkalinization, and OA efflux, but fluxes are assumed to occur approximately from 0.5–2.5 mm from the tip.

View larger version (16K): [in this window] [in a new window]   Fig. 3. Al3+ activity as a function of distance from the root surface and the length of the diffusion pathway. Each curve presents aAl3+ across an external diffusion pathway of length 100, 200, 400, 800, or 1200 µm.

 

View larger version (15K): [in this window] [in a new window]   Fig. 1. Scaled diagram of a root tip of Atlas 66 wheat drawn from photographs presented in Fig. 1A of Puthota et al. (1991) and reproduced with the kind permission of Springer Science and Business Media. On the drawn horizontal scale, R(x = 250 µm) denotes the root radius, and UL(x = 650 µm) denotes the assumed mean position of the outer edge of the unstirred layer.

 
It is assumed here that the mean diameter of the root over its sensitive region is approximately 0.5 mm and that the length of the external diffusion pathway is 400 µm. This length will be determined by mucilage and the unstirred layer near the surface of the root. OA diffusing away from the root surface will pass through imaginary cylindrical surfaces that are proportional to the radii of the cylinders. Thus the area through which the solute passes will be 2.6 times larger at 400 µm from the root surface (corresponding to a radius of 0.65 mm) than at the surface (corresponding to a radius of 0.25 mm).

The flux of solute j, at the root surface, is designated J_j, and fluxes away from the root are assigned negative values. The flux of j at position x exterior to the root is

(3)

where R is the root radius. As illustrated in Fig. 1, x increases from 0 at the centre of the root to R at the surface, to UL at the outer edge of the unstirred layer. The term R/x adjusts the flux for the changes in the areas of the cylindrical surfaces through which the solute passes.

The flux of total OA (TOA, the sum of all OA species), total Al (TAl, the sum of all Al species), and H⁺ at the root surface are designated J_TOA, J_TAl, and J_H+, respectively. Table 1 gives the values assumed for the standard model. J_TOA is not constant. OA secretion is small in the absence of Al and increases as Al in the medium increases until it achieves a maximum at concentrations above 100 µM Al (see references in Table 1). For that reason, J_TOA should be adjusted whenever a treatment causes a change in a_{Al3+, root surf}. Such treatments include those that change the length of the diffusion pathway. However, such adjustments cause small effects in the cases considered here (as will be seen), so J_TOA has been taken to be constant when a_{Al3+, medium} is constant. J_TAl is an Al accumulation term and is taken to be zero for the standard run, but is given positive values for other runs. J_H+ is large relative to other fluxes and is therefore not considered to be much affected by changes in proton consuming or releasing reactions (e.g. OA^2–+H⁺HOA^–). To express the flux of any of these solutes at position x, the flux at the root surface (designated by upper-case J) must be multiplied by R/x as in Equation 3.

Diffusion interior to the root surface:
Diffusion does not stop at the outer surface of the root. OA, Al³⁺, H⁺, and other solutes diffuse through, or around, the cells of the epidermis. Equation 3 describes flux through the diffusion pathway external to the surface of the root, so now it is necessary to describe flux through the interior of the root. In the case of OA, and some other solutes, it is necessary to account for the fact that these solutes are synthesized or consumed within the root. For OA, the assumption is made that synthesis is uniform throughout the volume of the sensitive region of the root. A term, S_TOA, is defined as the rate of synthesis of OA per unit volume. This will be 2.78x10^–13 mol apex^–1 s^–1/(3.93x10^–10 m³ apex^–1) = 0.000707 mol m^–3 s^–1. The flux at any position x within the root will equal the amount of OA synthesized within the cylinder, having x as its radius, divided by the area of the cylinder. The volume of the cylinder (V(x)) is x²l; the area (A(x)) is 2xl; and the length of the sensitive region (l) is 0.002 m. Thus,

(4)

Equations for concentration based upon fluxes:
Equations for concentration at position x external to the root may be derived from equations for the fluxes according to Fick's First Law of Diffusion,

(5)

which states that the flux of solute j along the axis x is proportional to the concentration gradient (Nobel, 1991). The proportionality constant D_j is the diffusion coefficient, and the negative sign signifies that flux is in the direction of decreasing concentration. The concentration of solute j may be obtained by integrating and rearranging Equation 5. In these equations c_j must be expressed in mol m^–3 when D is expressed in m² s^–1.

(6)

C_{j, UL} is the constant of integration which in this study's treatment will be the concentration of solute j at x = UL, that is, at the outer edge of the unstirred layer, and equal to the concentration in the external, bulk-phase medium. For cylindrical diffusion,

(7)

which, when integrated from x = x to x = UL, becomes

(8)

When Equation 4, which pertains to diffusion within the root tissue, is rearranged and integrated from x = x to x = R then

(9)

is obtained, where C_{TOA, R} is the constant of integration equal to the concentration of total OA at the root surface. It is assumed that D_{TOA, tissue} is greatly different from D, especially through the epidermis whose cells fit together tightly. If fluxes are channelled between the cells, the area through which the fluxes occur will be reduced. This will increase fluxes and concentration gradients (d(c_j)/dx) through the channels. These effects can be simulated by making changes in the diffusion coefficient. In any case, the apparent diffusion coefficient will decrease whether fluxes are mainly channelled between the cells or through the cells: the latter possibility being unlikely because of the large electrochemical gradient against OA entry into the symplasm from the apoplasm. Fluxes and concentrations of H⁺ within the root can be treated similarly.

Speciation:
The equations presented thus far are sufficient for the computation of c_TOA(x), c_TAl(x), and c_H+(x). To compute the concentrations of species such as c_Al3+(x) or c_OAAl+(x), values for c_TOA(x), c_TAl(x), and c_H+(x) may be linked to a chemical speciation programme. Other inputs to the speciation programme will include other solutes that do not change significantly with x or that do not react with Al or OA. These would include Cl^– and Na⁺. Solutes, such as that do react with Al, but whose concentrations are much greater than Al³⁺, may be considered invariant with x external to the root, but these solutes will generate species such as whose concentrations do vary with x.

