Calcium/aluminium interactions in the cell wall and ... - Springer Link

3 downloads 0 Views 739KB Size Report
Springer-Verlag 1995. Calcium/aluminium interactions in the cell wall and plasma membrane of Chara. Robert J. Reid 1, Mark A. Tester 2, F. Andrew Smith 1.
Planta (1995)195:362-368

P l a n t a @ Springer-Verlag 1995

Calcium/aluminium interactions in the cell wall and plasma membrane of Chara Robert J. Reid 1, Mark A. Tester 2, F. Andrew Smith 1 1Botany Department, University of Adelaide, Adelaide, S.A. 5005, Australia 2 Department of Plant Science, University of Cambridge, Downing Street, Cambridge CB2 3EA, UK Received: 1 April 1994 / Accepted: 8 May 1994

Abstract. The proposal that aluminium (A1) toxicity in plants is caused by either inhibition of Ca 2+ influx or by displacement of Ca 2+ from the cell wall, was examined. For this study the giant alga Chara corallina Klein ex Will. em. R.D. Wood was selected because it shows a similar sensitivity to A1 as in roots of higher plants and, more importantly, it is possible to use the large single internodal cells to make accurate and unambiguous measurements of C a 2+ influx and C a 2+ binding in cell walls. G r o w t h of Chara was inhibited by A1 at concentrations comparable to those required to inhibit growth of roots, and with a similar speed of onset and p H dependence. At A1 concentrations which inhibited growth, influx of calcium (Ca 2+) was only slightly sensitive to A1. The maxim u m inhibition of Ca 2+ influx at 0.1 m o l . m 3 A1 at p H 4.4 was less than 50%. At the same concentration, lanthanum (La 3+) inhibited influx of Ca 2+ by 90% but inhibition of growth was similar for both La 3+ and A1. Removal of C a 2+ from the external solution did not inhibit growth for more than 8 h whereas inhibition of growth by A1 was apparent after only 2.5 h. Ca 2+ influx was more sensitive to A1 when stimulated by addition of high concentrations of potassium (K +) or by action potentials generated by electrical stimulation. Other membrane-related activities such as sodium influx, rubidium influx and m e m b r a n e potential difference and conductance, were not strongly affected by A1 even at high concentrations. In isolated cell walls equilibrated in 0.5 m o l . m 3 Ca2+ at p H 4.4, 0.1 m o l . m 3 A1 displaced more than 80% of the bound Ca 2+ with a half-time of 25 min. F r o m the p o o r correlation between inhibition of growth and reduction in Ca 2+ influx, it was concluded that A1 toxicity was not caused by limitation of the Ca 2+ supply. Short-term changes in other membrane-related activities induced by A1 also appeared to be too small to explain the toxicity. However the strong displacement, and probAbbreviations: CPW = artificial pond water; PD = potential difference Correspondence to: R.J. Reid: FAX: 61(8)2323297; Tel.: 61(8)3035290; e-mail: [email protected]

able replacement, of cell wall Ca 2+ by A1 may be sufficient to disrupt normal cell development.

Key words: Aluminium toxicity - Calcium influx wall and calcium - Chara - L a n t h a n u m

Cell

Introduction In recent years there has been a substantial research effort directed towards understanding the mechanisms of aluminium toxicity and tolerance in plants. Much of this work has concentrated on roots in which toxicity symptoms first become apparent. The possible causes of toxicity and tolerance have been well reviewed, most recently by Taylor (1991) and Rengel (1992a, b). The latter emphasised the various levels of interaction of A1 with C a 2+, both apoplasmic and symplasmic, and proposed that disturbance of normal C a 2+ function is the primary cause of A1 toxicity. Rengel and Elliott (1992a, b) showed that A1 inhibited 45Ca influx in protoplasts and there have been a number of reports of inhibition of 4SCa influx into intact roots (Asp and Berggren 1990; Lindberg 1990; H u a n g et al. 1992). Obtaining a quantitative assessment of the alteration of membrane fluxes of Ca 2+ in intact roots is however difficult because of extensive binding of Ca 2+ in the cell wall (reviewed by Reid and Tester 1992). This problem can be overcome in the giant cells of the alga Chara corallina, for which reliable methods for measuring Ca 2+ fluxes have recently been described (Reid and Smith 1992a, b). In the current work it was found that growth of Chara was also sensitive to A1, with close parallels with roots in terms of the speed of onset and the concentration and p H dependence of the inhibition. Chara therefore appears to offer a simplified system in which to examine various aspects of A1 toxicity, particularly with respect to Ca2+/A1 interactions.

