Sorption of Aluminum to Plasma Membrane Vesicles Isolated from Roots of Scout 66 and Atlas 66 Cultivars of Wheat

To further elucidate the mechanisms of differential genotypic tolerance to Al, plasma membrane (PM) vesicles were isolated from whole roots, root tips, and tipless roots of Al3+-sensitive and Al3+-tolerant cultivars (cv) of wheat (Triticum aestivum L. cv Scout 66 and cv Atlas 66, respectively). Vesicles from cv Scout root tips sorbed more Al than vesicles prepared from any other source. The intrinsic surface-charge density of vesicles isolated from cv Scout was 26% more negative than vesicles from cv Atlas (-37.2 versus -29.5 millicoulombs m-2). Growth experiments indicated that cv Scout is slightly more sensitive to La3+ than is cv Atlas, that the cultivars are equally sensitive to H+, and that cv Atlas is slightly more sensitive to SeO42-. The difference in sensitivity to Al3+ was very large; for a 50% inhibition, a 16-fold greater activity of Al3+ was required for cv Atlas. Using a newly developed Gouy-Chapman-Stern model for ion sorption to the PM together with growth-response curves, we estimate that the difference in surface-charge density can account for the slightly greater sensitivity of cv Scout to cationic toxicants and the slightly greater sensitivity of cv Atlas to anionic toxicants. According to our estimates the differences in PM surface negativity and Al sorptive capacity probably account for some of the difference in sensitivity to Al3+, but the greater part of the difference probably arises from other tolerance mechanisms expressed in cv Atlas root tips that reduce the amount of Al3+ that can reach the PM.     

Aluminum Partitioning in Intact Roots of Aluminum-Tolerant and Aluminum-Sensitive Wheat (Triticum aestivum L.) Cultivars

Aluminum (Al) partitioning in intact roots of wheat (Triticum aestivum L.) cultivars that differ in sensitivity to Al was investigated. Roots of intact seedlings were exposed to Al for up to 24 hours and distribution of Al was assessed visually by hematoxylin staining or by direct measurement of concentration of Al by atomic absorption spectrophotometry or ion chromatography. Major differences in Al accumulation between Al-tolerant (Atlas 66) and Al-sensitive (Tam 105) cultivars were found in the growing regions 0 to 2 and 2 to 5 millimeters from the root apex. Al content was 9 to 13 times greater in the 0 to 2 millimeters root tips of cv Tam 105 than in the tips of cv Atlas 66 when exposed to 50 micromolar Al for 19 to 24 hours. The oxidative phosphorylation inhibitor carbonyl cyanide m-chlorophenylhydrazone and the protein synthesis inhibitor cycloheximide increased Al uptake by intact root tips of cv Atlas 66. Also, loss of Al from the roots of both cultivars was measured after the roots were “pulsed” with 50 micromolar Al for 2 hours and then placed in an Al-free nutrient solution for 6 hours. The 0 to 2 millimeter root tips of cv Tam 105 lost 30% of the absorbed Al, whereas the tips of cv Atlas 66 lost 60%. In light of these results, we conclude that the differential Al sensitivity in wheat correlates with the concentration of Al in the root meristems. The data support the hypothesis that part of the mechanism for Al tolerance in wheat is based on a metabolism-dependent exclusion of Al from the sensitive meristems.     

Calcium Transport in Membrane Vesicles Isolated from Maize Coleoptiles (Effect of Indoleacetic Acid and Fusicoccin)

Maize (Zea mays L.) coleoptile segments loaded with 45Ca released about 50% of the ion after 1 h when treated with indoleacetic acid (IAA). In contrast, fusicoccin (FC) had no effect. The same relation was found when ATP-dependent Ca2+ transport, measured as 45Ca uptake, was determined in a plasmalemma-rich membrane vesicle fraction isolated from coleoptiles treated or untreated for 1 h with IAA or FC. In fact, IAA-treated membranes showed an increase in ATP-dependent 45Ca uptake by more than 30% with respect to the control and the FC treatment. Ca2+ uptake in IAA-treated membranes was only slightly affected (+27%) by supplying calmodulin (Cam) exogenously. However, Ca2+ uptake in membranes from the control and FC-treated coleoptiles were stimulated (+80%) by exogenous Cam. Calmidazolium, a Cam antagonist, inhibited Ca2+ uptake in the IAA treatment (-48%) to a greater extent with respect to the control and FC treatment (-33 and -29%, respectively). A possible relationship between the effect of IAA on the ATP-dependent Ca2+ transport activity, the involvement of Cam, and their effect on growth are discussed.     

