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Keywords:

  • aluminium (Al);
  • calcium (Ca);
  • cytoplasmic Ca2+ homeostasis;
  • cytoskeleton;
  • callose;
  • H+-ATPase;
  • plasma membrane;
  • secondary messenger system

Abstract

  1. Top of page
  2. Abstract
  3. I. Introduction
  4. II. Symptoms of aluminium toxicity
  5. III. Calcium – aluminium interactions
  6. IV. The role of electrical properties of the plasma membrane in calcium–aluminium interactions
  7. V. Oxidative stress
  8. VI. Callose
  9. VII. Cytoskeleton
  10. VIII. Conclusions
  11. Acknowledgements
  12. References

Contents

  • I.
    Introduction  000
  • II.
    Symptoms of aluminium toxicity  000
  • III.
    Calcium – aluminium interactions  000
  • IV.
    The role of electrical properties of the plasma membrane in calcium–aluminium interactions  000
  • V.
    Oxidative stress  000
  • VI.
    Callose  000
  • VII.
    Cytoskeleton  000
  • VIII.
    Conclusions  000
  • Acknowledgements  000

  • References  000

Summary

This review is concentrating on the role of aluminium (Al)-calcium (Ca) interactions in Al toxicity syndrome in plants. Disruption of cytoplasmic Ca2+ homeostasis has been suggested as a primary trigger of Al toxicity. Aluminium causes an increase in cytosolic Ca2+ activity, potentially disrupting numerous biochemical and physiological processes, including those involved in the root growth. The source of Ca2+ for the increase in cytosolic Ca2+ activity under Al exposure is partly extracellular (likely to be due to the Al-resistant portion of the flux through depolarization-activated Ca2+ channels and fluxes through Ca2+-permeable nonselective cation channels in the plasma membrane) as well as intracellular (increased cytosolic Ca2+ activity enhances the activity of Ca2+ release channels in the tonoplast and the endoplasmic reticulum membrane). The effect on increased cytosolic Ca2+ activity of possible Al-related inhibition of the plasma membrane and endo-membrane Ca2+-ATPases and Ca2+ exchangers (CaX) that sequester Ca2+ out of the cytosol is insufficiently documented at present. The relationship between Al toxicity, cytoplasmic Ca2+ homeostasis and cytoplasmic pH needs to be elucidated. Technical improvements that would allow measurements of cytosolic Ca2+ activity within the short time after exposure to Al (seconds or shorter) are eagerly awaited.


I. Introduction

  1. Top of page
  2. Abstract
  3. I. Introduction
  4. II. Symptoms of aluminium toxicity
  5. III. Calcium – aluminium interactions
  6. IV. The role of electrical properties of the plasma membrane in calcium–aluminium interactions
  7. V. Oxidative stress
  8. VI. Callose
  9. VII. Cytoskeleton
  10. VIII. Conclusions
  11. Acknowledgements
  12. References

Aluminium is the most abundant metal and the third most abundant chemical element in the Earth"s crust. Most Al is incorporated into aluminosilicate soil minerals, with only small quantities appearing in soluble forms that can influence living organisms (May & Nordstrom, 1991). However, solubilization of Al-containing minerals is enhanced in acidic environments, where concentrations of Al species can reach levels toxic to organisms.

Aluminium toxicity has been a major factor limiting crop production and yield in many acid soils throughout the world (Foy, 1988). Soil acidification results from: imbalances in nitrogen, sulphur and carbon cycles (Bolan & Hedley, 2003; Tang & Rengel, 2003); excess uptake of cations over anions (Tang & Rengel, 2003); continuous use of ammonia- and amide-containing fertilizers (Mahler et al., 1985); and nitrogen-fixation by legumes (Bolan et al., 1991; Coventry & Slattery, 1991; Tang & Rengel, 2003). Aluminium toxicity is aggravated by soil acidification, the process that also contributes to an increase in the soil area affected by Al toxicity.

Despite decades of extensive efforts to decipher the mechanism(s) of Al phytotoxicity, the primary cause of Al toxicity remains largely speculative and controversial (Delhaize, 1995; Horst, 1995; Kochian, 1995; Rengel, 1996; Matsumoto, 2000; Barcelo & Poschenrieder, 2002). Nevertheless, interactions between Al3+ and Ca2+ have long been implicated in Al phytotoxicity because symptoms of severe Al toxicity in the field resemble those of Ca2+ deficiency, and supplementation of Ca2+ can substantially alleviate Al-stress symptoms (see reviews by Foy, 1988; Rengel, 1992b). Given the important and critical roles of Ca2+ in plant metabolism (Kauss, 1987; Grabski et al., 1998), development (Hepler & Wayne, 1985) and signal transduction (Gilroy et al., 1993; Bush, 1995; Trewavas, 1999; Knight, 2000; Pandey et al., 2000; Plieth, 2001; Sanders et al., 2002), it is not surprising that interactions between Al3+ and Ca2+ have drawn considerable attention in studying Al phytotoxicity. In this review, we will highlight recent progress in understanding the role of Ca2+ ions in Al toxicity to plants, paying particular attention to Al-induced disruption of cytosolic Ca2+ ([Ca2+]cyt) signalling cascades. In doing so, we will build upon previous reviews on the interactions between aluminium and calcium (Rengel, 1992a, 1992b). For other aspects of Al toxicity and resistance, readers are referred to a number of reviews that can be consulted for a more detailed background of the field (Delhaize, 1995; Horst, 1995; Kochian, 1995; Barcelo et al., 1996; Rengel, 1996; Ma, 2000; Matsumoto, 2000; Ma et al., 2001; Barcelo & Poschenrieder, 2002).