R.J. et al.: Calcium/aluminium interaction in Chara

Materials and methods Plant material. The giant-celled alga Chara corallina was grown in the laboratory in large plastic tanks on a substrate of garden soil and river sand in partially deionised water. The cultures were illuminated on a 16 h/8 h light/dark cycle at a photon flux density of approximately 35 gmol.m -2.s-1 at the surface of the solution. Before experiments, individual internodal cells (40-90 mm long and approximately 1 mm in diameter) or whole shoots were isolated from the plant and stored in an artificial pond water (CPW) consisting of 1 mol .m -3 NaC1, 0.1 mol.m 3 K 2 S O 4 and 0.5 mol . m 3 CaCI2. Solutions. The experimental solutions were based on CPW and were unbuffered to eliminate possible complexing of AI by organic ligands. Adjustment of pH was made using dilute solutions of NaOH or HCt. To minimise pH changes during experiments, large volumes of solution were used and they were gently agitated. Aluminium was added from a 10mol.m -3 stock of A1C13 dissolved in 100 m o l . m 3 HCI. Unless the valence is specifically stated, AI refers to the combined concentration of all aluminium species in solution, Aluminiumexists in solution as a number of hydrated mononuclear or polynuclear species. At low concentration and pHs less than about 5 the predominant species will be At 3+ and AI(OH)z+, according to the equation

363 internodal cells. However, for these small cells it was not practicable to isolate the cytoplasm and vacuole by the methods used for the larger internodal cells, so to obtain an estimate of the influx the following procedure was adopted. At the end of the rinse period the individual branches of the whorl were separated wit h a scalpel and the terminal and sub-terminal cells cut open and the contents released into 1 ml of deionised water. The solution was agitated for 10 s and the cell wall fragments removed. The 45Ca activity in the remaining solution was determined by liquid scintillation counting, Calcium influx measured in this way is probably mainly influx to the vacuole; some of the cytoplasmic 45Ca would have been effluxed to the external solution, some retained in slowly exchanging cytoplasmic compartments and some transported to the vacuole. This flux showed a similar sensitivity to La 3+ inhibition compared to plasmalemma and vacuolar influx in internodal cells. Influxes of 22Na and 86Rb (0.1 mol. m -3 in CPW) were measured over 20 min with a rinse of 2 min. The cells were not fractionated as for the 45Ca measurements but counted simply after rinsing.

Cell wall binding, Cell wall sleeves were prepared by flushing out internodal cells with deionised water to remove the cell contents, Replicate 40-mm lengths were incubated in 4SCa-CPW_+A1, then rinsed in several changes of deionised water to remove unbound 45Ca. Radioactivity was measured using liquid scintillation counting,

A13+ +H20 = AI(OH)2+ + H § The log K for this reaction is around --5. Thus A13+ represents 90% of the total activity at pH 4 and approximately 77% at pH 4.4, According to Kinraide and Parker (1990) and Kinraide (1991), polynucleation and precipitation becomes significant when {A13+}/ {H+}3> 10~8, where the braces indicate activity. From the speciation equations given in Kinraide and Parker (1990), this equates to a total A1 concentration of approximately 1 mol-m 3 at pH 4 and of approximately 0.08 mol,m -3 at pH 4,4. At higher concentrations and higher pH the solution composition becomes less well defined because of reduced solubility and the formation of polynuclear species. It should be stressed that these are theoretical computations of speciation in the bulk solution; conditions in the cell wall and adjacent to the plasma membranes of plant cells can obviously be different fi'om the solution, especially in unbuffered media as used here.

Growth. Plants of C. corallina generally consist of a column of internodal cells joined by clusters of small nodal cells. From each node there are usually six branches, collectively known as a whorl, with each branch comprising three to four cells. The most vigorous growth occurs in the topmost internodal cell and its associated whorls. By the third internode, and after about 14-20 d the cells are usually mature (i.e. fully grown). In this study, growth was measured in the terminal and sub-terminal cells of intact whorls from the top of the plant. Measurements of Ca 2+ influx were made on these cells and also on mature whorl ceils from the third internode and on mature internodal cells. Elongation of young cells in freshly isolated shoots was measured under a microscope at 40 x magnification using a graduated eyepiece. Influx measurements. The basic methods for measuring Ca 2+ influx in Chara internodal ceils using 45Ca are given in Reid and Smith (1992a, b) and in Reid et al. (1993). Plasmalemma influx was measured using a divided-chamber technique with an influx period of 20 min and a rinse in CPW+2 mol.m-3 LaC13 of 4 min, after which the cell contents were rapidly separated from the cell wall as described in Reid and Smith (1992a,b). Influx to the vacuole of internodal cells was measured by separating the vacuole from cells after a 45Ca-influx period of 2-3 h and a rinse of 1 h. For measurements of influx to whorl cells, the top three internodal cells with attached whorls were isolated from the plant and immediately incubated in 45Ca-CPW for 2 h and rinsed for 1 h in CPW + La 3+, as for measurements of Ca z+ influx to the vacuole of

Electrophysiology. The membrane electrical potential difference (PD) was measured by recording the voltage between a KCl-filled glass micropipette inserted into the vacuole and a reference electrode in the bathing medium. Membrane conductance was determined by the deflection of membrane PD caused by the injection of a current pulse of known amplitude into the vacuole via a second KCl-filled glass micropipette. Bipolar pulses were generated every 20 s. All results are presented as mean _+SE (number of replicates).

