Planta (1992) 188:414-421 Planta © Springer-Verlag 1992 Aluminum effects on the kinetics of calcium uptake into cells of the wheat root apex Quantification of calcium fluxes using a calcium-selective vibrating microelectrode Jianvei W. Huang! David L. Grune "and Leon V. Kochian®* 2 Department of Soil, Crop and Atmospheric Sciences, and * U.S. Plant, Soil und Nutrition Laboratory, USDA-ARS, Comell University, Inhaca, NY 14883, USA Received 18 February: accepted 3 July 1992 Abstract. The effects of aluminum on the concentration- dependent kinetics of Ca?* uptake were studied in two winter wheat (Triticum aestivum L.) cultivars, Al-toler- ant Atlas 66 and Al-sensitive Scout 66. Seediings were ‘grown in 100 uM CaCl, solution (pH 4.5) for 3 d. Subse- quently, net Ca?” fluxes in intact roots were measured using a highly sensitive technique, employing a vibrating, Ca?*-selective microelectrode. The kinetics of Ca** up- take into cells of the root apex, for external Ca** con- centrations from 20 to 300 uM, were found to be quite similar for both cultivars in the absence of external Al; Ca?* transport could be described by Michaelis-Menten Kinetics. When roots were exposed to solutions contain- ing levels of Al that were toxic to Al-sensitive Scout 66 but not to Atlas 66 (5 to 20 uM total Al), a strong correlation was observed between Al toxicity and Al- induced inhibition of Ca?’ absorption by root apices. For Scout 66, exposure to Al immediately and dramat cally inhibited Ca?* uptake over the entire Ca?* con- centration range used for these experiments. Kinetic analyses of the Al-Ca interactions in Scout 66 roots were consistent with competitive inhibition of Ca?’ uptake by AL. For example, exposure of Scout 66 roots to in- creasing Al levels (from 0 to 10 uM) caused the Ky for Ca?* uptake to increase with each rise in Al concentra- tion, from approx. 100 uM in the absence of Al to ap- prox. 300 uM in the presence of 10 uM Al, while having, no effect on the Vmay. The same Al exposures had little effect on the kinetics of Ca?* uptake into roots of Atlas 66. The results of this study indicate that AI disruption of Ca?” transport at the root apex may play an impor- tant role in the mechanisms of Al toxicity in Al-sensitive wheat cultivars, and that differential Al tolerance may be associated with the ability of Ca? transport systems in cells of the root apex to resist disruption by potentially toxic levels of Al in the soil solution. * To whom correspondence should be addressed; FAX: 1(607) 2552454 Key words: Aluminum toxicity ~ Calcium (channel, transport) ~ Microelectrode (calcium selective, vibrating) Triticum (calcium uptake) Introduction Extensive studies have been conducted concerning alu- ‘minum (Al) phytotoxicity in plants grown on acid soils; however, the cellular bases for the mechanisms of Al phytotoxicity and Al tolerance are still not well under- stood (for reviews, see Foy et al. 1978; Foy 1988; Taylor 1988, b). The carliest and most dramatic visual symp- toms of Al toxicity are inhibition of root growth and associated anatomical alterations localized in the root apex (e.g. swollen and stunted roots with disrupted or damaged epidermal cells). These visual symptoms, along with numerous reports of Al-induced alterations of min- eral nutrition, have led to frequent suggestions in the literature that Al toxicity involves disruption and dam- ‘age to the root-cell plasmalemma and-or plasmalemma transport proteins (Vierstra and Haug 1978; Suhayda and Haug 1985; Deleers 1986; Zhao ct al, 1987). How- ever, recent electrophysiological studies in both excised and intact roots showed that Al did not significantly affect root-cell membrane potentials or disrupt the oper- ation of the H*-translocating ATPase (Kinraide 1988; Miyasaka et al. 1989; Huang et al. 1992). These observa- tions indicate that although Al toxicity may involve Al- induced alterations in ion-transport processes, these ef- fects are more subtle than gross disruption of the plas- malemma. Indeed, it appears that in roots treated with toxic levels of Al, the root-cell plasmalemma remains relatively intact. The root-cell ion-transport systems involved in ca um homeostasis and calcium nutrition are potentially important sites for interaction with Al during the initial stages of Al toxicity. There is considerable circumstantial evidence indicating that there are important interactions between Al and Ca?