II. Symptoms of aluminium toxicity

  1. Top of page
  2. Abstract
  3. I. Introduction
  4. II. Symptoms of aluminium toxicity
  5. III. Calcium – aluminium interactions
  6. IV. The role of electrical properties of the plasma membrane in calcium–aluminium interactions
  7. V. Oxidative stress
  8. VI. Callose
  9. VII. Cytoskeleton
  10. VIII. Conclusions
  11. Acknowledgements
  12. References

Inhibition of root growth is one of the earliest and most dramatic symptoms exhibited by plants suffering from Al stress; this symptom has been observed within hours, or even minutes, of exposure to micromolar concentrations of Al in solution cultures (Ryan et al., 1993; Blancaflor et al., 1998; Sivaguru & Horst, 1998; Zhang et al., 1998; Vazquez et al., 1999; Zhang & Rengel, 1999; Ahn et al., 2001; Ma et al., 2002). However, with prolonged exposure to Al, plants exhibit a myriad of toxicity symptoms on both roots and shoots (Foy, 1988; Rengel, 1996). Indeed, many studies in literature have focused on long-term effects of Al on plants. However, the findings from these studies may shed little light on primary mechanisms of Al toxicity since the responses to long-term Al exposure may not be caused by Al directly, but may rather be a consequence of rapid Al-induced changes in other biochemical and physiological processes.

Aluminium-induced inhibition of root growth often precedes, or coincides with, a decline in cell division (Wallace & Anderson, 1984; Horst, 1995; Frantzios et al., 2001). Therefore, the rapid Al-induced inhibition of root growth is likely to be caused by inhibition of cell elongation rather than cell division. Nevertheless, disruption of cell division due to Al binding to nuclei of root tip cells (Silva et al., 2000), leading to decreased cell production (Lazof & Holland, 1999), is responsible for impedance of root growth during prolonged exposure to Al. Recent studies have shown that inhibition of root growth requires the root apex (Ryan et al., 1993), in particular the distal part of the elongation zone (DTZ) within the apex (Sivaguru & Horst, 1998; Kollmeier et al., 2000), to be directly exposed to Al. These findings indicate that the root apex is a critical site of perception and expression of Al toxicity and resistance. Unfortunately, measurements and observations on the root apex exposed to Al are relatively rare in the literature on Al toxicity and resistance. Most studies report on the whole root systems, providing limited information for understanding of the primary mechanisms of Al toxicity and resistance.

The extent of inhibition of root growth has been widely used as a measure of Al toxicity (Foy, 1988). However, root growth in its nature is a complex and dynamic process. It is therefore likely that a number of biochemical and physiological processes may have already been altered before Al-induced inhibition of root growth. Therefore, root growth per se might not be the most suitable parameter to measure in research of the primary causes of Al toxicity; instead, more direct and rapid responses of plants to toxic Al should be addressed in studying the primary mechanism(s) of Al phytotoxicity.

Numerous biochemical and physiological processes have been demonstrated to be affected within minutes to hours of exposure to Al, such as disturbance of cytoplasmic Ca2+ and pH homeostasis (Lindberg & Strid, 1997; Jones et al., 1998a, 1998b; Zhang et al., 1998, 1999; Plieth et al., 1999; Zhang & Rengel, 1999; Ma et al., 2002), inhibition of the H+-ATPase activity in the plasma membrane of the root tip cells (Ahn et al., 2001, 2002), changes in membrane surface charge (Kinraide et al., 1992, 1994, 1998; Ahn et al., 2002), exacerbated oxidative stress (Cakmak & Horst, 1991; Yamamoto et al., 2002), callose accumulation (Jorns et al., 1991; Zhang et al., 1994; Horst et al., 1997; Massot et al., 1999; Rengel, 2000; Frantzios et al., 2001; Ahn et al., 2002; Collet et al., 2002), disruption of cytoskeleton dynamics (Grabski & Schindler, 1995; Blancaflor et al., 1998; Horst et al., 1999; Sivaguru et al., 1999a; Frantzios et al., 2000, 2001; Alessa & Oliveira, 2001; Schwarzerova et al., 2002), inhibition of cation uptake (Ca2+, Mg2+, K+, NH4+) (Rengel & Robinson, 1989b; Huang et al., 1992; Rengel & Elliott, 1992a, 1992b; Nichol et al., 1993; Lazof et al., 1994; Reid et al., 1995; Ward et al., 1995; Van Praag et al., 1997; Van Oene, 1998), and inhibition of plasmodesmata-mediated intercellular transport (Sivaguru et al., 2000). These changes may directly or indirectly underlie the observed Al toxicity symptoms.

Among biochemical and physiological processes affected by Al, the four that are most likely to involve at least some degree of Al–Ca interaction (disruption of cytoplasmic Ca2+ and pH homeostasis; diminishing activity of H+-ATPase in the plasma membrane coupled to depolarization of the plasma membrane; callose accumulation; and alteration of the cytoskeleton dynamics) will be discussed in the present paper. Calmodulin, the intracellular Ca2+ binding protein, has been suggested as the target for Al binding in early literature (for a review see Haug, 1984), the view which was criticised later (Rengel, 1992a, 1992b). Indeed, more recent work suggested no evidence for involvement of calmodulin as the primary target in Al toxicity (Jorge et al., 2001). Calmodulin therefore will not be covered in the present paper; for research on calmodulin, readers are referred to recent review papers (Snedden & Fromm, 1998, 2001; Luan et al., 2002).

III. Calcium – aluminium interactions

  1. Top of page
  2. Abstract
  3. I. Introduction
  4. II. Symptoms of aluminium toxicity
  5. III. Calcium – aluminium interactions
  6. IV. The role of electrical properties of the plasma membrane in calcium–aluminium interactions
  7. V. Oxidative stress
  8. VI. Callose
  9. VII. Cytoskeleton
  10. VIII. Conclusions
  11. Acknowledgements
  12. References

1. Calcium in plant cells

Calcium is present in soil solutions in relatively high concentrations, the median values being around 1.5 mm (Runge & Rode, 1991). These concentrations far exceed what is considered required for optimal growth (e.g. less than 10 µm for monocots grown in flowing solution culture, Loneragan & Snowball, 1969, and between 10 and 1000 µm Ca for dicotyledonous species).