The Fructose Transport Mechanism in Microsomal Membrane Vesicles from Rye Roots

In a fructose-containing medium in which rye root-microsomal membrane vesicles had reached the equilibrium of uptake of fructose, the presence of both Mg²⁺ and ATP caused the efflux of fructose from the vesicles. Among nucleotides examined, ATP caused the largest efflux of fructose. The efflux of fructose dependent on Mg²⁺ and ATP was quite insensitive to a protonophore, carbonylcyanide m-chlorophenylhydrazone (CCCP), which actually abolished MgATP-dependent proton accumulation in the vesicles, while it was largely inhibited by vanadate, which inhibits ATP-binding cassette transporters (ABCTs). The Michaelis-Menten constant (Km) of the efflux of fructose was 0.4 mM. It was observed that fructose stimulated the ATPase activity of the vesicles and that vanadate markedly decreased the fructose-stimulated ATPase activity. This revised version was published online in July 2006 with corrections to the Cover Date.     

Polyamine Binding to Plasma Membrane Vesicles Isolated from Zucchini Hypocotyls

The general features of [14C]spermidine binding to plasmalemma vesicles isolated from zucchini (Cucurbita pepo L.) etiolated hypocotyls are reported in the present paper. The specific interaction of the polyamine with the plasma membranes was reversible and thermolabile, since it decreased by about 50% in the assay performed at 40[deg]C compared to that carried out on ice. On the contrary, nonspecific binding was unaffected by temperature. Specific spermidine binding showed a pH dependence with a maximum at pH 8.0 and it reached saturation between 0.75 and 1 mM external spermidine concentration. The value of the dissociation constant calculated from Scatchard analysis was 4.4 x 10-5 M. Specific spermidine interaction appeared to be sensitive to detergents and was markedly reduced by the presence of divalent cations, such as Mg2+ and Ca2+, whereas it was stimulated by monovalent cations. Polyamine binding sites were highly sensitive to pronase treatment. Competition experiments, performed using a series of compounds structurally related to spermidine, may provide some indication of the characteristics of spermidine binding sites. The results presented here suggest that specific spermidine binding occurs mainly with the protein component of the plasma membrane.     

Kinetics of Aluminum Uptake by Excised Roots of Aluminum-Tolerant and Aluminum-Sensitive Cultivars of Triticum aestivum L

Uptake of aluminum (Al) by excised roots of two Al-tolerant cultivars and two Al-sensitive cultivars of Triticum aestivum L. (wheat) was biphasic, with a rapid phase of uptake in the first 30 minutes followed by a linear phase of uptake up to 180 minutes. At the end of the uptake period, higher concentrations of Al were found in roots of the Al-sensitive cultivars (Neepawa and Scout-66) than in the Al-tolerant cultivars (Atlas-66 and PT-741), but differences were small. Experiments testing the effectiveness of several desorption agents demonstrated that citric acid was most effective in desorption of loosely bound Al (the putative apoplasmic compartment) followed by others in the order tartaric acid > EDTA > CaSO₄ = ScCl₃. In all cultivars, 30 minutes of desorption with citric acid depleted the rapidly exchanging, putative apoplasmic compartment, although some tightly bound Al remained in that compartment. The relationship between Al remaining after desorption and time in the uptake medium was nearly linear and no distinction was observed between Al-tolerant and Al-sensitive cultivars. However, uptake of Al by the Al-tolerant cultivars was increased by treatment with the protonophore 2,4-dinitrophenol (DNP), while uptake of Al by Al-sensitive cultivars was relatively unaffected. Such results suggest the possible involvement of an active exclusion mechanism in Al-tolerant cultivars of T. aestivum.     