* during the onset of Al toxicityJ.W-Huang etal: Aluminum effects on calcium uptake kinetics For example, Haug and his coworkers have suggested that Al-induced alterations in calmodulin could interfere with normal Ca?'-calmodulin interactions, and this ight be a key lesion of Al phytotoxicity (For a review, see Haug 1984; Haug and Caldwell 1985). Alternatively, wwe are intrigued by the possibility that early events lead ing to Al toxicity might involve AI disruption of Ca?” transport at the root-cell plasmalemma, which could in- terfere with cellular Ca** homeostasis. Such effects might be most noticeable at the root apex, since the initial symptoms of Al toxicity are primarily’ associated with the root apical region (Hecht-Buchholz and Foy 1981; Bennet etal, 1985; Taylor 1988a). Recently, working with a nonvibrating Ca?*-selective microelectrode, we demonstrated that Al significantly inhibited Ca?* influx in the A-sensitive wheat cultivar, Scout 66, but had a much smaller effect on Ca?" trans. port in the Al-tolerant cultivar, Atlas 66 (Huang etal 1992), The rapidity of this inhibition, and the fast rever- sal of inhibition upon removal of Al from the solution bathing the root indicated that the primary site of this Al inhibition of Ca?” transport is at the outer face of the root-cell plasmalemma, possibly via blockage of Ca?* channels (Huang et al. 1992). One of the difficul- ties encountered in measurement of Ca?* fluxes with the statie-microelectrode system was the small Ca?* con- centration gradients (ether depletion or accumulation) that were generated in the unstirred layer at the root surface. For the experiments previously described (Huang et al. 1992), this difficulty was overcome by ‘modifying the growth technique to include a plant pre- treatment for 5h in a solution containing a low level of Ca?* (20 wM CaCl,). With this modification, it was possible to measure a transient net Ca?* flux (lasting several hours) at specific sites along intact wheat roots, as long as the experiments were conducted in the same ow background Ca?" level (20 uM Ca®*). When we attempted to measure Ca?” fluxes either at higher exter- nal Ca?* concentrations (100 uM or greater), or in roots grown in higher Ca** levels (100 uM), the Ca?* concen- tration gradients generated at the root surface were t00 small to make reliable measurements using the static Ca?* microelectrode. Recently, a system utilizing an ultra-sensitive, Ca®*- selective, vibrating microelectrode was developed by Jaffe and his colleagues (Jaffe and Levy 1987; Kiihtreiber and Jaffe 1990). With the vibrating Ca?* microelectrode, it is possible to detect minute extracellu- lar Ca?* gradients, In a preliminary study using vibrat- ing K*, H*, and Ca** microclectroiies to measure ion fluxes in maize roots and maize suspension cells, we Found this system to be approximately 50 times more sensitive than the nonvibrating system previously em- ployed (Kochian et al. 1992) ‘The primary objective of the present study was to use the vibrating Ca**-selective microelectrode to inves- tigate Al effects on concentration-dependent and time- dependent kinetics for Ca?” uptake at the root apex of Al-tolerant and Al-sensitive wheat cultivars. The ef- fects of various Al levels on concentration-dependent Ca?*-transport kinetics were investigated to determine ats if Al was competitively inhibiting Ca?