Results Growth. I n p r e l i m i n a r y studies it was f o u n d t h a t 5 0 % of isolated m a t u r e i n t e r n o d a l cells died in 0.1 m o l - m -3 A1 at p H 4.4 w i t h i n 5 d (data n o t shown). E l o n g a t i o n of growing cells in isolated whorls was i n h i b i t e d w i t h i n 2 h b y 0.1 m o l , m 3 A1 at p H 4.4 a n d s t o p p e d c o m p l e t e l y w i t h i n 6 h (Fig. la), By c o m p a r i s o n , r e m o v a l of Ca2 + from the b a t h i n g m e d i u m did n o t i n h i b i t g r o w t h u n t i l 8 h after the s t a r v a t i o n c o m m e n c e d (Fig. lb). C o m p a r i s o n was m a d e b e t w e e n the effects o n g r o w t h of A1 a n d of a n o t h e r trivalent c a t i o n L a 3+, which is k n o w n to be a p o t e n t i n h i b i t o r of C a 2+ influx. It was f o u n d t h a t A1 a n d La 3+ h a d identical effects o n growth. A t 0.02 m o l . m 3 b o t h c a t i o n s i n h i b i t e d g r o w t h by app r o x i m a t e l y 4 0 % a n d at 0.1 m o l . m -3 b y m o r e t h a n 9 0 % (Fig, 2). Influx o f Ca 2+. I n a p r e v i o u s s t u d y (Reid a n d S m i t h 1992b) it was f o u n d that C a 2+ influx in Chara increased c o n s i d e r a b l y in s o l u t i o n s c o n t a i n i n g K + at c o n c e n t r a tions high e n o u g h to depolarise the p l a s m a m e m b r a n e or w h e n cells were s t i m u l a t e d to generate a c t i o n potentials. Influx was also higher in g r o w i n g cells. T h e effect of A1 o n C a 2+ influx in each of these states was investigated. W h e n m a t u r e i n t e r n o d a l cells were p r e t r e a t e d for i h in 0.1 m o l . m 3 A1 at p H 4.4, p l a s m a m e m b r a n e influx was reduced f r o m 0 . 4 0 + 0 . 0 6 (10) to 0.22_+0.06 (12)

R.J. et al.: Calcium/aluminium interaction in Chara

364 Table 1. Effects of La 3+ and A1 on Ca 2+ influx to terminal whorl cells of Chara corallina. Influx was measured using 45Ca

Cells

Growing

Influx time (h) 2

Treatment

Ca 2+ influx (nmol-m 2.s 1)

control

1.77 • 0.16 (4) 0.17+0.04 (13) 1.00_+0.10 (10)

10 56

1.23 _+0.13 (12) 0.15_+0.03 (12) 0.65_+0.15 (12)

12 53

0.44_+0.03 (12) 0.12+0.05 (6) 0.39+0.04 (12)

27 89

0.1 m o l . m -3 La 3+ 0.1 m o l . m 3 A1

Mature

2

control 0.1 m o l - m -3 La 3+ 0.1 m o l . m 3 AI

Mature

24

control 0.I m o l - m 3 La3+ 0.1 m o l - m 3 A1

15

(a)

14 A= O) C 9

% Control

Control

12 Lanthanum 0.02

Aluminium 0.02

1

10

5 O

c_ o

,

o

,

8

6

o

o

25

4

(b)

20

control

2 0

lo

Fig. 2. Inhibition of growth of whorl cells of Chara corallina by 0.02 and 0.1 m o l . m 3 La3+ or A1. Growth was measured over 24 h in

5

C P W p H 4.4

o

0

5

10

15

20

25

T (h) Fig. la, b. Effect of addition of 0.1 m o l . m 3 A1 (a) and removal of Ca 2+ (b) on the growth of whorl cells of Chara corallina in C P W pH 4.4. Elongation of individual cells in the shoot was measured under a microscope at 40 x . Each point is the mean + S E of 15-20 cells. Experiments shown in a and b were conducted using cells from different cultures