大麦根质膜微囊的制备及其H~ ,Ca~(2 )转运活性

用蔗糖密度梯度离心法制备出密闭程度较高的大麦根细胞质膜微囊。喹吖咽荧光猝灭和~(45)Ca~(2 )同位素示踪测定表明所制备的微囊具H~ ,Ca~(2 )转运活性。对制备出的质膜制剂纯度和膜朝向进行了分析,并探讨了质膜纯化中影响膜微囊密闭性的因素。匀浆液和悬浮液巾的单价离子盐有利于密闭膜微囊的形成。蔗糖密度梯度和葡聚糖密度睇度离心法均可得到密闭性较高的膜微囊,但后者的纯化效果较差。     

Tryptophan Transport into Plasma Membrane Vesicles Derived from Rat Brain Synaptosomes

Abstract: Tryptophan uptake by membrane vesicles derived from rat brain was investigated. The uptake is dependent on the Na⁺ gradient [Na⁺] outside > [Na⁺] inside and is maximal when both Na⁺ and Cl⁻ are present. The uptake represents transport into an os-motically active space and not a binding artifact, as indicated by the effect of increasing the medium osmo-larity. The uptake of tryptophan is stimulated by a membrane potential (interior negative) as demonstrated by the effects of the ionophores valinomycin and carbonyl cyanide m-chlorophenylhydrazone and anions with different permeabilities. Kinetic data show that tryptophan is accumulated by two systems with different affinities. Ouabain, an inhibitor of Na⁺, K⁺-activated ATPase, does not affect tryptophan transport. The uptake of tryptophan is inhibited by high concentrations of phenylalanine, tyrosine, leucine and 3, 4-dihydroxyphenylalanine.     

L-Aspartate Transport into Plasma Membrane Vesicles Derived from Rat Brain Synaptosomes

Abstract: Aspartate uptake by membrane vesicles derived from rat brain was investigated. The uptake is dependent on a Na⁺ gradient ([Na⁺] outside > [Na⁺] inside). Active transport of aspartate is strictly dependent upon the presence of sodium and maximal extent of transport is reached when both Na⁺ and Cl⁻ ions are present. The uptake is transport into an osmotically active space and not a binding artifact as indicated by the effect of increasing the medium osmolarity. The uptake of aspartate is stimulated by a membrane potential (negative inside), as demonstrated by the effect of the ionophore carbonyl cyanide m -chlorophenylhydrazone and anions with different permeabilities. The presence of ouabain, an inhibitor of (Na⁺+ K⁺)-ATPase, does not affect aspartate transport. The kinetic analysis shows that aspartate is accumulated by two systems with different affinities, showing K _m and V _max values of similar order to those found in slightly "cruder" preparations. Inhibition of the l -aspartate uptake by d -aspartate and d - and l -glutamate indicates that a common carrier is involved in the process, this being stereospecific for the d - and l -glutamate stereoisomers.     

Surfactant-Increased Glyphosate Uptake into Plasma Membrane Vesicles Isolated from Common Lambsquarters Leaves

Plasma membrane vesicles were isolated from mature leaves of lambsquarters (Chenopodium album L.) to investigate whether this membrane is a barrier to glyphosate uptake and whether surfactants possess differential abilities to enhance glyphosate permeability. Amino acids representing several structural classes showed [delta]pH-dependent transport, indicating that the proteins necessary for active, proton-coupled amino acid transport were present and functional. Glyphosate uptake was very low compared to the acidic amino acid glutamate, indicating that glyphosate is not utilizing an endogenous amino acid carrier to enter the leaf cells and that the plasma membrane appears to be a significant barrier to cellular uptake. In addition, glyphosate flux was much lower than that measured for either bentazon or atrazine, both lipid-permeable herbicides that diffuse through the bilayer. Glyphosate uptake was stimulated by 0.01% (v:v) MON 0818, the cationic surfactant used in the commercial formulation of this herbicide for foliar application. This concentration of surfactant did not disrupt the integrity of the plasma membrane vesicles, as evidenced by the stability of imposed pH gradients and active amino acid transport. Nonionic surfactants that disrupt the cuticle but that do not promote glyphosate toxicity in the field also increased glyphosate transport into the membrane vesicles. Thus, no correlation was observed between whole plant toxicity and surfactant-aided uptake. Current data suggest that surfactant efficacy may be the result of charged surfactants' ability to diffuse away from the cuticle into the subtending apoplastic space, where they act directly on the plasma membrane to increase glyphosate uptake.     