A pivotal role of cytosolic free Ca2+ ions, acting as the secondary messenger, in transduction of various hormonal and environmental signals to the responsive elements of cellular metabolism has been well established in plant cells in the last decade (Gilroy et al., 1993; Poovaiah & Reddy, 1993; Bush, 1995; Webb et al., 1996; Trewavas & Malho, 1997; Trewavas, 1999; Knight, 2000; Pandey et al., 2000; Plieth, 2001; Sanders et al., 2002). Free cytosolic Ca2+ activities in plant cells at rest are maintained at a very low level, i.e. c. 100–200 nm (Bush, 1995; Webb et al., 1996) to prevent a reaction of Ca2+ with inorganic phosphates; this reaction would otherwise result in a formation of insoluble precipitates severely affecting cell metabolism and thus making Ca2+ cytotoxic (Weber, 1976). By contrast, Ca2+ activities in the cell wall (apoplasm) and several organelles (e.g. vacuoles and endoplasmic reticulum) are three to four orders of magnitude higher than the cytosolic Ca2+ (Clarkson, 1984; DuPont et al., 1990; Evans et al., 1991). When Ca2+-permeable channels embedded in the plasma membrane (cf. Thuleau et al., 1994; Piñeros & Tester, 1995, 1997; Kiegle et al., 2000; Very & Davies, 2000; White et al., 2000; Demidchik et al., 2002a, 2002b) and endo-membranes (cf. Allen & Sanders, 1995; Klusener & Weiler, 1999) become activated in response to a given stimulus, Ca2+ ions can rapidly move down the steep electrochemical gradient into the cytosol through the Ca2+ channels, resulting in a rapid increase in [Ca2+]cyt. ATP-dependent Ca2+ pumps and Ca2+ exchangers (CaX) in the plasma membrane and the endo-membranes are involved in maintaining the low level of [Ca2+]cyt by sequestering cytosolic Ca2+ into different organelles and pumping Ca2+ into the apoplasm (Evans et al., 1991; Gilroy et al., 1993; White, 1998; Hirschi, 2001; Miedema et al., 2001).

2. Displacement of apoplasmic Ca2+ by Al

One of the most dramatic effects of Al on plants is inhibition of cell elongation, the process that involves cell wall relaxation (Cosgrove, 1997). Hence, interactions of Al3+ with the cell-wall components (Rengel & Robinson, 1989a; Blamey et al., 1990; Le Van et al., 1994; Horst, 1995; Reid et al., 1995; Rengel, 1996; Schmohl & Horst, 2000; Blamey, 2001), in particular, displacement by Al3+ of Ca2+ ions that occupy critical sites in the apoplasm, could be, at least partly, responsible for the observed Al toxicity symptoms (Kinraide & Parker, 1987; Caldwell, 1989; Rengel, 1992b; Reid et al., 1995; Ryan et al., 1997a). For example, in Chara internodal cells, between 90% (Reid et al., 1995) and up to 99.99% (Taylor et al., 2000) of cell-wall-bound Ca2+ is displaced by Al. The Ca2+-displacement hypothesis has also been used to explain toxicities of Na+ (Hanson, 1984; Cramer et al., 1985; Yermiyahu et al., 1997b) and H+ (Yan et al., 1992; Kinraide, 1998).

Aluminium binds more strongly to pectin than does Ca2+ (Blamey, 2001). Because Ca2+ plays a key role in cross-linking the pectic materials in the cell wall (Carpita & Gibeaut, 1993), the displacement of pectin-bound Ca2+ would inevitably alter physical properties of the cell wall, including extensibility, rigidity and permeability (Blamey & Dowling, 1995; Horst, 1995; Blamey, 2001), which would be detrimental to cell extension as well as division. Replacement of pectin-bound Ca2+ ions by Al would have a particularly detrimental impact on cells in which pectin is a dominant component of the cell wall (e.g. pollen tube apex, Heslop-Harrison, 1987). In this context, it is interesting to note that Al rapidly induces tip bursting of pollen tube of Chamelaucium uncinatum, and the tip-bursting is markedly reduced by increasing the Ca2+ activity of the incubation medium (Zhang et al., 1999).

Consistent with the Ca2+-displacement hypothesis is that the Al toxicity symptoms are effectively ameliorated by elevated external Ca2+ activities (cf. Kinraide & Parker, 1987; Kinraide et al., 1994; Kinraide, 1998). The ameliorative effect of Ca2+ on Al-treated plants is the results of a decrease in activity of Al3+ at the plasma membrane surface due to shielding or neutralizing of the membrane surface charges (Kinraide et al., 1994; Horst, 1995; Ryan et al., 1997a; Yermiyahu et al., 1997c; Kinraide, 1998). However, an addition of other cations that further decrease the apoplasmic activity of Ca2+ (e.g. Mg2+ or Na+) significantly alleviates the Al-induced inhibition of root growth (Kinraide et al., 1994; Ryan et al., 1997a). Moreover, Ca2+ in the root tips of Al-intoxicated Allium cepa was not displaced by Al to a significant degree as shown by the particle-induced X-ray emission technique (Schofield et al., 1998). These latter findings argue against the displacement of apoplasmic Ca2+ playing a major role in the early stages of Al toxicity. The apparent controversy in the literature on the role of displacement of cell wall Ca2+ by Al in the Al toxicity syndrome is likely to be due to different experimental systems and environmental conditions.

3. Ca2+-permeable channels

The plasma membrane and the endo-membranes contain Ca2+-permeable channels that are gated by voltage, stretch and ligands such as IP3, cADP-R, glutamate, G-proteins, cytoplasmic Ca2+ and calmodulin (Ding et al., 1993; Piñeros & Tester, 1995, 1997; Trewavas & Malho, 1997; White, 1998; Dennison & Spalding, 2000; Miedema et al., 2001; Demidchik et al., 2002a; White & Davenport, 2002). There are also voltage-independent rapidly activating Ca2+-permeable nonselective cation channels in the plasma membrane that can facilitate substantial Ca2+ fluxes (Demidchik et al., 2002a, 2003). In addition, increased [Ca2+]cyt acts as a channel modulator by activating the slow vacuolar Ca2+-release channels embedded in the tonoplast (e.g. Allen & Sanders, 1995; Sanders et al., 2002).