The following rooting medium was assumed for the standard run: 23.85 µM AlCl₃, 1 mM CaCl₂, and 2638 µM NaCl, adjusted to pH 4.3 with HCl. For this solution c_Al3+ = 20.75 µM, a_Al3+ = 10.00 µM, I = 5825 µM, and _Al3+ = 0.482 (where _Al3+ is an activity coefficient). This solution is moderately toxic to Atlas wheat and very toxic to Scout wheat (Parker and Pedler, 1998; this study), which does not secrete appreciable amounts of OA (Pellet et al., 1996; Nian et al., 2002). For some other solutions, AlCl₃ and NaCl were adjusted so that neither a_Al3+ nor I changed. In a solution containing Na₂SO₄, for example, the input concentrations were AlCl₃ = 35.54 µM, 1 mM CaCl₂, 0 µM NaCl, and 1 mM Na₂SO₄ adjusted to pH 4.3 with HCl so that a_AlSO4+ = 10.25 µM and a_Al3+ = 10.00 µM.

	Results

Top Abstract Introduction Materials and methods Results Discussion References

 
Modelling for external fluxes, concentrations, and activities
The standard run and some checks of the computations:
Figure 2 presents the results of the standard run of the diffusion model. Conditions for the standard run are presented in Table 1. As expected, concentrations of Al³⁺ and H⁺ decline toward the root surface, and concentrations of OA species increase toward the root surface. The sum of the concentrations of all Al species (essentially Al³⁺, Al-malate⁺, AlOH²⁺, and ) equals 23.85 µM at all distances from the root surface. Each of these species has its own flux as shown in Fig. 2, but the sum of all Al fluxes is zero at every position, because the standard run assumes no accumulation (i.e. J_TAl is small relative to J for Al³⁺ and other Al species). The sum of the fluxes of all OA species (malate^2–, Al-malate⁺, H-malate^–, and H₂-malate⁰) multiplied by x/R was –88.4 nmol m^–2 s^–1 at all positions. Thus, j_TOA is variable because of the cylindrical configuration, and c_TOA is variable too.

View larger version (21K): [in this window] [in a new window]   Fig. 2. Computed concentrations and fluxes of several solutes external to the root tip of Atlas wheat. Subscript TMA refers to the sum of all OA species (essentially, malate2–, Al-malate+, H-malate–, and H2-malate0). The sum of all concentrations of Al species (essentially, Al3+, Al-malate+, AlOH2+, and ) is constant at 23.85 µM, and the flux of total Al is zero in the standard run of the model.

 
The model predicts a reduced toxicity because of the conversion of Al³⁺ to non-toxic species rather than to a reduction in total Al. To judge the adequacy of reducing c_{Al3+, root surf} to 10.1 µM (a_{Al3+, root surf} = 4.90 µM) it may be considered that 4.90 µM a_{Al3+, medium} is very intoxicating to sensitive genotypes. Furthermore, the computed concentration of 21.13 µM total OA (malate) at the root surface is about 10-fold less than that required to alleviate significantly the Al³⁺ intoxication (Delhaize et al., 1993b; Parker and Pedler, 1998).

The influence of the length of the diffusion pathway:
Figure 3 presents runs of the standard model except that the length of the external diffusion pathway (UL) was varied. When the path length is short a_Al3+ declines steeply as the root surface is approached, but does not achieve values as small as when the path length is long. One might assume, therefore, that a long path length would reduce Al³⁺ intoxication, but a more detailed analysis indicates that intoxication may be quite independent of diffusion path length unless J_TOA is much greater than the –88.4 nmol m^–2 s^–1 of the standard run.

Figure 4A presents a_{Al3+, root surf} as a function of the diffusion path length, and Fig. 4B presents pH_{root surf}. Ions at the ‘root surface’ are still in the bathing medium; they are not at the plasma membrane (PM) surface; but it is well established that ion uptake, intoxication, or both, correlate well with ion activities at the PM surface and often correlate poorly with ion activities in the bathing medium (Gimmler et al., 2001; Zhang et al., 2001; Kinraide, 2003a, b).

View larger version (14K): [in this window] [in a new window]   Fig. 4. Conditions at the root surface as a function of the length of the external diffusion pathway. aAl3+, root surf refers to activity at the root surface, but not close enough to be influenced by the electrical potentials of the CWs or PMs. PM is the electrical potential and aAl3+, PM is the activity of Al3+ at the PM surface of the outer edge of epidermal cells. aAl3+, PM for cells beneath the epidermis is smaller but similarly unresponsive to the diffusion path length external to the root. Variable JTOA means that JTOA is proportional to aAl3+, root surf (with JTOA = –88.4 nmol m–2 s–1 when the diffusion path length is 400 µm and aAl3+, root surf is 4.90 µM).

 
Ion activities at the PM surface are computed by the Nernst Equation,

(10)

where Z_j is the charge of ion j, and 25.7 is a collection of constants appropriate when the PM surface electrical potential (_PM) is expressed in mV at 25 °C. _PM is computed with a Gouy–Chapman–Stern model (Yermiyahu et al., 1997; Kinraide, 2003b). Ions that bind strongly to the PM, such as Al³⁺ and H⁺, reduce the negativity of the PM surface and thereby reduce the activity of cations at the surface.

Because a_{Al3+, root surf} and a_{H+, root surf} increase with decreasing diffusion path length, _PM becomes less negative with decreasing path length (Fig. 4C). Consequently, cations become attracted less strongly, and Fig. 4D presents a_{Al3+, PM} as a function of path length. One can see that a_{Al3+, PM} is quite insensitive to path length under the conditions of the standard run, but if J_TOA is increased 10-fold (enough to elevate malate to 211 µM at the root surface when the unstirred layer is 400 µm thick) then a_{Al3+, PM} is sensitive to path length. Because root elongation is a function of a_{Al3+, PM}, rather than a_{Al3+, root surf}, root elongation may be expected to be insensitive to path length under conditions of the standard run.

The influence of pH:
The value for J_H+ (= 1000 nmol m^–2 s^–1) may be somewhat large for wheat roots (Table 1). In the standard run, pH approaches 4.58 as the root surface is approached (Fig. 4B). If the rooting medium were buffered, or if the proton flux were reduced, then the pH would not change as much. Figure 5A illustrates that the effects of reducing j_H+ to zero are to reduce pH_{root surf} to 4.30 and to reduce the decline of a_Al3+ toward the surface of the root. By contrast, the effects of increasing J_H+ 1.5-fold are to increase pH_{root surf} to 4.83 and to increase the decline in a_Al3+. However, a_{Al3+, PM} actually increases as pH_{root surf} increases and a_{Al3+, root surf} declines (see the values for a_{Al3+, PM} written in Fig. 5A).