* transport into cells of the root apex. If Al is blocking Ca?” channels, at the outer surface of the plasmalemma of root cells, it would be expected that such a competitive interaction, would occur. Materials and methods Plant culture. Seeds of two winter wheat (Triticum aesti- tum L.) cultivars (Al-tolerant Atlas 66, and Al-sensitive Scout 66) were germinated on filter paper saturated with 100 uM CaCl, solution (pH 4.5). Two germinated seeds, with radicals emerged, were placed at the bottom of a polyethylene cup having a polyethylene mesh bottom, and covered with black polyethylene beads. Four seed- Jing cups were placed into holes eut into black polyethyl- ene covers that fitted tightly over black polyethylene pots containing 2.8 L of aerated 100 yM CaCl, solution (pH 4.5), The seedlings were grown for 3 in a light- and temperature-controlled growth chamber with a 16- h, 20°C/8-h, 15°C day-night regime. The photon fluence rate at the level of the wheat shoots was 580 jrmol-m=?+s Vibrating-Ca?* microelectrode system. Technical details of the Ca?*-selective vibrating-microclectrode system have been reported by Kithtreiber and Jaffe (1990). The following is a brief description of the technique. Micro- pipettes with a tip diameter of yum were pulled from 1.5-mm-diameter borosilicate-glass capillaries (Catalog ‘No. TW150-4; WP Instruments Inc., Sarasota, Fla., USA) with a horitzontal electrode puller (Model P-87; Sutter Instruments Ine., Novato, Cal., USA) using a two-step pulling procedure. After silanization, the mi cropipettes were front-filled with a 100-mM CaCl, solu- tion. The tip of the micropipette was then front-filed with, a liquid-membrane-type, neutral, carrier-based Ca? -selective sensor (Catalog No. 21048; Fluka Chem- ical Co., Haupaugge, N.Y., USA), until a 40- to 80-ym- Jong column of sensor was produced ‘The Ca?*-selective microelectrode is vibrated by an assembly of three piezoelectric microstages (PZS-100; Burleigh Instruments, Inc., Fishers, N.Y., USA) stacked in orthogonal directions, which are in turn held by New- pport translation stages (Newport Corp., Fountain Valley, Cal,, USA). The above set-up is mounted on the stage of an inverted microscope (IM 35; Zeiss, Oberkochen, FRG) equipped with a video camera. The microstages are driven by a damped square wave at a low frequency (routinely 0.5 Hz). The Ca** microelectrode can be vi- brated at any angle in a two-dimensional plane by con- trolling the amplitude of vibration of the microstages separately ‘Measurement of the voltage differences at the two extreme positions of vibration, representing detection of a Ca?*-activity gradient, was achieved by digitizing the Ca? microelectrode signal and computing the potential difference with an IBM PC-XT computer. The move- ment of the microelectrode is controlled by the com- puter. The Ca?* microelectrode moves back and forth416 [ 4 wl Le He [Net Ca** fluxes in intact roots of Scout 66 wheat measured ata position 2mm back from the root apex. Net Ca?" fhoxes Tor treatments 4, B, or C were measured in 100, 200, oF 400 uM CaCl, solutions (pH 4.5), respectively. For cach bathing solution, the Ca**-flux measurements began after the roots were equilbrat” cd in the corresponding solution for 20 min. RS denotes the Ca! Sloctive microelectrode positioned at the root surface (vibrating between $0 und 80 um from the root surface) and BG denotes the microelectrode positioned in the background bulk solution {about 10 mm away [rom the root surface). Insert: means -£SES For net Ca?* Muxes between two pre-set positions. At each extreme of vibra tion, the computer takes 1000 samples, blanks out any transitional points, and calculates the average. Then, the computer calculates the difference between this average and the previous one at the other extreme position, and finally calculates a moving average of these differences ‘over any desired time period. The results can be recorded ‘on a timelapse videorecorder as well as on disk. Measurement of net Ca?* flux. Intact 3-d-old wheat seedlings were set up in plastic Petri dishes. The roots were anchored at the bottom of the dish by small Plexi- las blocks with silicone grease, and the shoots were held onto the petri dish wall by a small piece of dental wax. The Plexiglas blocks (8-4-4 mm) had a notch cut into one face to allow them to be placed over the root without causing physical damage. The plants were set in the Petri dishes, which held 100 xM CaCl, solution (pH 4.5), for 30 min before taking Ca? *-flux measure- ments. Calcium fluxes were measured at various dis- tances back from the root apex. In studying Al effects on Ca?