Patch-clamping studies have identified two types of voltage-gated Ca2+-permeable channels in the plasma membrane of higher plants: hyperpolarization-activated (Kiegle et al., 2000; Pei et al., 2000; Very & Davies, 2000; Murata et al., 2001; Klusener et al., 2002; Lecourieux et al., 2002; Perfus-Barbeoch et al., 2002) and depolarization-activated (Thuleau et al., 1994; Thion et al., 1998). A similar depolarization-activated Ca2+-permeable channel has also been characterized in cereal root plasma membranes inserted in artificial planar lipid bilayers (PLBs) (White, 1994, 1997; Piñeros & Tester, 1995, 1997). Depolarization-activated Ca2+-permeable as well as voltage-independent Ca2+-permeable nonselective cation channels have been suggested to play a role in signal transduction (White et al., 2000; Miedema et al., 2001; White & Davenport, 2002), while hyperpolarization-activated Ca2+ channels (Miedema et al., 2001), together with voltage-independent Ca2+-permeable nonselective cation channels (Demidchik et al., 2002a, 2003), are important in acquisition of Ca2+. In mature root epidermal cells, Ca2+ fluxes from the apoplasm through the nonselective cation channels would predominate, while in root hairs and cells in the root elongation zone, Ca2+ fluxes through hyperpolarization-activated channels would be most important for Ca2+ acquisition (Demidchik et al., 2002a).

The depolarization-activated Ca2+-permeable channels are modulated by microtubules in Daucus carota (Thion et al., 1996) and Arabidopsis thaliana cells (Thion et al., 1998). By contrast, activity of the hyperpolarization-activated plasma membrane Ca2+ channels is enhanced by reactive oxygen species (Pei et al., 2000; Murata et al., 2001; Klusener et al., 2002; Lecourieux et al., 2002) and by increased [Ca2+]cyt (Demidchik et al., 2002a). Similarly, the Ca2+-permeable nonselective cation channels in the plasma membrane of Arabidopsis root cells are activated by free oxygen radicals (Demidchik et al., 2003).

Both the hyperpolarization-activated Ca2+ channels (Kiegle et al., 2000; Very & Davies, 2000) and depolarization-activated Ca2+ channels (Piñeros & Tester, 1995; Rengel et al., 1995) are sensitive to Al, but sensitivity of the former is greater (87 ± 7% inhibition by Al, Kiegle et al., 2000) than the sensitivity of the latter (only 44% decrease in the Ca2+ current by Al et al. 1995). However, the sensitivity to Al of depolarization-activated Ca2+ channels has only been tested in the artificial planar lipid bilayers (PLBs). Hence, the effects of Al on the activity of these channels need to be tested in protoplasts. It is important to note that the activity and regulation of Ca2+-permeable channels may be influenced by the cytoplasmic content (present in protoplasts, but absent in plasma membrane patches inserted in the PLBs). The second concern arising from the usage of Triticum aestivum root-cell plasma membrane patches inserted in PLBs is that the artificial membrane was voltage-clamped (see Piñeros & Tester, 1995, 1997). Hence, the Al-caused depolarization of the plasma membrane, as measured in intact Beta vulgaris roots (Lindberg et al., 1991), Al-sensitive Zea mays root elongating cells (by 55 mV, Sivaguru et al., 1999a) and Nicotiana tabacum suspension cells in the log phase of growth (Rengel, 2000), could not have occurred. Given that the recent research has shown that Al depolarizes plasma membrane also in the T. aestivum root tip cells (Ahn et al. unpublished results), it appears possible that this Al-related plasma membrane depolarization may activate Ca2+-permeable channels to mediate influx of apoplasmic Ca2+ and thus influence the level of inhibition (or mitigate it completely) that was measured (Piñeros & Tester, 1995, 1997) upon Al treatment of T. aestivum root-cell membrane patches inserted in the voltage-clamped artificial PLBs. This hypothesis, however, remains to be experimentally tested.

4. The relationship between inhibition of Ca2+ influx into plant cells and Al toxicity

Aluminium rapidly and effectively inhibits Ca2+ influx into intact plant cells (Huang et al., 1992; Ryan & Kochian, 1993; Jones et al., 1995; Reid et al., 1995), protoplasts (Rengel & Elliott, 1992a, 1992b; Rengel, 1994) and the membrane vesicles (Huang et al., 1996; White, 1998). The blockade of Ca2+-permeable channels in the plasma membranes of plant cells could account for the Al-induced inhibition of Ca2+ influx into the plant cells. Indeed, Al has been shown to be a potent antagonist of hyperpolarization-activated Ca2+-permeable channels (Kiegle et al., 2000; Very & Davies, 2000), including hyperpolarization-activated mechano-sensitive Ca2+-permeable channels (Ding & Pickard, 1993; Ding et al., 1993), and also to decrease the current through depolarization-activated Ca2+ channels to about half of the Al-free control (Piñeros & Tester, 1995, 1997; Rengel et al., 1995).

The inhibition of Ca2+ influx into the root apex precedes the inhibition of root growth and any visible symptoms of Al toxicity to plants (Huang et al., 1992; Ryan & Kochian, 1993; Jones et al., 1995; Reid et al., 1995). Moreover, Al inhibits Ca2+ influx into the root apex of Al-sensitive genotypes more than Al-resistant ones (Huang et al., 1992; Ryan & Kochian, 1993). These findings have led to the hypothesis that inhibition of Ca2+ influx by Al could be a primary trigger of Al toxicity to plants (Huang et al., 1992; Rengel, 1992a). However, further studies have revealed that inhibition of root growth in the presence of low concentration of Al3+ may occur without the inhibition of Ca2+ influx, while the addition of other cations (Mg2+ and Na+) improves root growth, even though the Ca2+ influx remains markedly inhibited (Ryan & Kochian, 1993; Kinraide et al., 1994; Jones et al., 1995; Ryan et al., 1997a). These findings indicate that the Al-induced inhibition of Ca2+ influx alone cannot be a critical factor in triggering the Al toxicity syndrome in plants. However, there is no doubt that prolonged inhibition of Ca2+ uptake into Al-treated root cells will disrupt Ca nutrition, which in turn will exacerbate the Al toxicity syndrome.