View larger version (27K): [in this window] [in a new window]   Fig. 5. The effect of varying several model parameters on the profile of computed Al3+ activity (see Table 1 and the text for a description of the parameters). The numbers written at the left end of some of the curves refer to aAl3+, PM at the outer surfaces of the epidermal cells.

 
The influence of Al accumulation:
For the standard run it is assumed that J_TAl = 0, but some Al accumulation must occur in roots that elongate in Al solutions. This accumulation may be apoplastic, symplastic, or both. Figure 5B presents the case where J_TAl = –J_TOA/2 = 44.2 nmol m^–2 s^–1, rather than zero as in the case of the standard run. The effect is to reduce a_{Al3+, root surf}, but it is estimated that the assumed accumulation rate of 44.2 nmol m^–2 s^–1 is unreasonably large and would lead to a concentration of Al of about 2.55 mmol l^–1 root volume or about 688 µg Al g^–1 root DW, a concentration that would be very inhibitory to Atlas wheat (Samuels et al., 1997).

The influence of the parameters J_TOA, D, K_OAAl+, and K_HOA+:
Changing some of the parameters of the model will affect a_{Al3+, root surf}. Increasing J_TOA 2-fold and decreasing D 2-fold have identical effects; both reduce a_{Al3+, root surf} to 2.95 µM (Fig. 5C). The standard run incorporates malate as the OA, but the citrate and oxalate secreted by some species have larger association constants (Ryan et al., 2001). The effect of increasing the association constant, K_OAAl+, to 10⁷ M^–1 reduces a_{Al3+, root surf} to 2.35 µM (Fig. 5C).

The influence of buffering Al³⁺:
Just as H⁺ may be buffered, so too may Al³⁺. Figure 5D presents a run in which Na₂SO₄ = 1 mM. NaCl was reduced to maintain constant I, and AlCl₃ was adjusted so that a_{Al3+, medium} = 10 µM (see speciation section above). The effect was to increase the Al³⁺ buffering capacity from 2.40 for the standard run to 3.55 in the presence of Na₂SO₄ (because of the reaction ). The buffering capacity was computed as (added AlCl₃)/a_{Al3+, medium}. As expected, the buffered solutions resisted declines in a_{Al3+, root surf}.

Modelling for internal activities of Al³⁺
Figure 6 illustrates the profile of a_Al3+ through the external diffusion pathway and 30 µm into the root tissue. The middle curve from distance 0–400 µm is the curve of the standard run presented in Fig. 5. The continuation of the curve into the tissue (distance <0) uses Equation 9 and related equations for Al³⁺ and H⁺. To use Equation 9, values must be assigned to D_{TOA, tissue}, D_{Al3+, tissue}, and D_{H, tissue}. For the initial assignments, D and D_H were similarly reduced so that D_{TOA, tissue} = D_{Al3+, tissue} = D/9 and D_{H, tissue} = D_H/9. With these changes, a_Al3+ = 0.278 µM and pH = 5.83, 30 µm into the root.

View larger version (21K): [in this window] [in a new window]   Fig. 6. Computed Al3+ activities across the root epidermis and the unstirred layer. The calculations for Atlas wheat are according to the standard run for distances 0–400 µm. For distances <0 (i.e. the root interior), the diffusion coefficients have smaller values (DTOA, tissue = DAl3+, tissue = D/72 and DH, tissue = DH/6.7). The model parameters for Scout are similar to those for Atlas except that OA secretion is zero. The concentrations of AlCl3 for the middle and bottom curves ensure equal, moderate intoxication of the roots. The reductions in aAl3+ seen for Scout are attributable to pH only while the reductions in aAl3+ for Atlas show the added effect of OA secretion.

 
These new values for a_Al3+ and pH would alleviate inhibition, but a problem arises. The reduction of a_Al3+ to 0.278 µM is almost entirely the consequence of the elevated pH because c_{TOA, tissue} is only 43.5 µM. If it is assumed that J_TOA = 0, as is the case for Scout wheat, then the pH increase alone is enough to reduce a_Al3+ to 0.415 µM. These results indicate that Scout would be only 1.4-fold more sensitive than Atlas, rather than the 16-fold more sensitive that is known (Kinraide, 1997; Parker and Pedler, 1998).

An additional problem is that a 9-fold reduction of D to obtain D_{TOA, tissue} is probably much too small. If OA diffuses around, rather than through, the epidermal cells, then the area through which OA must pass will be closer to 1% of the total surface area. If c_TOA is to rise to the estimated required values (Delhaize et al., 1993b; Parker and Pedler, 1998), D_{TOA, tissue} must be a similarly small fraction of D. Furthermore, apoplastic pH just beneath the epidermis may not rise to 5.82. If these adjustments are made, D_{TOA, tissue} = D_{Al3+, tissue} = D/72 and D_{H, tissue} = D_H/6.7, then a_Al3+ = 0.290 µM, pH = 5.10, and c_{TOA, tissue} = 201 µM, 30 µm into the root of Atlas wheat.

What would be the subepidermal conditions in Scout exposed to the same medium as Atlas or to a medium containing 1/16 the Al (and therefore intoxicated similarly to Atlas)? In order to estimate that, a similar pH profile, but no secretion of OA, was assumed. Under those conditions a_Al3+ = 4.56 µM (Fig. 6, top curve) or 0.286 µM (Fig. 6, bottom curve), 30 µm into the root of Scout wheat.

Finally, it was considered whether the outer cell walls of the epidermis could themselves provide a substantial protective barrier. If they could, then it would not be necessary to propose, as we shall, that the epidermal cells are either more resistant to Al³⁺ than other tissues or that the epidermis plays a minor role in whatever events, essential for growth, are disrupted by Al³⁺. According to these calculations, a 2 µm thick cell wall would need diffusion coefficients for OA and Al³⁺ that were nearly 1000 times smaller than those in water, if the pH at the inner face of the cell walls were allowed to rise to 5.5 (i.e. D_H 119-fold smaller than its value in water). Nobel (1991), in his calculations of diffusion across cell walls assumed a thickness of 1 µm and a 5-fold decrease in the value of diffusion coefficients.