1.6 E E

1.2

0.8

K

._~

n m o l . m 2.s ~. Influx to the vacuole was reduced by approximately 30% under similar conditions (Fig. 3). However, the K+-stimulated influx was much more sensitive to A1 and was virtually abolished by 0.2 m o l - m 3 A1 (Fig. 3). In order to make valid comparisons between effects of A1 on growth and o n C a 2+ influx, 45Ca-influx measurements were made on shoot cells similar to those used for measuring growth. Influx of C a 2+ to growing whorl cells over the first 2 h of exposure to A1 was inhibited by 50% by 0.1 m o l . m 3 A1 at p H 4 . 4 (Table 1). In comparison, La 3+ inhibited the influx in these cells by 90% at the same pH (Table 1). Qualitatively similar effects of A1 and La 3+ were found for mature whorl cells from lower down on the plant (Table I). The Ca 2+ influx in whorl cells was less affected when measured over a 20 h incubation in 45Ca + La 3+ or 45Ca + A1 ; Ca 2 + influx under these conditions was inhibited by 73% and 11%, respectively. In Chara, Ca2+-permeable channels in the plasma membrane which open during an action potential allow

0.4 0

.......... o ~ ~ . _ ~ .

~ - ?-PF '

0

'

0.05

,

0.10

,

0,15

0.20

AI (tool m 3) Fig. 3. Effect of A1 on Ca 2+ influx to the vacuole of mature internodal cells of Chara corallina. Cells were pretreated in C P W + A1 at p H 4 . 4 with (0 o) or without n---n supplementary K + (20 mol- m 3) for 1 h. 45Ca-influx time = 2 h + K +

free Ca 2+ in the cytoplasm to rise t o a concentration high enough to stop protoplasmic streaming. It appears that A1 blocks these channels because following pretreatment with high concentrations of A1 (0.2 m o l . m 3, pH 4.0), protoplasmic streaming no longer stopped when cells were electrically stimulated, indicating either that the action potential itself was inhibited or that the Ca 2+ channels were blocked (Fig. 4). The effect of A1 on the action potential required pretreatment for 0.5 h and was re-

R.J. et al. : Calcium/aluminium interaction in Chara

365

2.0 ~:

100

g

80

.g E (3 i1)

60

~

40

o

20

"

1.6

1.2 c::3 o cl

0.8

o 0

0.4

0.05 Ca~ " ~ ' " - - - - - - o ~ ~ 1

2

3

0

i

0

Time (h)

Fig. 4. Effect of A1 (0.2 mol.m-3, pH 4.0) on the transient cessation of protoplasmic streaming that normally follows an action potential (AP) in internodal cells of Chara. The cells were electrically stimulated at each point and observed under a microscope to determine whether streaming had ceased. Rinse solution = CPW pH 4.0. n = 14 cells

4" E -5 E vE

120 o o

~6

100

i

i

r

,

i

0.1 AI (mol m 3)

i

i

i

P 0.2

Fig. 6. Displacement of Ca 2+ from cell walls of Chara by A1. Isolated cell wall segments were incubated for 24 h in CPW pH 4.4 containing either 0.05 or 0.5 mol.m 3 45Ca+A1. Non-bound 45Ca was removed by rinsing in deionised water

160 140

i

"D c

8O

g

_~

6o

o 0

--

40

c~

2.5

(a) +

2.0 1.5 1.0 +AI ~

0.5 0

i

i

i

4.0

i

4.4

i

i

4.8

i 5.2

pH

20 0

i

0

i

0.05 AI

i

0.10 0.15 (molm 3)

i

0.20

Fig. 5. Effect of A1 on the influxes of 22Na and 86Rb into mature internodal cells of Chara corallina. Cells were pretreated in CPW_A1 pH 4.4 for 1 h. Influx time=20min. Control rates (nmol'm-2"s-1): Na + (1 mol.m3) =6.8 +0.7 (10); Rb § (0.1 mol.m -3 +0.2 mol.m 3 K + ) = 1.6+0.2 (8)

'~

(

)

3

5 E S

2

c_

1

b

)

o O

versible with a similar time lag to the onset (Fig. 4). The A1 effect also tended to disappear if the bathing solution w a s n o t agitated occasionally, an o b s e r v a t i o n perhaps related to the fact that the A1 effect could be quickly reversed by increasing the pH.

0

, 4.0

.

.

.

.