5. Disturbance of cytoplasmic Ca2+ homeostasis by Al

Environmental stimuli trigger a change, usually an increase, in the [Ca2+]cyt resulting from activation of the plasma membrane and/or endo-membrane Ca2+ channels (Rengel, 1992a, 1992c; Gilroy et al., 1993; Ward et al., 1995; Holdaway-Clarke et al., 2000; Knight, 2000; White et al., 2000; DeWald et al., 2001; Knight, 2002). The increased [Ca2+]cyt acts as a signal to elicit changes in a series of biochemical and physiological processes (Poovaiah & Reddy, 1987; Bush, 1995; Webb et al., 1996; Pandey et al., 2000). With advances in cytosolic Ca2+ imaging and measuring techniques in the last decade (Read et al., 1992; Knight et al., 1993; Webb et al., 1996; Felle & Hepler, 1997; Gilroy, 1997; Rudd & Franklin-Tong, 1999; Roos, 2000; Plieth, 2001), direct involvement of [Ca2+]cyt in transduction of abiotic signals to changes in cellular metabolism has been demonstrated for a range of environmental stresses: salinity (Rengel, 1992c; Bush, 1996; Knight et al., 1997; DeWald et al., 2001), cold (Knight et al., 1991, 1996; Jian et al., 1999; Holdaway-Clarke et al., 2000; Knight, 2002; Moore et al., 2002), heat shock (Gong et al., 1998; Larkindale & Knight, 2002), hypoxia (Bush, 1996; Sedbrook et al., 1996), oxidative stress (Price et al., 1994; McAinsh et al., 1996; Knight et al., 1998; Cessna & Low, 2001), mechanical stimulus (Knight et al., 1992; Bibikova et al., 1997; Legue et al., 1997; Jones et al., 1998a), osmotic shock (Cessna & Low, 2001) as well as Al toxicity (Nichol & Oliveira, 1995; Jones et al., 1998a, 1998b; Zhang et al., 1998; Plieth et al., 1999; Zhang & Rengel, 1999; Rengel, 2000; Ma et al., 2002). Thus, early hypothesis that disruption of Ca2+-related signalling cascades may be a key event in perception of Al toxicity to plants (Rengel, 1992a, 1992b; Rengel et al., 1995) has been substantiated. Similarly, in animal cells, Al interactions with the phosphoinositide signalling system and subsequent disturbance of cytoplasmic Ca2+ homeostasis have been demonstrated to be a primary cause of Al toxicity (Shi et al., 1993; Haug et al., 1994).

An increase in [Ca2+]cyt would have an important role to play in expression of Al toxicity because of increased [Ca2+]cyt making the cell responsive elements unable to respond to transient spikes in [Ca2+]cyt caused by a variety of signals. For example, Al-induced increase in [Ca2+]cyt accounts for the observations that Al inhibits the plasmodesmata-mediated cell-to-cell transport in Al-sensitive T. aestivum roots (Sivaguru et al., 2000) because the closure of plasmodesmata by an increase in [Ca2+]cyt has been elegantly demonstrated (Holdaway-Clarke et al., 2000). The Al-related increase in [Ca2+]cyt could disrupt a range of Ca2+-dependent metabolic processes that are directly or indirectly involved in regulation of cell division and elongation, leading to the observed growth inhibition. However, much work remains to be done to demonstrate unequivocally the causal relationships, if any, between dynamics of [Ca2+]cyt and dynamics of root growth under Al toxicity.

The most direct assessment of the role of disruption of the cytosolic Ca2+ homeostasis by Al in the overall Al toxicity syndrome might arise from experimentally manipulating cytosolic Ca2+ activities using caged Ca2+ probes (i.e., Malho & Trewavas, 1996; Bibikova et al., 1997; Fricker & Oparka, 1999; Greulich et al., 2000) combined with Ca2+-channel modulators, while monitoring the growth and callose response of roots to Al. In addition, manipulation of intracellular inositol trisphoshates (IP3) can be achieved by using polyamine carriers (Ozaki et al., 2000). These experimental approaches are currently being pursued in the first author's laboratory.

6. Aluminium–calcium interactions in various experimental systems

The involvement of [Ca2+]cyt in Al phytotoxicity has been studied in a range of experimental systems (Nichol & Oliveira, 1995; Lindberg & Strid, 1997; Jones et al., 1998a,b; Zhang et al., 1998; Plieth et al., 1999; Zhang & Rengel, 1999; Rengel, 2000; Ma et al., 2002). Most of these studies used Ca2+-sensitive fluorescent dyes and Ca2+ imaging techniques to detect Al-induced changes in [Ca2+]cyt in several plant species (Table 1). All except one of the reports published so far demonstrated an increase in cytosolic Ca2+ activity as a consequence of Al toxicity, e.g. in excised Hordeum vulgare roots (Nichol & Oliveira, 1995), A. thaliana root hairs (Jones et al., 1998a), intact T. aestivum (Zhang et al., 1998; Zhang & Rengel, 1999) and Secale cereale roots (Ma et al., 2002), and protoplasts isolated from T. aestivum roots (Lindberg & Strid, 1997) (Table 1). However, regardless of increase or decrease in [Ca2+]cyt, the fact remains that all plant experimental system tested so far responded to the Al toxicity stress by altering the cytosolic Ca2+ homeostasis (as predicted by Rengel, 1992a). Even the response of pollen to Al toxicity (tube tip bursting) is consistent with a disturbance of cytosolic Ca2+ homeostasis caused by Al ions (Zhang et al., 1999).

Table 1.  Results of published papers on the effects of Al on [Ca2+]cyt and cytoplasmic pH in various experimental systems
SpeciesPlant partsCa2+ measurement techniqueChanges in [Ca2+]cytChanges in cytoplasmic pHReferences
  1. nd, not determined.