*-uptake kinetics, Ca?* fluxes were measured between 1 and 2mm back from the root apex. The ex- perimental solutions employed were the factorial combi- nations of four Al levels (0, 5, 10, and 20 uM AICI) and six Ca?* levels (20, 50, 70, 100, 200, and 300 uM CaCl). The Al concentrations presented here represent the total Al in solution, and not the Al?* activity. For each experimental solution, Ca?’ lux measurements be- gan after the roots were equilibrated in the solution for at least 30 min. The solutions were changed every 20 to 30 min, Calcium fluxes were calculated from Fick's first law of diffusion: = Des (Cp-C2) ean Jes! J.W.Fuang etal: Aluminum effects on calm uptake kinetics oe |; protem'e"t Fig. 2. Micrograph depiting Ca?-flux veetors at various positions along the apex of an intact root of Seout 66 wheat. The Ca” Selective microelecrode can be seen vibrating ata postion approx. mm back from the root tip and 70 um from the root surface. The Mux measurements were made in a solution consisting of 100 uM CaCl, (p14.5), The inward vectors denote @ net Ca” influx. The distance bar in the micrograph indicates a distance 60300 jm, while the Cau bar denotes a Mux of | paol-em * Net Cathe (poms) ‘itanoe tam 00 ape Fig. 3 Spatial variation in Ca?* uptake along intact roots of Atlas 66 and Scout 66 wheat, The Ca?" Muxes were measured in 100 jM CaCI, solution (pH45) where Joy is the net Ca?* Mux (in pmol-em=*-s~"); Dex is the self-diffusion coefficient for Ca** (in em*-s""); Cy, and C, are the Ca?" activities at the two positions, and AX is the amplitude of vibration (usually 30 wm). ‘The microelectrode was positioned so that its long axis made an angle of approx. 45° with the long axis of the root, and the electrode was vibrated so that the axis of vibration was perpendicular to the root. The electrode was vibrated at radial distance of 50 um from the root surface, so that at is extreme of vibration the microelec- trode tip was between 50 and 80 ym from the surface of the root4J.W.Huang et a: Aluminum effects on calcium uptake kinetics Rs, ‘Scout of 204M CaCl RS 5 i 5 a 3 z a7 Fig. 4A, B. Elfects of addition and ce- moval of 20 yM AICI, on net Ca®* FTuxes at the apex of intact roots of Scout &6 wheat. The Ca** fluxes were ‘measured at a position 1 mm back from “0 the root apex, in solutions containing ei- ther 20 uM CaCl, (A) o 100 uM CaCl (B). See legend in Fig. 1 for the defini- tion of RS and BG 1Omie Voltage difference (iV) Results Calcium fluxes. The sensitivity of the vibrating Ca?” selective’ microelectrode was examined by measuring Ca?* fluxes in intact roots in a series of solutions con- taining increasing levels of calcium. A representative trace is shown in Fig. 1. In the background solution (about 10mm away from the root surface), the output signal of the Ca?* microelectrode (system noise) was about £5. When the vibrating Ca®” microelectrode ‘was moved into the unstirred layer near the root surface (between 50 and 80 um from the root surface), the out- put signal increased immediately, yielding a voltage dif- ference between the extremes of vibration of the Ca?* microelectrode that was due to detection of a Ca**- contrat © 45M Al 2 +10uM AL = +20,M ai | 080 100 150 200 250 300 {s] (um) Fig. 7. Hanes-Woolf plots for the Ca**-uptake kinetic data pre: senied in Fig. tative traces are presented for Scout 66 (Fig. 4) and Atlas 66 (Fig. 5). In a solution containing 20 uM Ca”, addi- tion of 20 uM Al caused Ca?* fluxes into roots of Scout 66 to be rapidly reversed from a net influx to a net efflux (Fig. 4). However, for the same Al treatment Atlas 66 still maintained a substantial net influx (about 50% of the control; Fig. 5). When Al was removed From the bathing solution, net Ca** uptake immediately recovered for both Scout 66 and Atlas 66 (Figs. 44, 5A). Ina 100 4M CaCl, solution, following the exposure Of roots to 20M Al, net Ca?” uptake by Scout 66 was decreased by 50% (Fig. 4B). When the Al was re- moved, the Ca?