4.4

, 4,8

, 5.2

pH

Fig. 7a, b.. pH dependence of the effects of 0.1 mol .m 3 A1 on cell wall binding of Ca 2+ (a) and K+-stimulated Ca 2§ influx (b) in Chara. For the measurement of influx the CPW was supplemented with 20 mol.m -3 K § Influx time=2.5 h. n = 5 (a) and n= 10 (b)

Fluxes o f N a + and Rb +. Influx of N a + was n o t inhibited by A1. At A1 c o n c e n t r a t i o n s b e t w e e n 0.02 and 0.1 m o l m -3 N a + influx was stimulated (Fig. 5). We have previously observed stimulations o f N a + influx in Chara in the presence of La 3+ at c o n c e n t r a t i o n s b e t w e e n 5 and 30 m m o l . m 3. Influx of Rb + was partially inhibited by A1 at concentrations greater than 0.05 m o l . m -3 (Fig. 5)

contained less C a 2+ and this w a s reduced by m o r e than 90% by 0.1 m o l . m -3 A1 (Fig. 6). A time-course of the displacement of Ca 2+ by A1 s h o w e d that at 0.5 m o l . m -3 Ca 2+, the half-time for release was a p p r o x i m a t e l y 25 m i n (data n o t shown).

Effect ofpH. The effects of A1 on the K +-stimulated Ca 2 + Calcium binding in ceil walls. In n o r m a l C P W containing 0 . 5 m o l . m -3 Ca 2+, a p p r o x i m a t e l y 1 . 7 m m o l . m -2 Ca 2+ was b o u n d to cell walls. A d d i t i o n of 0.1 m o l . m -3 A1 reduced the cell wall Ca 2+ content by m o r e than 80% and at 0.2 m o l . m -3 by a l m o s t 90% (Fig. 6). W h e n the Ca 2+ c o n c e n t r a t i o n was reduced to 0.05 m o l . m 3, the cell walls

influx and on cell wall binding of Ca 2+ were strongly dependent on pH. At 0.1 m o l - m 3 A1 binding of Ca 2+ was n o t significantly affected at p H 5.2 but was m a x i m a l ly inhibited at p H 4.4 (Fig. 7a). Similarly the K + - s t i m u lated Ca 2+ influx was n o t inhibited at p H 5.2 but w a s strongly inhibited at l o w e r p H (Fig. 7b). The inhibition

R.J. et al.: Calcium/aluminium interaction in Chara

366 Table 2. Effect of 0.1 m o l . m 3 A1 p H 4.4 on m e m b r a n e potential difference and m e m b r a n e conductance in young internodal cells of Chara corallina. (n = 6)

Control 0.1mol.m3A1

Membrane PD ( m V )

Conductance (S. m 2)

- 122 + 8 --143_+7

0.55 +_0.05 0.44_+0.05

by A1 was complicated by the fact that Ca 2+ influx in the absence of A1 was also sensitive to pH.

Effects of A1 on membrane PD and conductance. Addition of 0.1 m o l - m 3 A1 at pH 4.4 caused cells to hyperpolarise by between 10 and 40 mV and the conductance to fall slightly (Table 2). The changes in PD and conductance occurred gradually over approximately 30 min and were reversible over a similar period (data not shown). Discussion

Chara cells clearly differ from roots in terms of morphology, habitat and growth pattern. However, both are essentially 'organs' for the absorption of nutrients, including Ca 2+, whose main route of entry is almost certainly through CaZ+-permeable channels. Comparison of the characteristics of Ca 2+ influx across the plasma membrane of Chara (Reid and Smith 1992b; Reid et al. 1993) with Ca 2+ channels isolated from the plasma membrane of maize (Marshall et al. 1994) and wheat (Pifieros and Tester 1993) indicates basic similarities between the two plant types. It is perhaps therefore not surprising that the growth of Chara shows precisely the same sensitivity as roots to A1 in terms of concentration and pH dependence and speed of onset. The true value of using Chara to study A1 toxicity lies in the ability to examine directly and unambiguously, the interaction of A1 and Ca z+ in the cell wall and in the plasma membrane, with a high degree of temporal resolution. Inhibition of growth by A1 was measurable in 3 h and probably occurred within a much shorter time span. During this period we have noted changes in three separate CaZ+-related functions, (1) inhibition of Ca 2+ influx, (2) displacement of Ca 2+ from the cell wall, and (3) membrane hyperpolarisation and decrease in membrane conductance. Do any of these short-term changes point to the primary cause of A1 toxicity? Inhibition of Ca 2+ influx. Clearly, A1 can inhibit C a 2+ influx in Chara. It is less clear though that reduced Ca 2+ influx was the principal cause of the inhibition of cell growth. Under normal growing conditions, C a 2+ influx was inhibited by less than 50% by 0.1 m o l . m 3 A1 and the degree of inhibition appeared to diminish with longer exposure to A1. It seems unlikely, for several reasons, that even a 50% reduction in Ca 2+ influx, by itself, would be sufficient to inhibit growth so rapidly. Firstly, the amount of Ca 2+ absorbed by a cell in the time it takes to inhibit growth would be small compared to the intracellular reserves and, secondly, removal of Ca 2+ from the