Comparison of DABA and GABA Transport into Plasma Membrane Vesicles Derived from Synaptosomes

Abstract: Transport of GABA by a high-affinity transport system ( K _m≃ 10⁻⁵ M) is thought to terminate the action of this postulated neurotransmitter. 2,4-Diaminobutyric acid (DABA), a structural analogue, is taken up by neuronal elements and inhibits GABA uptake. Localization of [³H]DABA by auto-radiography has been used to identify neurons with the GABA high-affinity transport system. After reconstitution of lysed synaptosomal fractions in potassium salts, transfer of these membrane vesicles to sodium salts produces sodium and potassium ion gradients which drive [³H]GABA and [³H]DABA transport. For each, transport requires external sodium, is abolished by ionophores that dissipate the Na⁺ gradient, and is enhanced by conditions which make the intravesicular electromotive force more negative. Some characteristics of the transport of these substances, however, differ. For example, external chloride is required for GABA, but not DABA, transport. Internal potassium is required for DABA, but not GABA, transport. DABA is a competitive inhibitor ( K _i≃ 0.6 MM) of GABA transport into membrane vesicle and synaptosomes. GABA, however, is a feeble inhibitor of DABA uptake into the membrane vesicles. These differences suggest that the two substances are transported by different mechanisms and possibly by different carriers. In addition to these experiments, using enzymatic-fluorometric techniques, it was shown that the artificially imposed ion gradients drive net chemical transport of GABA into the vesicles.     

Effect of Zinc Deficiency on Proton Fluxes in Plasma Membrane-Enriched Vesicles Isolated from Bean Roots

Plasma membrane-enriched vesicles were isolated by density gradientcentrifugation from roots of zinc-sufficient and zinc-deficientbean (Phaseolus vulgaris L. cv. Prélude) plants. Thetwo populations of vesicles had similar activities of specificmembrane marker enzymes and ATP hydrolysis and were competentfor proton transport. However, vesicles from zinc-deficientroots showed lower rates of ATP-dependent intravesicular acidificationand increases in passive permeability to protons as well asin the rate, of dissipation of a non-metabolic transmembranepH gradient. The decrease of the rate of proton accumulationin isolated vesicles closely paralleled the increase in potassiumleakage from intact roots and the appearance of visual zincdeficiency symptoms in the shoots. Re-supply of zinc to deficientplants for 24 h promoted shoot growth, reduced potassium leakagefrom roots and led to partial recovery of the proton accumulationcapacity and to a decrease in passive permeability to protonsin isolated vesicles. The results obtained with isolated vesiclesconfirm the previously observed ‘in vivo’ effectsof zinc deficiency and are consistent with the idea that analteration of plasma membrane lipids leads to an increase inpermeability and an impairment in trans-plasma membrane protongradient. Key words: Plasma membrane, H⁺ fluxes, ATPase, zinc deficiency     

水稻幼苗根细胞质膜和液泡膜微囊Ca2+-ATP酶的特性

水稻幼苗根质膜和液泡膜Ca2 -ATP酶对ATP的Km值分别为7.1和4.5 μ mol·L-1;反应的最适pH分别为8.0和7.0.两者活性均受Na3VO4和曙红B(EB)抑制;CPZ抑制质膜Ca2 -ATP酶活性,但促进液泡膜Ca2 -ATP酶活性.30mmol·L-1CaCl2浸种和CaCl2浸种结合低温锻炼预处理,均可提高此酶的活性和冷稳定性.     

水稻幼苗根细胞质膜和液泡膜微囊Ca^2＋－ATP酶的特性

水稻幼苗根质膜和液泡膜Ca2+-ATP酶对ATP的Km值分别为7.1和4.5 μ mol·L-1;反应的最适pH分别为8.0和7.0.两者活性均受Na3VO4和曙红B(EB)抑制;CPZ抑制质膜Ca2+-ATP酶活性,但促进液泡膜Ca2+-ATP酶活性.30mmol·L-1CaCl2浸种和CaCl2浸种结合低温锻炼预处理,均可提高此酶的活性和冷稳定性.     

Evidence that Electrostatic Interactions between Vesicle-associated Membrane Protein 2 and Acidic Phospholipids May Modulate the Fusion of Transport Vesicles with the Plasma Membrane

The juxtamembrane domain of vesicle-associated membrane protein (VAMP) 2 (also known as synaptobrevin2) contains a conserved cluster of basic/hydrophobic residues that may play an important role in membrane fusion. Our measurements on peptides corresponding to this domain determine the electrostatic and hydrophobic energies by which this domain of VAMP2 could bind to the adjacent lipid bilayer in an insulin granule or other transport vesicle. Mutation of residues within the juxtamembrane domain that reduce the VAMP2 net positive charge, and thus its interaction with membranes, inhibits secretion of insulin granules in β cells. Increasing salt concentration in permeabilized cells, which reduces electrostatic interactions, also results in an inhibition of insulin secretion. Similarly, amphipathic weak bases (e.g., sphingosine) that reverse the negative electrostatic surface potential of a bilayer reverse membrane binding of the positively charged juxtamembrane domain of a reconstituted VAMP2 protein and inhibit membrane fusion. We propose a model in which the positively charged VAMP and syntaxin juxtamembrane regions facilitate fusion by bridging the negatively charged vesicle and plasma membrane leaflets.     