Experimental
Variable agitation:
The results of the modelling inspired experiments in which Atlas wheat seedlings were cultured so as to reduce the length of the diffusion pathway. The reduced path length will increase the a_{Al3+, root surf} (Fig. 4A) but not necessarily increase the sensitivity to a_{Al3+, medium} (Fig. 4D). In five experiments the concentrations of AlCl₃ ranged from 0 to 200 µM in 1 mM CaCl₂ and 5320 µM NaCl at pH 4.3 or 4.5. Aeration treatments were no aeration, gentle aeration, and vigorous aeration and were assigned the categorical values 0, 1, or 2. Root lengths for the pooled data conformed to this equation.

(11)

where a_{Al3+, medium} is expressed in µM, and RL_S, defined in the Materials and methods, is specific for each experiment, and the expression (37.3/(1+0.265Aeration)) is equivalent to RL_C, the Al-free control. Thus aeration was moderately inhibitory to root elongation in the absence of Al so that the 2-d elongation was 37.3 mm in still solution and 24.4 mm in vigorously aerated solution. However, aeration had no effect on Al³⁺ sensitivity. That is, the strength coefficient, 0.0385, was unaffected by aeration.

Figure 7A presents relative root lengths in response to a_{Al3+, medium} for the pooled data. The drawn curve conforms to Equation 2 for which p = 0.0416 and q = 0.734. Aeration could not be incorporated into this equation in a statistically significant way, and Fig. 7B illustrates the absence of an effect upon Al³⁺-sensitive elongation.

View larger version (13K): [in this window] [in a new window]   Fig. 7. Relative root length of Atlas wheat in response to Al3+ activity and aeration. The values 0, 1, and 2 refer to no aeration, gentle aeration, and vigorous aeration, respectively.

 
For readers who fear that something may be hidden by the use of RRLs, or question the incorporation of aeration into the computation of RL_C, unadjusted RL versus a_{Al3+, medium} was analysed over the interval of 0–10 µM, where the relationship is linear. For the equation

(12)

a_{Al3+, medium} is expressed in µM, and the coefficient a was positive and significant and coefficients b and d were negative and significant for the pooled data and for each experiment individually. The coefficient c was never significantly negative, thereby indicating no aeration-enhanced sensitivity to Al³⁺.

To confirm that aeration does reduce the unstirred layer, and presumably the length of the diffusion pathway, two simple experiments were performed. Strips of dye-soaked paper were suspended in unaerated and aerated water. The dye washed out of the strips in the aerated water much more rapidly than in the unaerated water. In addition, a cube of ice melted much faster in the aerated water.

Removal of mucilage:
If the length of the external diffusion pathway is determined by mucilage, then the model predicts that removal of mucilage will cause an increase in a_{Al3+, root surf}, but not necessarily an increase in sensitivity to a_{Al3+, medium} as noted above. To investigate that proposition, three experiments were performed. Culture solutions contained variable concentrations of AlCl₃ and other solutes. Within each experiment the solutions were prepared in duplicate so that the roots for half the solutions could be wiped free of mucilage, under magnification, three times daily.

Datum points in Fig. 8 are scattered. All data are presented in the interests of objectivity, but the reason for the five low values represented by the open circles across the bottom of Fig. 8A is now known. They refer to the first experiment in which mucilage was removed. In that experiment, and to a lesser degree in the second experiment, removal of mucilage appeared to injure the root tips generally. Eventually mucilage was removed so as to avoid root injury, as described in the Materials and methods.

View larger version (13K): [in this window] [in a new window]   Fig. 8. Relative root length of Atlas wheat in response to Al3+ activity and the removal of mucilage.

 
With or without general injury, removal of mucilage failed to enhance sensitivity to Al³⁺. Whether the experiments were analysed individually or collectively, whether the data were analysed over the entire range of a_{Al3+, medium} (Fig. 8A) or the region of linear response (Fig. 8B), or whether the data were analysed in terms of RL or RRL, roots with intact mucilage were just as sensitive to Al³⁺ as the mucilage-free roots. This equation was applied to the pooled data.

(13)

where RL_S was specific for each experiment, and Mucilage was assigned the value 0 (not removed) or 1 (removed). The coefficients a, f, p, and q were positive and significant, indicating that removal of mucilage reduced root elongation (at least in two of three experiments); and g was non-significant, indicating that removal of mucilage did not enhance Al³⁺ sensitivity. Previously published results regarding the protective effect of mucilage are mixed (Horst et al., 1982; Li et al., 2000). Removal of the mucilage-secreting root cap of maize had no effect upon sensitivity (Ryan et al., 1993).

Al³⁺ buffering
Experiments were performed to test the effects of Al³⁺ buffering capacity upon Atlas seedlings. Two experiments included variable concentrations of AlCl₃, Na₂SO₄ (0 or 2000 µM), and NaCl (5320 or 0 µM) in a background of 1 mM CaCl₂ at pH 4.3. Two additional experiments included variable concentrations of AlCl₃, succinic acid (0–4000 µM), and NaCl (approximately 4000–0 µM) in a background of 1 mM CaCl₂ at pH 4.5. Succinate was used because the possible formation of jurbanite (AlSO₄OH.5H₂O) imposes a limit to the combined activities of Al³⁺ and (Nordstrom, 1982), but the use of succinate is problematic as well because of the pH buffering that will reduce root surface pH and thereby reduce the effects of increased Al³⁺ as noted in the text relating to Figs 4 and 5A.

Figure 9A presents relative root lengths in response to a_{Al3+, medium} for the pooled data. The drawn curve conforms to Equation 2 for which p = 0.0395 and q = 0.737. Al³⁺ buffering capacity could not be incorporated into this or other equations in a statistically significant way. Figure 9B illustrates the absence of an effect of Al³⁺ buffering upon root elongation.

View larger version (14K): [in this window] [in a new window]   Fig. 9. Relative root length of Atlas wheat in response to Al3+ activity and Al3+ buffering capacity.