bathing medium did not cause a noticeable reduction in growth for more than 8 h. It may be argued that true Ca 2+ starvation is difficult to achieve even with thorough washing because of the amount bound in the cell wall. However, we have shown previously (Reid and Smith 1992a) that Ca 2+ influx is not saturated at 0.5 m o l . m 3 (as in CPW) and the rinse regime employed here would almost certainly have reduced the Ca 2+ around the cells to a concentration which would reduce Ca 2+ influx by more than 50%. Thirdly, La 3+ was shown to be a much more potent inhibitor of Ca 2+ influx than A1, but at equivalent concentrations, La 3+ and A1 were equally effective at inhibiting growth. Thus there appears to be little correlation between Ca 2+ supply and inhibition of growth. This is contrary to the suggestion by Rengel et al. (1993) that rapid growth inhibition by A1 is due to inhibition of Ca 2+ uptake necessary for maintenance of high total intracellular C a 2+ levels. It is also inconsistent with a direct role of AI toxicity in perturbation of cytosolic C a 2+ homeostasis proposed by Rengel (1992a, b). Why then is A1 such an effective inhibitor of K +-stimulated Ca 2+ influx and Ca 2+ influx during the action potential? Unfortunately, our understanding of how Ca 2+ enters cells is far from complete. It seems likely that Ca 2+ entry is through ion channels, but the number of channel types, their selectivity for Ca 2+ and what controls their individual openings have hardly been described (Pifieros and Tester 1993, 1994). A simple explanation would be that since both high K + and action potentials cause membrane depolarisation, A1 blocks a voltage-gated class of C a 2+ channels but is less effective at blocking the channels which allow Ca 2+ entry in the hyperpolarised state.

Disruption of cation binding in cell walls. Calcium is an important structural component, providing cross-links between negatively charged side-groups of cell-wall polymers. Aluminium was highly effective at displacing cellwall Ca 2+, reducing the amount bound by up to 90% at the A1 concentrations tested, over a time period which correlates well with the inhibition of growth. Presumably the charged sites vacated by Ca 2+ are then occupied by an equivalent charge density of A1, which from the data shown in Fig. 6 would give a ratio of A13+/Ca z+ in the cell wall of approximately 3 at 0.5 m o l . m 3 Ca2+ and more than 6 at 0.05 m o l . m 3 Ca2+ for an A1 concentration of 0.1 m o l . m 3 at pH 4.4. At this pH however, a significant fraction of the A1 is present as hydroxy-A1 species of lower charge (see Materials and methods). If these species were also bound then the total A1 content of the cell wall would be even higher. There appear to be two different ways in which A1 could disrupt normal cell wall growth, (i) by reducing the Ca 2+ concentration below the minimum requirement for cross-linking of pectic residues and (ii) formation by A1 of cross-linkages which alter the normal cell wall structure. In order to test the former proposition it would be necessary to reduce the cell-wall Ca 2+ content to a similar level to that caused by A1. In practice, this is difficult to achieve, but a small number of treatments were found which could reduce Ca 2+ to this level in Chara. These

367

R.J. et al.: Calcium/aluminium interaction in Chara were E G T A (Reid and Overall 1992), Zn 2+ and La 3+, but in each case, at the concentrations required, cell growth was inhibited or the cells died. Kinraide et al. (1993) used various agents to vary the Ca 2+ activity at the plasma m e m b r a n e and in the cell wall of wheat roots and concluded that Ca 2+ displacement was not the cause of A1 rhizotoxicity. The alternative proposition, that replacement of cell wall Ca 2+ by A1 causes structural deformities remains untested, although distortion of normal root m o r p h o l o g y (tissue lesions and swollen epidermal cells) is a characteristic s y m p t o m of A1 toxicity in roots (Fleming and Foy 1968; Wagatsuma et al. 1987; Kinraide 1988). Attempts to quantify the effects of A1 on Ca 2+ influx in roots have involved the use of stationary and vibrating Ca-selective microelectrodes to monitor changes in Ca 2+ concentrations at the surface of the root resulting from changes in the fluxes of Ca 2+ in (i), the cell wall and (ii), across cell membranes. Because these two fluxes occur in series/parallel, care needs to be exercised in discriminating between A1 effects on the two different processes. Using this technique, H u a n g et al (1992) found that in dead wheat roots, the net Ca 2+ flux returned to zero approximately 15 min after the addition of A1 whereas in live roots, net fluxes continued. In Al-tolerant roots the Ca 2+ influx from 20 m m o l - m 3 Ca2+ was inhibited by 50% by 2 0 m m o l - m 3 A1 whereas in Al-sensitive roots a net efflux was observed. Ryan and Kochian (1993) used a similar but more-sensitive system to examine the effect of A1 at more realistic Ca 2+ concentrations ( C a 2 + = 180 m m o l . m 3; A1 = 4 m m o l .m 3) and found that the cell wall flux appeared to settle after about 15 20 min. They found that the inhibition of the subsequent Ca 2+ influx by A1 was small and in some cases influx was stimulated by A1 under conditions where root growth was inhibited by more than 50%. Interpretation of the Chara influxes, measured using techniques for separation of cell wall, is not complicated by exchanges in the cell wall and the true effects of A1 on m e m b r a n e transport of Ca 2+ are therefore less ambiguous. Nevertheless the conclusions concerning the effects of A1 on Ca 2+ influxes in Chara are basically the same as those of R y a n and Kochian (1993) and R y a n et al. (1992) for wheat roots.