Membrane Transport in Isolated Vesicles from Sugarbeet Taproot: III. Effects of Fluoride on ATPase Activity and Transport

The effects of fluoride on the tonoplast type ATPase and transport activities associated with sealed membrane vesicles isolated from sugarbeet (Beta vulgaris L.) storage tissue were examined. This anion had two distinct effects upon the proton-pumping vesicles. When ATP hydrolysis was measured in the presence of gramicidin D, significant inhibition (approximately 50%) only occurred when the fluoride concentration approached 50 millimolar. In contrast, the same degree of inhibition of proton transport occurred when the fluoride concentration was about 24 millimolar. Effects on proton pumping at this concentration of fluoride could be attributed to an inhibition of chloride movement which serves to dissipate the vesicle membrane potential. Valinomycin could partially restore ATPase activity in sealed vesicles which were inhibited by fluoride and this restoration occurred with a reduction in the membrane potential. Fluoride demonstrated a competitive interaction with chloride-stimulation of proton transport and inhibited the uptake of radioactive chloride into sealed vesicles. When the vesicles were allowed to develop a pH gradient in the absence of KCl, and KCl was subsequently added, fluoride reduced enhancement of the existing pH gradient by KCl. The results are consistent with a chloride carrier that is inhibited by fluoride.     

Characterization of Ca Transport in Purified Endoplasmic Reticulum Membrane Vesicles from Lepidium sativum L. Roots

The characteristics of Ca²⁺ transport into endoplasmic reticulum vesicles isolated from roots of Lepidium sativum L. cv Krause have been investigated. The concentration of free Ca²⁺ and ATP needed for half-maximal activity were 2.5 and 73 micromolar, respectively, and the enzyme obeyed Michaelis-Menten-like kinetics. The pH maximum occurred at 7.5 and the activity was greatly reduced at either pH 7.0 or 8.0.

The Ca²⁺-dependent modulation protein, calmodulin, was tested for its effect on Ca²⁺ transport into endoplasmic reticulum vesicles. Although the phenothiazine inhibitors chlorpromazine, fluphenazine, and trifluoperazine all inhibited Ca²⁺ transport activity with a half-maximal effect at approximately 35 micromolar, authentic bovine brain calmodulin did not alter the activity at concentrations of 0.5 to 8 micrograms per milliliter. Calmodulin also showed no influence on the time-dependent accumulation of Ca²⁺ into vesicles. The membranes did not contain endogenously bound calmodulin since washing with (ethylenebis[oxyethylenenitrile])tetraacetic acid or fluphenazine, treatments which disrupt calmodulin binding, did not alter Ca²⁺ transport activity. The inhibition of Ca²⁺ transport by phenothiazine drugs was likely related to their nonspecific interaction with the membrane. Thus, there was no indication that calmodulin regulated Ca²⁺ uptake into root endoplasmic reticulum.

     