 
Co-ordinated growth and staining experiments
A 50% inhibition of root elongation was observed in Atlas in a medium containing 50 µM AlCl₃ in 1 mM CaCl₂ at pH 4.5 (a_{Al3+, medium} = 22.3 µM), but Scout was inhibited 50% in 3 µM AlCl₃ (a_{Al3+, medium} = 1.36 µM). According to our computations, the a_{Al3+, root surf} was 6.88 µM for Atlas and 0.609 µM for Scout. It was considered that these greatly differing values for a_{Al3+, root surf} would be observable using the stain haematoxylin. Specifically, it was considered that the root surface of Atlas would stain darkly relative to Scout and thereby confirm the modelled inability of Atlas (inhibited 50%) to reduce a_{Al3+, root surf} to the activities that inhibit Scout 50%.

Figure 10 illustrates that these expectations were met. For Scout, neither the root surface, nor the root cap, nor the epidermal cells stained appreciably. The cell walls of the interior tissues did stain with a particularly abrupt transition from unstained to stained just behind the root cap and at the outer cortex, but nowhere were nuclei seen to stain. For Atlas, the cortex stained weakly, but the root surface and the root cap stained much more intensely. Staining was particularly intense in the nuclei of the border cells of the root cap and surface cells in the region of transition between cap and main root. Hand-cut longitudinal sections through the tip confirmed the surface staining in Atlas and the subsurface staining in Scout. At Al concentrations sufficient to inhibit Atlas more than 50% but less than 100% (e.g. 100 µM) some cortical staining was observed, but the surface staining was always more intense. When Scout was very severely inhibited (Al >6 µM) staining of the surface and of nuclei does occur, but the cortex was always stained more intensely than the epidermis.

View larger version (68K): [in this window] [in a new window]   Fig. 10. Haematoxylin stained root tips of Atlas and Scout wheat. Atlas roots were exposed to 50 (A) or 60 (C) µM AlCl3 in 1 mM CaCl2 at pH 4.5. Scout roots (B, D) were exposed to 3 µM AlCl3 in 1 mM CaCl2 at pH 4.5. These media induced approximately 50% inhibition of root elongation in each case, and that may explain why the Atlas roots in this figure are somewhat thicker than the zero-Al root represented in Fig. 1. These photographs are representative in that surface staining was always more intense than interior staining in Atlas, while the opposite was true for Scout, irrespective of Al concentration.

 
For Atlas and Scout the anatomical responses in the epidermis to 50% inhibition were quantitatively different. Whether Al intoxicated or not, the surface of the first 2–3 mm of the root was smooth and the cells were small and tightly packed. In non-intoxicated roots the cells farther back than 2–3 mm from the tip enlarged in a typical elongated pattern, and the surface remained smooth except for the developing hairs. In roots expressing 50% inhibition (50 µM AlCl₃ for Atlas, 3 µM AlCl₃ for Scout), the cells farther than 2–3 mm from the tip became swollen, especially in the epidermis, but these effects were greater for Atlas than for Scout. These anatomical effects, together with the results of staining, indicate greater a_{Al3+, root surf} for Atlas than for Scout at 50% inhibition.

	Discussion

Top Abstract Introduction Materials and methods Results Discussion References

 
The authors accept that Al resistance is almost certainly related to OA secretion, but the idea that OA secretion reduces a_{Al3+, root surf} to very small values (comparable to the <1 µM values tolerated by the non-secreting sensitive genotypes) is probably wrong. Neither modelling nor experimentation supports that view. Apparently, the resistance mechanism requires a component other than (the apparently inadequate) reduction of a_{Al3+, root surf}. We propose that the additional component is a substantial reduction in a_Al3+ across the epidermis, caused by a resistance to OA diffusion through the epidermis that is much greater than the resistance through the unstirred layer. This increased resistance allows the OA concentration to increase to the needed values (about 200 µM). Thus the a_Al3+ in the free space of the cortex will be small (<1 µM) for both resistant and sensitive genotypes when each is incubated in solutions causing equal and modest inhibitions of growth (see Fig. 6).

Our ‘biphasic diffusion hypothesis’ for Al resistance embodies several implications about the nature of Al intoxication. First is the implication that the site of Al action lies principally in the cortex, or root interior, and not in the epidermis. Readers are referred to a growing body of literature supporting the hypothesis that Al³⁺ acts in the transition zone of the root (lying between the meristematic zone and the elongation zone) to inhibit elongation in the elongation zone (Ryan et al., 1993; Hasenstein and Evans, 1988; Sivaguru and Horst, 1998; Kollmeier et al., 2000, and reviewed in Baluka et al., 2001). The elongation zone, when it alone was exposed to Al³⁺, continues to elongate. A candidate for the Al-initiated effect in the transition zone is the inhibited translocation of auxin through the cortex into the elongation zone from the meristematic zone into which the auxin had been transported through the phloem from the shoot. It strikes the authors as more than likely that exposure of the cortex to Al³⁺ is more important to the inhibition of auxin transport through the cortex than is exposure of the outer surface of the epidermis. An intriguing observation may relate to this proposition: the outermost cortical cells, rather than the epidermal cells, are the most sensitive to disruption of microtubules by Al³⁺ (Sivaguru et al., 1999).

There is no direct evidence that epidermal cells are not involved in the train of events leading to inhibited root elongation, but in many respects these cells, and others, appear to be individually resistant to Al³⁺. a_Al3+ sufficient to inhibit elongation causes epidermal cells behind the transition zone to swell, whether applied to whole roots or to the zone of elongation alone, in which case elongation continues but swelling occurs (Ryan et al., 1993). These swollen cells exhibit vigorous cytoplasmic streaming and typical negative transmembrane potential differences that hyperpolarize normally in response to stimulated proton extrusion (Kinraide, 1988). That is, these cells appear to be healthy in many respects and capable of enlargement, albeit disordered, to the lengths they would have achieved in the absence of Al. Thus the anatomical changes (e.g. swelling and altered microtubule orientation) may be local effects and may not be related to the inhibition of root elongation. Furthermore, the fact that Atlas (inhibited 50%) exhibits greater anatomical lesions in the epidermis than Scout (inhibited 50%) may be indicative of a small role for Al intoxication of the epidermis in the inhibition of root elongation.