sults are difficult to interpret. It has been argued (e.g. Keifer and Lucas 1982) that Ca 2+ occupies binding sites on the plasma m e m b r a n e that control the opening of m e m b r a n e channels and that displacement of Ca 2+ by other cations (e.g. N a +, K +) causes channels to open. This proposal is supported by the fact that removal of Ca 2 + causes a n u m b e r of changes in the properties of the plasma membrane, most notably m e m b r a n e depolarisation and increased m e m b r a n e conductance (Bisson 1984; Reid et al. 1993), and increased permeability to N a + and K + (Whittington and Smith 1992). The short-term m e m brane responses reported here are opposite to those expected from removal of Ca 2+ and suggest either that A1 displaces Ca 2+ but maintains control over channel activity, or that the responses observed are unrelated to Ca 2 +. In any event, the complexity of transport processes operating across the plasma m e m b r a n e means that it will be difficult to isolate direct effects of A1 at this level.

Disruption of membrane transport. It was shown, that under some circumstances, A1 can inhibit the influx and accumulation of Ca 2+ and reduce transport of Rb +, which in Chara at least can enter through K +-permeable channels (Tester 1988). The small hyperpolarisation of the plasma m e m b r a n e and the decrease in m e m b r a n e conductance following application of A1 is consistent with partial blocking of K + channels. Although in this study we were only interested in the short-term effects of A1, Lindberg et al. (1991) found in sugar-beet roots that while the plasma m e m b r a n e hyperpolarised immediately following addition of A1, the plasma m e m b r a n e in roots cultivated in the presence of A1 was depolarised. However as the m e m b r a n e PD is the net result of a n u m b e r of active and passive ion transfers across the plasma membrane, any of which m a y be either affected directly by A1 or by replacement of m e m b r a n e - b o u n d Ca 2+, such re-

References

Conclusions. Aluminium inhibits growth of Chara at concentrations and over a similar time to those that inhibit growth of roots. The strong p H sensitivity of several key processes points to the trivalent AI as the toxic species. Inhibition of Ca 2+ influx by A1 did not appear to be sufficient to explain the rapid cessation of growth, especially when compared to other treatments which inhibit Ca influx such as removal of external Ca 2+ or addition or La 3+. Similarly, the short-term changes in m e m b r a n e fluxes of N a + and R b + were small, as were effects on m e m b r a n e PD and conductance. Of the three areas of Ca2+/A1 interaction considered, the most likely cause of the inhibition of growth appears to be the disruption of the cell wall due to displacement of Ca 2+ and/or the binding of large amounts of AI. An alternative proposition which has not been considered here is the possible disruption by AI of intracellular Ca 2+ homeostasis. This would require a significant m e m b r a n e penetration by A1, and this has yet to be clearly demonstrated. The technical assistance of Dawn Verlin is gratefully acknowledged. This work was supported by the Australian Research Council.

Asp, H., Berggren, D. (1990) Phosphate and calcium uptake in beech (Fagus sylvatica) in the presence of aluminium and natural fulvic acids. Physiol. Plant. 80, 307-314 Bisson, M.A. (1984) Calcium effects on electrogenic pump and passive permeability of the plasma membrane of Chara corallina. J. Membr. Biol. 81, 59-67 Fleming, A.L., Foy, C.D. (1968) Root structure reflects differential aluminium tolerance in wheat varieties. Agron. J. 60, 172-175 Huang, J.W., Shaft, J.E., Grunes, D.L., Kochian, L.V. (1992) Calcium fluxes in Al-tolerant and Al-sensitive wheat roots measured by Ca-selective microelectrodes. Plant Physiol 98, 230-237 Keifer, D.W., Lucas, W.J. (1982) Potassium channels in Chara corallina. Control and interaction with the electrogenic H+-pump. Plant Physiol. 69, 781 788 Kinraide, T.B. (1988) Proton extrusion by wheat roots exhibiting severe aluminium toxicity symptoms. Plant Physiol. 88, 418-423 Kinraide, T.B. (1991) Identity of the rhizotoxic aluminium species. Plant Soil 134, 167-178