*-uptake rates of Scout 66 once again returned to the values measured prior to the addition of AL The same Al treatment did not dramatically affect net Ca?* fluxes in roots of Atlas 66 (Fig. 5B). Effects of Al on Ca?* uptake kinetics. Net Ca* uptake measured over a range of Ca?* concentrations (20 to 300 uM Ca?*) yielded concentration-dependent kinetics that could be described by the Michaelis-Menten equa- tion (Fig. 6). Exposure to Al (5 to 20 uM) had little effect on the Ca**-uptake kinetics for Atlas 66 roots, while dramatically altering the kinetic curves for Scout 66 (Fig. 6. B). Addition of 5 or 10 4M Al consistentlyJ.W.Huang et al: Aluminum effects on calcium uptake kineties dcereased net Ca?* uptake into Scout 66 roots over the entire Ca?* concentration range. When Scout 66 roots ‘were exposed to a solution containing 20 uM_Al, net Ca?* efflux was observed at the lowest Ca~ level (20 uM), while Ca?* influx was dramatically reduced at all other Ca?~ levels (50 to 300 uM Ca?*), By con- ast, addition of 5 or 10 4M Al did not significantly inhibit net Ca?” uptake by Atlas 66 at any Ca?” concen- tration. At the highest Al level (20 uM), a moderate de- crease in net Ca?” uptake was observed for Atlas 66. However, the magnitude of inhibition of Ca®* uptake by 20 uM Al was much less in Atlas 66 than in Scout 66 (compare Figs. 6A, 6B). The Ca*-uptake kinetic data were analyzed using 4 linear trnsformation of the kinetic curves, via the Hanes-Woolf equation (Segel 1976). As seen in Fig. 7A, Hanes-Woolf transformation of the Ca?*-transport netics obtained for roots of Scout 66 exposed to different Alllevels yielded results consistent with competitive inhi- bition of Ca?* absorption by Al. The series of parallel lines with increasing Y intercepts as the Al level was increased from 0 to 104M are indicative of an Al-in- duced inerease in the Ky, for Ca?* uptake, while having litle affect om the Vaua- This is verified by determination of the kinetic constants for Ca?* uptake in the presence of the different Al concentrations. The values in Table { indicate that for Ca?* transport in Scout 66, the Ky values increased from 98 to 278 uM as the external Al concentration was increased from zero to 10 uM, while the Vay, values were unchanged. The same Al exposures. had litte effect on the kinetic parameters for Ca?* up- take into roots of Al-tolerant Atlas 66 (Fig. 7B, Table 1). For Al exposures up to 10M for Scout 66, and 204M for Atlas 66, the kinetic curves for Ca?* uptake fit classical Michaelis-Menten enzyme kinetics. The values of R? indicate that the Hanes-Woolf equation could describe the variations of Ca?* uptake by more than 97% for both cultivars (Table 1). However, at the highest Al level employed (20 uM), Ca?” uptake by Scout 66 deviated from Michaelis-Menten kinetics f Al on kinetic parameters for Ca** uptake at a Ka R (pinol-em=*-s uy Altolerant Atlas 66 ° 445 103 099 5 410 100 099 10 429 131 098 2 433 168 058 Absensitive Scout 66 0 4.08, 099 5 48 098 10 435, 097 20 410 03 * Values of Vu: and Ky, were obiained by regression analysis of| the Ca?*-uptake kinete data using linear transformation via the Hanes: Woolf equation, The data were means of replicate measur ‘ments from atleast four plants 419 (Fig. 6A, Table 1). This is probably a consequence of the alteration of Ca?" transport at the lowest Ca? level, from an influx to an efflux, when roots were exposed to 20 uM AL. This type of response will confound kinetic analysis of the data at low external Ca?* concentrations. This also points out a shortcoming of applying the rapi equilibrium kinetic approach of Henri, Michaelis, and Menten, that is based on unidirectional (Forward) reac- tions where the reverse reaction is insignificant, to net ionic fluxes where the reverse reaction (unidirectional efflux) might be significant under certain conditions (see Discussion). Discussion By using a vibrating Ca**-selective microelectrode we were able to measure directly net Ca?* fluxes in roots of intact plants at external Ca?