Why, at 50% inhibition of root elongation, does the interior of Scout roots stain while the surface does not? Before providing an interpretation here, readers are directed to several previous studies of Al localization in roots that indicate a greater accumulation of Al in the cortex than in the epidermis of sensitive genotypes incubated in moderate to low concentrations of Al. Compare Fig. 8A to Fig. 8B and Fig. 9A to Fig. 9B in Delhaize et al. (1993a). Tracing the root surface in the A figures onto clear plastic and superimposing them onto the B figures is helpful. See also Fig. 6 in Kataoka et al. (1997); Figs 3 and 6 in Silva et al. (2000); Fig. 3 in Silva et al. (2001); Fig. 5d in Tice et al. (1992); and compare Fig. 3C and 3D with Figs 3C' and 3D' in Ahn et al. (2002). It is concluded that the immature cortex has a greater capacity to accumulate Al than other regions of the root. Perhaps the cell walls there contain more binding sites for Al³⁺ or that Al³⁺ enters the cell interiors more readily. It is proposed here that it is the cortex, rather than the root surface, that must be protected from Al³⁺.

A second implication of the ‘biphasic diffusion hypothesis’ is that resistant and sensitive genotypes (at least in wheat) have similar intrinsic sensitivities to Al³⁺. That is, both are similarly intoxicated by a given a_Al3+ in the sensitive region of the root. If the sensitive region is the root surface, then a_{Al3+, root surf} must be similarly small (<1 µM) for Scout and Atlas, but if the sensitive region is the root interior, then a_{Al3+, root surf} may be different for the two cultivars.

Some support for similar intrinsic sensitivities is provided by Samuels et al. (1997) who measured growth responses and Al uptake by Al-resistant Atlas and Al-sensitive Tam 105 cultivars of wheat. For the former, 105 µM AlCl₃ in the medium inhibited growth 50% and caused a tissue accumulation of 377 µg Al g^–1 root DW. For the latter, 11 µM AlCl₃ in the medium inhibited growth 50% and caused a tissue accumulation of 546 µg Al g^–1 DW. These values (377 and 546) may indicate that Atlas actually has a greater intrinsic sensitivity than Tam. Further support is presented by Tice et al. (1992) from whose data one may see that equal root-tip content of Al (in µmol g^–1 FW) results in equal inhibition in the resistant Yecora Rojo and the sensitive Tyler cultivars of wheat.

A third implication of the ‘biphasic diffusion hypothesis’ is that the metabolic cost of Al resistance is small relative to the popularly assumed alternative. Only one-tenth the amount of OA need be synthesized to maintain adequate concentrations of OA beneath the epidermis, compared with the amount needed to maintain similar concentrations at the root surface.

How may the ‘biphasic diffusion hypothesis’ be tested further? It is acknowledged that much of the argument is based upon modelling. Therefore, it is hoped that readers will examine the model critically, including this study's selection of parameter values and the extent to which they have been altered for variations of the standard run. The absence of direct measurements of root-surface OA and Al³⁺ concentrations is also acknowledged. Eventually, these measurements must be obtained. It is thought that the experiments with removal of mucilage, agitation of the medium, and Al³⁺ buffering all provided results consistent with the biphasic diffusion hypothesis, but it is acknowledged that the complex interaction of Al³⁺, H⁺, and cell-surface electrical charges makes an interpretation of those experiments difficult (refer to Fig. 4 and related text). In the authors' view, the haematoxylin staining experiments (as well as the observations of anatomical effects) provide the best experimental complement to the modelling because they are a rough, qualitative measure of a_{Al3+, root surf}. Nevertheless, further anatomical studies and studies to localize tissue Al, especially at root surfaces, may be productive. Finally, the sufficiency of the outer cell walls of the epidermis (together with closely adhering mucilage not easily removed by wiping) as an effective barrier cannot be entirely ruled out, although it is considered to be an unlikely prospect.

	Acknowledgements

 
We wish to thank Frantisek Baluka, Pax Blamey, Walter Horst, Peter Ryan, Mayandi Sivaguru, and Peter Wenzl for reading the manuscript and making many helpful suggestions.

	Footnotes

 
Abbreviations: a_{Al3+, root surf} and a_{Al3+, PM}, Al³⁺ activity at the root surface and at the plasma membrane surface, respectively; OA, organic acid; OAAl, organic acid–Al complexes; PM, plasma membrane; RL, root length; RRL, relative root lengths.

	References

Top Abstract Introduction Materials and methods Results Discussion References

 
Ahn SJ, Sivaguru M, Chung GC, Rengel Z, Matsumoto H. 2002. Aluminium-induced growth inhibition is associated with impaired efflux and influx of H⁺ across the plasma membrane in root apices of squash (Cucurbita pepo). Journal of Experimental Botany 53, 1959–1966.[Abstract/Free Full Text]

Baluka F, Volkmann D, Barlow PW. 2001. A polarity crossroad in the transition growth zone of maize root apices: cytoskeletal and developmental implications. Journal of Plant Growth Regulation 20, 170–181.[CrossRef][Web of Science]

Degenhardt J, Larsen PB, Howell SH, Kochian LV. 1998. Aluminium resistance in the Arabidopsis mutant alr-104 is caused by an aluminium-induced increase in rhizosphere pH. Plant Physiology 117, 19–27.[Abstract/Free Full Text]

Delhaize E, Craig S, Beaton CD, Bennet RJ, Jagadish VC, Randall PJ. 1993a. Aluminium tolerance in wheat (Triticum aestivum L.). I. Uptake and distribution of aluminium in root apices. Plant Physiology 103, 685–693.[Abstract]

Delhaize E, Ryan PR, Hebb DM, Yamamoto Y, Sasaki T, Matsumoto H. 2004. Engineering high-level aluminium tolerance in barley with ALTM1 the gene. Proceedings of the National Academy of Sciences, USA 101, 15249–15254.[Abstract/Free Full Text]

Delhaize E, Ryan PR, Randall PJ. 1993b. Aluminium tolerance in wheat (Triticum aestivum L.). II. Aluminium-stimulated excretion of malic acid from root apices. Plant Physiology 103, 695–702.[Abstract]

Gimmler H, de Jesus J, Greiser A. 2001. Heavy metal resistance of the extreme acidotolerant filamentous fungus Bispora sp. Microbial Ecology 42, 87–98.[Web of Science][Medline]

Hasenstein KH, Evans ML. 1988. Effects of cations on hormone transport in primary roots of Zea mays. Plant Physiology 86, 890–894.[Abstract/Free Full Text]

Horst WJ, Wagner A, Marschner H. 1982. Mucilage protects roots from aluminium injury. Zeitschrift für Pflanzenphysiologie 105, 435–444.