T. aestivumIntact root tipsFluo-3 imaging via confocal microscopyIncreasendZhang et al. (1998); Zhang & Rengel (1999)
S. cerealeIntact root tipsFluo-3 imaging via confocal microscopyIncreasendMa et al. (2002)
A. thalianaRoot hairsIndo-1 imaging via confocal microscopyIncreasendJones et al. (1998a)
A. thalianaExcised whole rootsFluorometryAl/low pH: transient increase. Long-term: Al diminished an increase caused by low pHIncrease by AlPlieth et al. (1999)
H. vulgareExcised whole rootsFluo-3 imaging via confocal microscopyIncreasendNichol & Oliveira (1995)
N. tabacumSuspension cellsFluo-3 imaging via confocal microscopyIncreasendRengel (2000)
SL line
N. tabacumSuspension cellsIndo-1 imaging via confocal microscopyDecreasendJones et al. (1998b)
BY-2 line
T. aestivumProtoplastsFura-2 imaging via confocal microscopyIncreaseDecreaseLindberg & Strid (1997)

Cell suspensions In cell suspensions, the SL cell line of N. tabacum showed an increase in [Ca2+]cyt (Rengel, 2000), while BY-2 suspension-cultured cells of N. tabacum decreased [Ca2+]cyt from 256 nm to 64 nm within 10 min of exposure to 200 µm AlCl3 (at pH 4.5) (Jones et al., 1998b). It should however, be mentioned that the nonchlorophyllous SL cell line derived from N. tabacum cv. Samsun (Nakamura et al., 1988) has been much better characterized with respect to responses to Al toxicity (e.g. Yamamoto et al., 1994, 1996, 1997; Chang et al., 1999; Sivaguru et al., 1999b) compared to the BY-2 N. tabacum cell line used by Jones et al. (1998b). Prolonged exposure to Al caused a marked increase in Fluo-3 fluorescence in the log-phase N. tabacum SL cells (Rengel, 2000, Sivaguru et al. unpublished results). The response of [Ca2+]cyt in N. tabacum cells to Al was concentration-dependent (a greater increase in [Ca2+]cyt was found after exposing log-phase cells to 100 µM compared to 50 µM Al). Interestingly, the growth phase has an obvious influence on the relationship between [Ca2+]cyt and Al, with stationary-phase cells not showing a measurable change upon Al exposure (Rengel, 2000, Sivaguru et al. unpublished results).

Protoplasts The treatment with AlCl3 (80 µm, pH 5.0) elicited a transient (2 min in duration) increase in [Ca2+]cyt from 165 nm to 224 nm in protoplasts derived from T. aestivum root apices (Lindberg & Strid, 1997). The similar increase in [Ca2+]cyt was concurrent with a decrease in cytoplasmic pH. The Al-induced increase in [Ca2+]cyt was noted in protoplasts derived from either the Al-resistant or Al-sensitive T. aestivum genotypes (Lindberg & Strid, 1997).

Given that interactions between Al and the cell wall could play an important role in Al phytotoxicity to plants (Rengel & Robinson, 1989a; Blamey et al., 1990; Le Van et al., 1994; Horst, 1995; Reid et al., 1995; Rengel, 1996; Schmohl & Horst, 2000; Blamey, 2001), the Al-related changes in [Ca2+]cyt of wall-free protoplasts are not physiologically relevant to Al-induced changes in the root growth. Hence, studies on protoplasts (e.g. Lindberg & Strid, 1997) may provide limited information about the role of [Ca2+]cyt in Al-related changes in biochemical and physiological processes of intact root cells that ultimately lead to cessation of root growth.

Cells with polar growth There was a sustained increase in tip-localized [Ca2+]cyt of root hairs of wild-type and Al-sensitive, but not of Al-resistant mutant of A. thaliana when the root hairs were treated with 0.1 mm AlCl3 (pH 4.5) (Jones et al., 1998a). Lanthanum, a commonly used Ca2+ channel blocker, induced a reduction in the tip-localized [Ca2+]cyt and a concomitant inhibition of root-hair growth. The decrease in tip-localized cytoplasmic Ca2+ activity and inhibition of growth in the presence of La3+ was reversible upon removal of La3+. By contrast, the Al-induced increase in tip-localized cytoplasmic Ca2+ activity and inhibition of growth was not readily reversible when Al was removed (Jones et al., 1998a).

It has been well established that elongation of root hairs and pollen tubes is strongly dependent upon the existence of a tip-to-base Ca2+ gradient that results from Ca2+ influx into the tips (Pierson et al., 1994; Herrmann & Felle, 1995; Holdaway-Clarke et al., 1997; Taylor & Hepler, 1997; Wymer et al., 1997). It was therefore unexpected that the inhibition of root hair growth in A. thaliana by Al was accompanied by enhancing rather than diminishing the tip-localized Ca2+ gradient (Jones et al., 1998a). However, the tip growth in Lilium longiflorum pollen tubes was inhibited by the treatment with Yariv phenylglycoside [(β-d-Glc)3], which binds arabinogalactan proteins (AGPs), while the tip-localized [Ca2+]cyt was increased (Roy et al., 1999). Therefore, disruption of assembly of the cell wall material may account for the inhibition of tube elongation. Whether a similar mechanism is responsible for the Al-induced inhibition of root-hair growth and an increase in tip-localized [Ca2+]cyt in A. thaliana remains to be tested.

The Al-induced increase in tip-localized [Ca2+]cyt of root hairs may only be restricted to cells exhibiting tip growth and may not necessarily occur in apical cells of main roots that exhibit diffuse growth, thus decreasing the importance of studying [Ca2+]cyt in root hairs in relation to Al-caused root growth inhibition. There are other instances of one signal causing different Ca-related responses in different cell types in roots (e.g. mechanical stimulus induced a transient increase in [Ca2+]cyt in main root cells Legue et al., 1997), but elicited a decrease in [Ca2+]cyt in root hair cells (Bibikova et al., 1997; Jones et al., 1998a).

Aequorin-expressing Arabidopsis Effects of Al and low pH on [Ca2+]cyt were studied in excised roots of A. thaliana expressing the calcium-activated photoprotein, aequorin (Plieth et al., 1999). The authors demonstrated a rapid increase in [Ca2+]cyt of roots upon exposure of excised roots to low pH (pH < 4.5), but this low-pH-elicited increase in [Ca2+]cyt was markedly inhibited by AlCl3 (100 µm, pH 4.0). When the roots were treated with low pH and Al simultaneously, a very small transient increase in [Ca2+]cyt was observed. These authors argued that an impairment of the Ca2+-mediated plant defence responses to low pH is a primary phytotoxic effect of Al. However, the authors did not compare effect of low pH on root growth in the absence and presence of Al.