* levels up to 400 uM. ‘One of our concerns has been whether the root pretreat ment in low external Ca?* (20 wM Ca?*) used in the previous study by Huang et al. (1992), had any artificial effects on Ca** uptake. We were particularly concerned with the large Ca?* fluxes measured at the root apex (0 to Smm from the root tip), that were significantly higher than the Ca?* fluxes in mature root regions. Be- cause this former root region, which was the focus of the previous and current study, was comprised primarily of new tissue grown during the 5-h pretreatment period in low external calcium, we wanted to know if the higher Ca? fluxes were an artifact of cell division and growth in solutions containing very low levels of calcium. In the current study, we measured net Ca? fluxes along roots in the same solution in which the plants were grown. The Ca?*-flux pattern was almost identical to that obtained previously using a static Ca** selective microvelectrode, Therefore, the observation that the largest net Ca?* uptake occurs at the root apex is characteristic of Ca?* uptake in wheat plants, and is possibly characteristic of other species. ‘There were dramatic varietal differences in the Al effects on Ca*” influx between Scout and Atlas (com- pare Figs. 4, 5, and 6). Exposure to Al caused dramatic inhibitions of Ca? uptake in Scout, often inducing a significant Ca?* efflux. For the same’Al treatments, At- las 66 still maintained a significant net Ca?* influx at all external Ca?* and Al concentrations (Fig. 6B), indi- cating that Atlas 66 had a greater ability to resist Al inhibition of Ca?” uptake. These results are in good agreement with those obtained by the static-microelec- trode technique (Huang et al. 1992). In a 100 yM CaCls solution (pH 4.5), addition of 20 yM Al did not dramati- cally affect net Ca?* uptake by Atlas 66, but decreased net Ca?* uptake into roots of Scout 66 by 50% (com- pare Figs. 4B, SB). This same Al exposure in 100 «M. CaCl, is toxic to Scout, dramatically inhibiting root elongation within 2-3 h after initial Al exposure, wale having little effect on Atlas 66 root growth. The results, presented here indicate that the dramatic differences in Altolerance for Atlas 66 and Scout 66 correlate well with the differences in Al inhibition of Ca?* uptake.20 The rapidity of the Al-induced inhibition of Ca?* uptake, and rapid reversal of this inhibition when Al is removed from the solution bathing the root (see Fig. 4), indicate that Al is acting extracellularly, possibly at the outer surface of the plasmalemma. It was neces- sary to consider the less-likely possibility that Al was. displacing Ca** from the cell wall, and thus apparently reducing net Ca?” uptake by increasing Ca*” efflux from the cell wall. This possibility was tested in the pre- vious study (Huang et al. 1992), by determining the in- fluence of the same Al exposures on morphologically intact root cell-wall preparations from Scout 66 and At- las 66, In that study, it was found that Al-induced Ca?* efflux from the cell wall was negligible, These results strongly indicate that AP is acting on Ca** transport systems at the root-cell plasmalemma, which would in- clude Ca?” channels mediating Ca** influx, and the Ca?* ATPase involved in Ca?” efflux. It should be not- ed at this time that the vibrating Ca** probe directly ‘measures the size of the extracellular Ca?* gradient, and from this gradient determination we indirectly compute values for Ca?* fluxes. Thus, any discussion concerning. plasmalemma Ca?*-transport systems is necessarily speculative. Nonetheless, we feel that the rapid inhibition and recovery of Ca?” influx upon AP* exposure and removal, along with the apparent competitive inhibition of Ca?" uptake by exogenous Al demonstrated in the current study, are consistent with Al blockage of Ca? channels facilitating Ca?