368 Kinraide, T.B., Parker, D.R. (1990) Apparent phytoxicity of mononuclear hydroxy-aluminium to four dicotyledonous species. Physiol. Plant. 79, 283-288 Kinraide, T.B., Ryan, P.R., Kochian, L.V. (1993)A13+-Ca 2+ interactions in aluminium rhizotoxicity. II. Evaluating the Ca 2 ~ -displacement hypothesis. Planta 192, 104 109 Lindberg, S. (1990) Aluminium interactions with K + (86Rb+) and 45Ca fluxes in three cultivars of sugar beet (Beta vulgaris). Physiol. Plant. 79, 275-282 Lindberg, S., Szynkier, K., Greger, M. (1991) Aluminium effects on transmembrane potential in cells of fibrous roots of sugar beet. Physiol. Plant. 83, 54-62 Marshall, J., Corzo, A., Leigh, R.A., Sanders, D. (1994) Membrane potential-dependent calcium transport in right-side-out plasma membrane vesicles from Zea mays L. roots. Plant J. 5, 683 694 Pifieros, M., Tester, M. (1993) Plasma membrane Ca 2+ channnels in roots of higher plants and their role in aluminium toxicity. Plant Soil 155/156, 119-122 Pifieros, M., Tester, M. (1994) Characterisation of a voltage-dependent Ca2+-selective channel from wheat roots. Planta, in press Reid, R.J., Overall, R.L. (1992) Intercellular communication in Chara. Factors affecting transnodal electrical resistance and solute fluxes. Plant Cell Environ. 15, 507-517 Reid, R.J., Smith, F.A. (1992a) Measurement of calcium fluxes in plants using 45Ca. Planta 186, 558-566 Reid, R.J., Smith, F.A. (1992b) Regulation of calcium influx in Chara. Effects of K +, pH, metabolic inhibition and calcium channel blockers. Plant Physiol. 100, 637-643 Reid, R.J., Tester, M. (1992) Measurements of Ca 2+ fluxes in intact plant cells. Phil. Trans. R. Soc. Lond. B 338, 73 82 Reid, R.J., Tester, M., Smith, F.A. (1993) Effects of salinity and turgot on calcium influx in Chara. Plant Cell Environ. 16, 547554

R.J. et al.: Calcium/aluminium interaction in Chara Rengel, Z. (1992a) Role of calcium in aluminium toxicity. New Phytol. 121,499-513 Rengel, Z. (1992b) Disturbance of cell Ca z+ homeostasis as a primary trigger of A1 toxicity syndrome. Plant Cell Environ. 15, 931 938 Rengel, Z., Elliott, D.C. (1992a) Aluminium inhibits net 45Ca uptake by Amaranthus protoplasts. Biochem. Physiol. Pflanz. 188, 177186 Rengel, Z., Elliott, D.C. (1992b) Mechanism of AI inhibition of net 45Ca2~ uptake by Amaranthus protoplasts. Plant Physiol. 98, 632 638 Rengel, Z., Pifieros, M., Tester, M. (1993) Transmembrane calcium fluxes during A1 stress. In: Proceedings of the Third International Symposium on Plant-Soil Interactions at Low pH. Kluwer Ryan, P.R., Kochian, L.V. (1993) Interaction between toxicity and calcium uptake at the root apex in near isogenic lines of wheat (Triticum aestivum L.) differing in aluminium tolerance. Plant Physiol. 102, 975 982 Ryan, P.R., Kinraide, T.B., Kochian, L.V. (1993) AI3+-Ca 2+ interactions in aluminium rhizotoxicity. I. Inhibition of root growth is not caused by reduction of calcium uptake. Planta 192, 98 103 Taylor, G.S. (1991) Current views on the aluminium stress response; the physiological basis of tolerance. Curr Top Plant. Biochem. Physiol. 10, 57 93 Tester, M. (1988) Blockade of potassium channels in the plasmalemma or Chara corallina by tetraethylammonium, Ba 2+, Na + and Cs +. J Membr. Biol. 105, 77-85 Wagatsuma, T., Kaneko, M., Hayasaka, Y. (1987) Destruction process of plant root cells by aluminium. Soil Sci. Plant Nutr. 33, 161-175 Whittington, J., Smith, F.A. (1992) Calcium-salinity interactions affect ion transport in Chara corallina. Plant Cell Environ. 15, 727 733