The use of fluorometry and transgenic, aequorin-expressing plants to monitor response of [Ca2+]cyt to Al has some advantages over Ca2+ imaging technique in which a Ca2+-sensitive fluorescent dye has to be loaded into the cells (cf. Zhang et al., 1998), but the changes in [Ca2+]cyt measured by fluorometry in aequorin-expressing plants represent an average of [Ca2+]cyt of all root cells. Because it is root apex that is a critical site of Al toxicity to plants (Ryan et al., 1993; Sivaguru & Horst, 1998), Al-induced changes in [Ca2+]cyt of root apical cells, rather than the average responses of the whole roots, should be more pertinent to Al-induced alterations of the root growth. Root apical cells account for a small percentage of root cells; therefore, if a response of [Ca2+]cyt to Al in root apical cells differs from that of mature root cells, a true change in [Ca2+]cyt of root apical cells will not be reflected in measurements of [Ca2+]cyt in the whole-root cells, especially because the same stimulus may have a different effect on the [Ca2+]cyt in different root cell types (e.g. mechanical stimulus, Bibikova et al., 1997; Legue et al., 1997; Jones et al., 1998a).

Intact root apices Direct evidence showing the involvement of [Ca2+]cyt in Al phytotoxicity comes from a recent studies with intact root apical cells of: near-isogenic T. aestivum lines differing in Al resistance at a single locus (Zhang & Rengel, 1999); and S. cereale (Ma et al., 2002), by visualizing the Ca2+-sensitive probe Fluo-3 with confocal laser scanning microscopy (Zhang et al., 1998). A sustained increase in the [Ca2+]cyt of root apical cells in the Al-sensitive near-isogenic T. aestivum line (Fig. 1) was observed when roots were exposed to 50 µm AlCl3 (pH 4.2). Exposure of the Al-resistant T. aestivum line to the same Al concentration resulted in only a slight increase in [Ca2+]cyt (Fig. 2), and 100 µm AlCl3 was required for a response similar to that observed in the Al-sensitive line at 50 µm Al (Fig. 3). The response in S. cereale root tips exposed to 100 µm AlCl3 was similar to the response of Al-resistant T. aestivum line at the same Al concentration (Ma et al., 2002). In both plant species, the increase in [Ca2+]cyt was closely correlated with Al-induced inhibition of root growth. Furthermore, the increase in [Ca2+]cyt was relatively slow (e.g. detectable after 10 min of Al exposure) and moderate (50 µm Al treatment increased [Ca2+]cyt by 48% and 27% in Al-sensitive and Al-resistant T. aestivum lines, respectively) (Zhang & Rengel, 1999), while [Ca2+]cyt was increased by 46% after 10 min exposure of S. cereale roots to 100 µm Al (Ma et al., 2002). Such a slow response may be due either to real slowness of the Al effect on the cell metabolism, or inadequate technical capacity at present to detect and measure Al-related increase in [Ca2+]cyt after a short Al exposure (milliseconds to seconds).

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Figure 1. Confocal images of Fluo-3 fluorescence showing the Al-induced increase in the fluorescence intensity (i.e. an increase in intracellular Ca2+ activity) of meristematic cells in roots of Al-sensitive near-isogenic Triticum aestivum line (ES8) treated with 50 µm AlCl3 (pH 4.2). The first layer of cortical cells (30–50 µm from the root surface) within 500 µm of the root apex was shown. Pseudocolour was used to enhance visualisation of Fluo-3 fluorescence intensity, with dark being minimum and white the maximum intensity. For further details see Zhang & Rengel (1999). Images were taken before exposure to Al (a), and after exposure to Al for 10 min (b), 20 min (c), 30 min (d), 40 min (e) and 60 min (f).

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image

Figure 2. Confocal images of Fluo-3 fluorescence showing the Al-induced increase in the fluorescence intensity (i.e. an increase in intracellular Ca2+ activity) of meristematic cells in roots of Al-resistant near-isogenic Triticum aestivum line (ET8) treated with 50 µm AlCl3 (pH 4.2). The first layer of cortical cells (30–50 µm from the root surface) within 500 µm of the root apex was shown. Pseudocolour was used to enhance visualisation of Fluo-3 fluorescence intensity, with dark being minimum and white the maximum intensity. For further details see Zhang & Rengel (1999). Images were taken before exposure to Al (a), and after exposure to Al for 20 min (b), 30 min (c), 40 min (d), 50 min (e) and 60 min (f).

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image

Figure 3. Confocal images of Fluo-3 fluorescence showing the Al-induced increase in the fluorescence intensity (i.e. an increase in intracellular Ca2+ activity) of meristematic cells in roots of Al-resistant near-isogenic Triticum aestivum line (ET8) treated with 100 µm AlCl3 (pH 4.2). The first layer of cortical cells (30–50 µm from the root surface) within 500 µm of the root apex was shown. Pseudocolour was used to enhance visualisation of Fluo-3 fluorescence intensity, with dark being minimum and white the maximum intensity. For further details see Zhang & Rengel (1999). Images were taken before exposure to Al (a), and after exposure to Al for 40 min (b) and 60 min (c).

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7. Sources of Ca2+ for an Al-caused increase in cytosolic Ca2+ activity

Given the high activity of Ca2+ in the apoplasm as well as in intracellular organelles (endoplasmic reticulum and vacuole), the involvement of both extracellular Ca2+ influx and Ca2+ release from intracellular stores were suggested to play a role in increasing [Ca2+]cyt upon challenges by oxidative burst and osmotic shock (Cessna & Low, 2001). Similarly, salinity and drought induced an increase in [Ca2+]cyt resulting from both extracellular Ca2+ influx and intracellular Ca2+ release (Knight et al., 1997). IP3-mediated Ca2+ release from vacuoles (cf. Alexandre & Lassales, 1992; Canut et al., 1993; Allen & Sanders, 1995) could partly be responsible for a drought-induced increase in [Ca2+]cyt (DeWald et al., 2001), as suggested by inhibition of the [Ca2+]cyt increase with inhibitors of phospholipase C (neomysin) and G-protein-activated phospholipase C (U-73122) (Knight et al., 1997).