Interactions of DNA with Biofilm-Derived Membrane Vesicles

The biofilm matrix contributes to the chemistry, structure, and function of biofilms. Biofilm-derived membrane vesicles (MVs) and DNA, both matrix components, demonstrated concentration-, pH-, and cation-dependent interactions. Furthermore, MV-DNA association influenced MV surface properties. This bears consequences for the reactivity and availability for interaction of matrix polymers and other constituents.The biofilm matrix contributes to the chemistry, structure, and function of biofilms and is crucial for the development of fundamental biofilm properties (46, 47). Early studies defined polysaccharides as the matrix component, but proteins, lipids, and nucleic acids are all now acknowledged as important contributors (7, 15). Indeed, DNA has emerged as a vital participant, fulfilling structural and functional roles (1, 5, 6, 19, 31, 34, 36, 41, 43, 44). The phosphodiester bond of DNA renders this polyanionic at a physiological pH, undoubtedly contributing to interactions with cations, humic substances, fine-dispersed minerals, and matrix entities (25, 41, 49).In addition to particulates such as flagella and pili, membrane vesicles (MVs) are also found within the matrices of gram-negative and mixed biofilms (3, 16, 40). MVs are multifunctional bilayered structures that bleb from the outer membranes of gram-negative bacteria (reviewed in references 4, 24, 27, 28, and 30) and are chemically heterogeneous, combining the known chemistries of the biofilm matrix. Examination of biofilm samples by transmission electron microscopy (TEM) has suggested that matrix material interacts with MVs (Fig. ​(Fig.1).1). Since MVs produced in planktonic culture have associated DNA (11, 12, 13, 20, 21, 30, 39, 48), could biofilm-derived MVs incorporate DNA (1, 39, 40, 44)?Open in a separate windowFIG. 1.Possible interactions between matrix polymers and particulate structures. Shown is an electron micrograph of a thin section through a P. aeruginosa PAO1 biofilm. During processing, some dehydration occurred, resulting in collapse of matrix material into fibrillate arrangements (black filled arrows). There is a suggestion of interactions occurring with particulate structures such as MVs (hollow white arrow) and flagella (filled white arrows) (identified by the appearance and cross-dimension of these highly ordered structures when viewed at high magnification), which was consistently observed with other embedded samples and also with whole-mount preparations of gently disrupted biofilms (data not shown). The scale bar represents 200 nm.     

Calcium Transport in Sealed Vesicles from Red Beet (Beta vulgaris L.) Storage Tissue : II. Characterization of Ca Uptake into Plasma Membrane Vesicles

Calcium uptake was examined in sealed plasma membrane vesicles isolated from red beet (Beta vulgaris L.) storage tissue using (45)Ca(2+). Uptake of (45)Ca(2+) by the vesicles was ATP-dependent and radiotracer accumulated by the vesicles could be released by the addition of the calcium ionophore A23187. The uptake was stimulated by gramicidin D but slightly inhibited by carbonylcyanide m-chlorophenylhydrazone. Although the latter result might suggest some degree of indirect coupling of (45)Ca(2+) uptake to ATP utilization via deltamuH(+), no evidence for a secondary H(+)/Ca(2+) antiport in this vesicle system could be found. Following the imposition of an acid-interior pH gradient, proton efflux from the vesicle was not enhanced by the addition of Ca(2+) and an imposed pH gradient could not drive (45)Ca(2+) uptake. Optimal uptake of (45)Ca(2+) occurred broadly between pH 7.0 and 7.5 and the transport was inhibited by orthovanadate, N,N'-dicyclohexylcarbodiimide, and diethylstilbestrol but insensitive to nitrate and azide. The dependence of (45)Ca(2+) uptake on both calcium and Mg:ATP concentration demonstrated saturation kinetics with K(m) values of 6 micromolar and 0.37 millimolar, respectively. While ATP was the preferred substrate for driving (45)Ca(2+) uptake, GTP could drive transport at about 50% of the level observed for ATP. The results of this study demonstrate the presence of a unique primary calcium transport system associated with the plasma membrane which could drive calcium efflux from the plant cell.     

Kinetics of Aluminum Uptake in Triticum aestivum L: Identity of the Linear Phase of Aluminum Uptake by Excised Roots of Aluminum-Tolerant and Aluminum-Sensitive Cultivars

The identity of a linear phase of aluminum (Al) uptake in Triticum aestivum was investigated by analysis of the kinetics of Al uptake by excised roots and purified cell wall fractions. Classical interpretation of kinetic data suggests that a linear phase of uptake with time reflects uptake across the plasma membrane; however, in studies with Al the possibility that the linear phase of uptake includes accumulation of Al in both the symplasm and the apoplasm has not been discounted. In our experiments, we observed a linear phase of Al uptake at both ambient and low temperatures, although the rate of uptake at 0°C was 53 to 72% less than at 23°C, depending on cultivars. This nonsaturable phase of uptake at low temperature suggests that a portion of the linear phase of Al uptake is nonmetabolic. Furthermore, analysis of Al in cell wall fractions isolated from excised roots pretreated with Al suggests that the linear phase of uptake includes a cell wall component. When excised roots were pretreated with Al, accumulation of Al in purified cell wall material included a linear phase that could not be desorbed with a 30 minute wash in citrate. The rates of linear-phase accumulation of Al by cell wall material and cell contents were similar. In contrast, the linear phase of in vitro uptake of Al by purified cell wall material was completely desorbed by a 30 minute wash with citrate. These results suggest that the linear phase of Al uptake observed in excised roots of T. aestivum included metabolism-dependent binding of Al in apoplasm.