Kataoka T, Mori M, Nakanishi TM, Matsumoto S, Uchiumi A. 1997. Highly sensitive analytical method for aluminium movement in soybean root through lumogallion staining. Journal of Plant Research 110, 305–309.[CrossRef]

Kinraide TB. 1997. Reconsidering the rhizotoxicity of hydroxyl, sulphate, and fluoride complexes of aluminium. Journal of Experimental Botany 48, 1115–1124.[Abstract/Free Full Text]

Kinraide TB. 1988. Proton extrusion by wheat roots exhibiting severe aluminium toxicity symptoms. Plant Physiology 88, 418–423.[Abstract/Free Full Text]

Kinraide TB. 2003a. The controlling influence of cell-surface electrical potential on the uptake and toxicity of selenate (). Physiologia Plantarum 117, 64–71.[CrossRef]

Kinraide TB. 2003b. Toxicity factors in acidic forest soils: attempts to evaluate separately the toxic effects of excessive Al³⁺and H⁺ and insufficient Ca²⁺ and Mg²⁺ upon root elongation. European Journal of Soil Science 54, 323–333.[CrossRef][Web of Science]

Kochian LV, Hoekenga OA, Piñeros MA. 2004. How do crop plants tolerate acid soils? Mechanisms of aluminium tolerance and phosphorus efficiency. Annual Review of Plant Biology 55, 459–493.[CrossRef][Medline]

Kollmeier M, Felle HH, Horst WJ. 2000. Genotypical differences in aluminium resistance of maize are expressed in the distal part of the transition zone. Is reduced basipetal auxin flow involved in inhibition of root elongation by aluminium? Plant Physiology 122, 945–956.[Abstract/Free Full Text]

Li XF, Ma JF, Hiradate S, Matsumoto H. 2000. Mucilage strongly binds aluminium but does not prevent roots from aluminium injury in Zea mays. Physiologia Plantarum 108, 152–160.[CrossRef]

Lindsay WL. 1979. Chemical equilibrium in soils. New York: John Wiley and Sons.

Miyasaka SC, Buta JG, Howell RK, Foy CD. 1991. Mechanism of aluminium tolerance in snapbeans. Root exudation of citric acid. Plant Physiology 96, 737–743.[Abstract/Free Full Text]

Nian H, Yang ZM, Ahn SJ, Cheng ZJ, Matsumoto H. 2002. A comparative study on the aluminium- and copper-induced organic acid exudation from wheat roots. Physiologia Plantarum 116, 328–335.[CrossRef]

Nobel PS. 1991. Physicochemical and environmental plant physiology. San Diego: Academic Press.

Nordstrom DK. 1982. The effect of sulphate on aluminium concentrations in natural waters: some stability relations in the system Al₂O₃-SO₃-H₂O at 298 K. Geochimica et Cosmochimica Acta 46, 681–692.[CrossRef][Web of Science]

Nordstrom DK, May HM. 1989. Aqueous equilibrium data for mononuclear aluminium species. In: Sposito G, ed. The environmental chemistry of aluminum. Boca Raton, FL: CRC Press, 29–53.

Ojima K, Miyazawa M, Sasaki M, Ohira K. 1987. Exudation of malic acid in germfree culture of an aluminium-resistant variety of wheat. Journal of Germfree Life and Gnotobiology 17, 363–366 (in Japanese).

Papernik LA, Bethea AS, Singleton TE, Magalhaes JV, Garvin DF, Kochian LV. 2001. Physiological basis of reduced Al tolerance in ditelosomic lines of Chinese Spring wheat. Planta 212, 829–834.[CrossRef][Web of Science][Medline]

Parker DR, Pedler JF. 1998. Probing the ‘malate hypothesis’ of differential aluminium tolerance in wheat by using other rhizotoxic ions as proxies for Al. Planta 205, 389–396.[CrossRef]

Parker DR, Reichman SM, Crowley DE. 2005. Metal chelation in the rhizosphere. In: Zobel RW, Wright SF, eds. Root effects on the rhizosphere. ASA Monograph Series, Madison, WI (in press).

Pellet DM, Papernik LA, Kochian LV. 1996. Multiple aluminium-resistance mechanisms in wheat. Roles of root apical phosphate and malate exudation. Plant Physiology 112, 591–597.[Abstract]

Piñeros MA, Shaff JE, Manslank HS, Alves VMC, Kochian LV. 2005. Aluminium resistance in maize cannot be solely explained by root organic acid exudation. A comparative physiological study. Plant Physiology 137, 231–241.[Abstract/Free Full Text]

Puthota V, Cruz-Ortega R, Johnson J, Ownby J. 1991. An ultrastructural study of the inhibition of mucilage secretion in the wheat cap by aluminium. In: Wright RJ, Baligar VC, Murrmann RP, eds. Plant–soil interaction at low pH. Dordrecht: Kluwer Academic Publishers, 779–787.

Ryan PR, Delhaize E, Jones DL. 2001. Function and mechanism of organic anion exudation from plant roots. Annual Review of Plant Physiology and Molecular Biology 52, 527–560.[CrossRef][Web of Science][Medline]

Ryan PR, Delhaize E, Randall PJ. 1995a. Characterization of Al-stimulated efflux of malate from the apices of Al-tolerant wheat roots. Planta 196, 103–110.[Web of Science]

Ryan PR, Delhaize E, Randall PJ. 1995b. Malate efflux from root apices and tolerance to aluminium are highly correlated in wheat. Australian Journal of Plant Physiology 22, 531–536.[Web of Science]

Ryan PR, Ditomaso JM, Kochian LV. 1993. Aluminium toxicity in roots: an investigation of spatial sensitivity and the role of the root cap. Journal of Experimental Botany 44, 437–446.[Abstract/Free Full Text]

Ryan PR, Shaff JE, Kochian LV. 1992. Aluminium toxicity in roots. Correlation among ionic currents, ion fluxes, and root elongation in aluminium-sensitive and aluminium-tolerant cultivars. Plant Physiology 99, 1193–1200.[Abstract/Free Full Text]

Samuels TD, Küçükakyüz K, Rincón-Zachary M. 1997. Al partitioning patterns and root growth as related to Al sensitivity and Al tolerance in wheat. Plant Physiology 113, 527–534.[Abstract]

Sasaki T, Yamamoto Y, Ezaki B, Katsuhara M, Ahn SJ, Ryan PR, Delhaize E, Matsumoto H. 2004. A wheat gene encoding an aluminium-activated malate transporter. The Plant Journal 37, 64–653.