* influx across the root-cell plasmalemma. It has been shown that a number of diva- lent and trivalent inorganic cations block Ca** channels (Hille 1992), and it would not be surprising to find that AL*, which is the dominant form of Al in our solutions, also functions in this manner. In plant cell systems, it hhas been previously shown that external AL" blocks * influx via a K* channel in the guard-cell plasmalem- ma (Schroeder 1988). However, in order to determine directly if AP* is blocking plant plasmalemma Ca?* channels, it will be necessary to conduct biochemical and biophysical studies with isolated plasmalemma vesi- cles and root protoplasts, which will be the subject of future research. The correlation between Al toxicity and Alinduced inhibition of root apical Ca?” uptake is intriguing, par- ticularly because the blockage of Ca" uptake precedes visual symptoms of Al toxicity by several hours. Is it possible that prolonged blockage of root-cell plasmalem- ma Ca?" channels will cause damage to the root apex? If'so, is Al blockage of Ca?* channels an important component of Al toxicity? It is easier to envision disrup- tion of cellular function by processes that tend to in- crease cytoplasmic calcium. In this case, one would ex- pect blockage of Ca?" channels to decrease cytoplasmic calcium. However, itis not unreasonable to expect that continued blockage of Ca?* entry into cells in a region of intense metabolic and mitotic activity such as the root apex could result in disfunction of the apex. In support of this, exposure of wheat roots of La", which is @ known Ca®*-channel blocker, elicits root toxicity symp- toms quite similar to those caused by Al (Kinraide et al 4992) J.W.Huang etal: Aluminum effects on calcium uptake kinetics The other question raised by this research concerns mechanisms of differential Al tolerance. If blockage of Ca?" channels in the plasmalemma of root apical cells isa critical aspect of Al toxicity, then cellular properties, that help resist this blockage could impart Al tolerance to higher plants. Several different mechanisms could be involved. These possibilities include differences in the orientation or structure of Ca?*-channel proteins, and differences in the lipid bilayer, particularly if Al is inhi- biting Ca?*-channel function indirectly by interacting with the lipid bilayer to change its physical properties Alternatively, there could be no differences in Ca?*~ channel proteins or the plasmalemma lipid bilayer be- tween sensitive and tolerant cultivars, and. tolerance could be imparted by an exclusion mechanism that re- duces the “effective” Al activity in the rhizosphere and- or cell-wall solution surrounding cells of the root apex. At this time, a reasonable candidate for such an exclu: sion mechanism would be release of Al-chelating organic compounds (c.g. organic acids) by root apical cells of the tolerant cultivar. Evidence in support of organic-acid release playing a role in Al tolerance has been presented by Miyasaka et al. (1991) to explain AI tolerance in snapbeans Conclusions Calcium transport in intact wheat roots is characterized, by a large net Ca?* influx into cells of the root apex (0 to S mm from the root tip), and a much smailer Ca?* uptake in mature root regions (5 mm or more back from the root apex). At exogenous Ca?" levels up to 300 iM, Al did not appreciably affect the Vu, but dramatically increased the Ky for Ca?" uptake, especially for Al- sensitive Scout 66. These results indicate that Al compe- Uitively inhibits Ca®* uptake into the root apex of wheat plants, possibly via blockage of Ca** channels. The dra matic varietal differences in Al inhibition of Ca?* up- take indicates that Al disruption of Ca?” transport at the root apex may play an important role in Al toxicity in Al-sensitive wheat cultivars, and the ability to resist Abinduced inhibition of Ca?* uptake may be linked to Al tolerance, ‘We would lke to thank Dr. Lionel F Jaffe, Director of the Nation- al Vibrating Probe Facility, Marine Biological Laboratory, Woods Hole, Mass., USA. for making his calium-selective vibrating-mi- croclectrode system available for a portion of this work. The re search presented here was supported in part by USDA/NRI Com pative Grant number 91-37100-6630 to Leon Kechian, Contibu- tion from the USDA-ARS, U.S. Plant, Soil and Nutrition Labor tory, Comell University, ithaca, N-Y. This esearch was part of the program of the Center for Root Soil Reseurch, Cornell Univer~ sil, Ithaca, N.Y. Department of Sol, Crop and Atmosphere Sc= cence, paper No, 1741 References Bennet, R.J., Breen, C.M., Fey, M.V. (1988) Aluminum uptake sites in the primary roots of Zea may L. S. Aft. J. Plant Soil 217{.W-Huang et al.; Aluminum effects on calcium uptake kinetics Deleers, M. (1986) Cationic atmosphere and cation competition binding at negatively charged membranes: Pathological impli- «ations of aluminum. Res. Commun. Chem, Pathol. Pharmacol, 9,277-294 Foy, C:D. (1988) Plant adaptation to ac, aluminum-toxic sols ‘Commun, Soil Sei, Plant Anal. 19, 959-87 Foy, C.D., Chaney, R.L., White, M.C. (1978) The physiology of ‘metal ‘oxicity implants. Annu, Rev. Plant Physiol. 29, 511-566 Haug, A.R. (1984) Molecular aspects of aluminum toxicity. CRC Cit, Rev, Plant Sei, 1, 345-373 Haug, A.R, Caldwell, CR. (1985) Aluminum toxicity in plants the role of the root plasma membrane and calmodulin. In Frontiess of membrane research in agriculture, pp. 359-382, St John, J.B, Berlin, B, Jackson, P.C., eds. Rowman and AI. lanheld, Totowa Hecht-Bucholz, C., Foy, C.D. (1981) Effect of aluminum toxicity ‘on r00t morphology of barley. Plant Soil 63, 93-95 Hille, B. (1992) Tonic channels of excitable membranes. 2nd Edi- tion, Sinauer Associates, Inc, Sunderland, Mass, USA Huang, J.W,, Sha, J.E,, Grunes, D-L., Kochian, LV. (1992) Alu- ‘minum effets of calcium fluxes at the root apex of aluminum- tolerant and aluminum-senstive wheat cultivars, Plant Physio. 98, 230-287 Jaffe, LF, Levy, $. (1987) Calcium gradients measured with @ vibrating calcium-slective electrode. Inst, Elect. Eng. Mod. Biol, Soe. Cont. 9, 779-781 Kinraide, T-B. (1988) Proton extrusion by wheat root exhibiting severe aluminum toxicity symptoms. Plant Physiol. 88, 418-423 Kinraide,T-B., Ryan, P-R., Kochian, LY. (1992) Interactive eects ‘of Al", H°, and other eations on root elongation considered in terms of cell-surface eletrical potential, Plant Physiol. 98, 1461-1468 Kochian, LV, Shaff, LE, Kihtreiber, WML, Jaffe, LP., Lucas, ‘WJ. (1992) The use of an extracellular, ion-selective, vibrating imieroelectrode system for the quantification of K*, H", and a (Ca?* flutes in maize roots and maize suspension cells. Planta, in press Kahtreiber, W.M,, Jaffe, LLF_ (1990) Detection of extracellular calcium gradients with « calcium-specfic vibrating electrode, J.Colt Biol. 10, 1565-1573. Miyasaka, S.C., Kochian, LV, haf, E., Foy, C.D. (1989) Mech: ‘anisms of aluminum tolerance in wheat. An investigation of ‘genotypic dilerences in rhizosphere pH, K*, and H transport, and root-cell membrane potentials. Plant Physiol. 91, 1188 1196 Miyasaka S.C., Buta, B.G., Howell, RK., Foy. C.D. (1991) Mech- ‘nism of aluminum toierance in snapbeans. Root exudation of citi acd. Plant Physiol, 96, 737-743, Schroeder, J.1 (1988) K* transport properties of K~ channels in the plasma membrane of Vicia faba guard cells. Gen. Physiol. 92, 657-683 Segel, 1H. (1976) Biochemical calculations: How to solve mathe- ‘matical problems in general biochemistry, 2nd Edo, John Wiley & Sons, Inc, USA, Suhayda, C.G, Haug, A. (1985) Citrate chelation as a potential ‘mechanism against aluminum toxicity in cells: the tole of cal- ‘edulin. Can. J. Biochem. Cell Biol. 63, 1167-1175, ‘Taylor, G.J_ (19888) The physiology of aluminum phytotoxiciy. In: Metal ions in biological systems, vol. 24: Aluminum and its role in biology, pp. 123-163, Sige, H., ed. Marcel-Dekker, New York Taylor, G.J. (19886) The physiology of aluminum tolerance. In Metal ions in biological systems, vol. 24: Aluminum and its role in biology, pp. 165-198, Sigel, H., ed. Marcel-Dekker, New York Vierstra, R., Haug, A. (1978) The effet of AI* on the physical properties of membrane lipids in Thermoplasma acidophitum. Biochem. Biophys. Res. Commun. 84, 138-143 Zhao, XJ, Sucoil, E., Stadelmann, EF. (1987) Al and Ca? ‘lication of membrane permeability of Quercus rubra r00% cor~ tex cells, Plant Physiol. 83, 159-162