Silva IR, Smyth TJ, Moxley DF, Carter TE, Allen NS, Rufty TW. 2000. Aluminium accumulation at nuclei of cells in the root tip. Fluorescence detection using lumogallion and confocal laser scanning microscopy. Plant Physiology 123, 543–552.[Abstract/Free Full Text]

Silva IR, Smyth TJ, Raper CD, Carter TE, Rufty TW. 2001. Differential aluminium tolerance in soybean: an evaluation of the role of organic acids. Physiologia Plantarum 112, 200–210.[CrossRef][Medline]

Sivaguru M, Baluka F, Volkman D, Felle HH, Horst W. 1999. Impacts of aluminium on the cytoskeleton of the maize root apex. Short-term effects on the distal part of the transition zone. Plant Physiology 119, 1073–1082.[Abstract/Free Full Text]

Sivaguru M, Horst W. 1998. The distal part of the transition zone is the most aluminium-sensitive apical root zone of maize. Plant Physiology 116, 155–163.[Abstract/Free Full Text]

Tice KR, Parker DR, DeMason D. 1992. Operationally defined apoplastic and symplastic aluminium fractions in root tips of aluminium-intoxicated wheat. Plant Physiology 100, 309–318.[Abstract/Free Full Text]

Wolterbeek HT. 1987. Determination of the surface area of fine tomato roots via cation uptake experiments. Physiologia Plantarum 69, 704–708.[CrossRef]

Yermiyahu U, Rytwo G, Brauer DK, Kinraide TB. 1997. Binding and electrostatic attraction of lanthanum (La³⁺) and aluminium (Al³⁺) to wheat root plasma membranes. Journal of Membrane Biology 159, 239–252.[CrossRef][Web of Science][Medline]

Weast RC. 1990. CRC handbook of chemistry and physics. Boca Raton, Florida: CRC Press.

Zhang Q, Smith FA, Sekimoto H, Reid RJ. 2001. Effect of membrane surface charge on nickel uptake by purified mung bean root protoplasts. Planta 213, 788–793.[CrossRef][Web of Science][Medline]

CiteULike    Connotea    Del.icio.us    What's this?

This article has been cited by other articles:

				P. R. Ryan, H. Raman, S. Gupta, W. J. Horst, and E. Delhaize A Second Mechanism for Aluminum Resistance in Wheat Relies on the Constitutive Efflux of Citrate from Roots Plant Physiology, January 1, 2009; 149(1): 340 - 351. [Abstract] [Full Text] [PDF]

				Y. Kobayashi, O. A. Hoekenga, H. Itoh, M. Nakashima, S. Saito, J. E. Shaff, L. G. Maron, M. A. Pineros, L. V. Kochian, and H. Koyama Characterization of AtALMT1 Expression in Aluminum-Inducible Malate Release and Its Role for Rhizotoxic Stress Tolerance in Arabidopsis Plant Physiology, November 1, 2007; 145(3): 843 - 852. [Abstract] [Full Text] [PDF]

				A Stass, Y Wang, D Eticha, and W. Horst Aluminium rhizotoxicity in maize grown in solutions with Al3+ or Al(OH)4- as predominant solution Al species J. Exp. Bot., December 1, 2006; 57(15): 4033 - 4042. [Abstract] [Full Text] [PDF]

				A. Ligaba, M. Katsuhara, P. R. Ryan, M. Shibasaka, and H. Matsumoto The BnALMT1 and BnALMT2 Genes from Rape Encode Aluminum-Activated Malate Transporters That Enhance the Aluminum Resistance of Plant Cells Plant Physiology, November 1, 2006; 142(3): 1294 - 1303. [Abstract] [Full Text] [PDF]

				T. Sasaki, P. R. Ryan, E. Delhaize, D. M. Hebb, Y. Ogihara, K. Kawaura, K. Noda, T. Kojima, A. Toyoda, H. Matsumoto, et al. Sequence Upstream of the Wheat (Triticum aestivum L.) ALMT1 Gene and its Relationship to Aluminum Resistance Plant Cell Physiol., October 1, 2006; 47(10): 1343 - 1354. [Abstract] [Full Text] [PDF]

				M. WATT, W. K. SILK, and J. B. PASSIOURA Rates of Root and Organism Growth, Soil Conditions, and Temporal and Spatial Development of the Rhizosphere Ann. Bot., May 1, 2006; 97(5): 839 - 855. [Abstract] [Full Text] [PDF]

This Article Abstract FREE Full Text (PDF) OA All Versions of this Article: 56/417/1853    most recent eri175v1 E-letters: Submit a response Alert me when this article is cited Alert me when E-letters are posted Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in ISI Web of Science Similar articles in PubMed Alert me to new issues of the journal Add to My Personal Archive Download to citation manager Search for citing articles in: ISI Web of Science (21) Disclaimer Google Scholar Articles by Kinraide, T. B. Articles by Zobel, R. W. Search for Related Content PubMed PubMed Citation Articles by Kinraide, T. B. Articles by Zobel, R. W. Agricola Articles by Kinraide, T. B. Articles by Zobel, R. W. Social Bookmarking          What's this?

Online ISSN 1460-2431 - Print ISSN 0022-0957

Copyright © 2005 Society for Experimental Biology

Oxford Journals Oxford University Press

• Site Map
• Privacy Policy
• Frequently Asked Questions

Other Oxford University Press sites: Oxford University Press Oxford Journals China Oxford Journals Japan American National Biography Booksellers' Information Service Children's Fiction and Poetry Children's Reference Corporate & Special Sales Dictionaries Dictionary of National Biography Digital Reference English Language Teaching Higher Education Textbooks Humanities International Education Unit Law Medicine Music Online Products Oxford English Dictionary Reference Rights and Permissions Science School Books Social Sciences Very Short Introductions World's Classics