It is possible that the source of Ca2+ for an increase in [Ca2+]cyt during Al exposure is either extracellular or intracellular (Fig. 4, see also Zhang & Rengel, 1999). A hyperpolarization-activated Ca2+ current through Ca2+-permeable plasma membrane channels (no. 1 in Fig. 4) from the root tip cells of A. thaliana (Kiegle et al., 2000) was inhibited by Al. A likely reason for such Al-related inhibition could be due to depolarization of the plasma membrane, e.g. in intact B. vulgaris roots (Lindberg et al., 1991), Al-sensitive Z. mays root elongating cells (Sivaguru et al., 1999a) and N. tabacum suspension cells in the log growth phase (Rengel, 2000), suggesting that the influx of extracellular Ca2+ into the cytoplasm through hyperpolarization-activated channels would have been inhibited. However, hyperpolarization-activated Ca2+ channels are activated by increased [Ca2+]cyt (Demidchik et al., 2002a). The findings that Al blocks hyperpolarization-activated channels as well as induces an increase in [Ca2+]cyt that, in turn, can activate hyperpolarization-activated Ca2+ channels raise some interesting questions about interactions of the two opposing modulators of channel activity. Further work on testing the activity of hyperpolarization-activated Ca2+ channels in the presence of Al with or without caged Ca2+ probes is warranted. This work will be particularly relevant because hyperpolarization-activated Ca2+ channels predominate in root hairs and cells of the root elongation zone, at least in A. thaliana (Demidchik et al., 2002a), where effects of Al are quick and intense.

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Figure 4. The schematic diagram showing processes affected by Al and sources of Ca2+ for increasing intracellular Ca2+ activity ([Ca2+]cyt) in the early stage of Al stress. The hyperpolarization-activated Ca2+ channels are inhibited by Al (1), while depolarization of the plasma membrane (PM) by Al may enhance Ca2+ fluxes through depolarization-activated Ca2+ channels (2) (these channels are only partially inhibited by Al in the absence of membrane depolarization) (note: thicker arrows indicate a greater flux than thin arrows). The putative effect of Al on the Ca2+-permable nonselective cation channels (3) is unknown at present. An increased flux of Ca2+ from the apoplast would result in an initial increase in [Ca2+]cyt that would activate Ca2+ release channels in the tonoplast (4) and the endoplasmic reticulum (ER) membrane (5), thus increasing ([Ca2+]cyt even further. Aluminium (either extracellular or intracellular) can block the IP3-formation and thus diminish the IP3-generated signal for activation of specific Ca2+ release channels in the tonoplast (6). The effect of Al on Ca2+-ATPases located in the endoplasmic reticulum (7) and the plasma membrane (8) is inadequately documented at present, but preliminary experiments indicate that Al may inhibit these pumps, which would result in increasing [Ca2+]cyt. No knowledge currently exists on the potential effects of Al on the Ca2+ exchangers (CaX) in the tonoplast (9).

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The alternative pathway for Ca2+ influx from the apoplasm is via depolarization-activated Ca2+-channels (Thion et al., 1998) (no. 2 in Fig. 4), which would likely be activated in depolarized Al-treated cells. Indeed, depolarization-activated Ca2+ channels have been suggested to play a role in signal transduction (Miedema et al., 2001). These channels were found to be only partly inhibited by Al (44%, Piñeros & Tester, 1995) (see Ca2+-permeable channels in section Calcium–aluminium interactions). The role of Ca2+-permeable nonselective cation channels in the plasma membrane (no. 3 in Fig. 4) is unclear at present because no assessment of their sensitivity to Al has been reported as yet.

The initial increase in [Ca2+]cyt due to influx of apoplasmic Ca2+ as well as relatively more positive cytosolic environment due to depolarization of the plasma membrane caused by Al would activate the Ca2+-release channels in the tonoplast (Allen & Sanders, 1995; Dobrovinskaya et al., 1999) (no. 4 in Fig. 4) and the endoplasmic reticulum membrane (Klusener & Weiler, 1999) (no. 5 in Fig. 4), allowing more Ca2+ to enter the cytosol from the vacuole and endoplasmic reticulum. However, Al-related inhibition of IP3 production (Jones & Kochian, 1995, 1997) would diminish the activity of the IP3-gated Ca2+ release channels (Alexandre & Lassales, 1992; Canut et al., 1993; Allen & Sanders, 1995) embedded in the tonoplast (no. 6 in Fig. 4), thus decreasing the capacity to elevate [Ca2+]cyt.

Inhibition of Ca2+ efflux pump (Ca2+-ATPase) in endoplasmic reticulum membrane (no. 7 in Fig. 4) and the plasma membrane (no. 8 in Fig. 4) could be an alternative explanation for the Al-induced increase in the [Ca2+]cyt. An increase in [Ca2+]cyt in the log-phase N. tabacum cells treated with cyclopiazonic acid (CPA, an inhibitor of the endoplasmic reticulum Ca2+-ATPase) (Rengel, 2000, Sivaguru et al. unpublished results) and preliminary experiments with Al-related inhibition of the plasma membrane Ca2+-ATPase in Cucurbita pepo (Ahn S J, personal communication) suggests that a decrease in sequestration of cytosolic Ca2+ into the endoplasmic reticulum or into the apoplast may indeed contribute to elevated activity of Ca2+ in the cytoplasm. Therefore, the intracellular stores of Ca2+ and/or lack of sequestration of cytoplasmic Ca2+ into the endo-membranes or out into the apoplast may be the most important sources of Ca2+ for increased [Ca2+]cyt in Al-treated intact cells and organs. A possibility could also not be excluded that the inhibition of CaX exchangers in the tonoplast (no. 9 in Fig. 4) may contribute to increased cytosolic Ca2+.

BY-2 suspension cells of N. tabacum By contrast to other experimental systems where an increase in [Ca2+]cyt was measured (Nichol & Oliveira, 1995; Lindberg & Strid, 1997; Zhang et al., 1998; Zhang & Rengel, 1999; Rengel, 2000; Ma et al., 2002), [Ca2+]cyt was reduced in the BY-2 suspension-cultured cells of N. tabacum treated with 200 µm AlCl3 (Jones et al., 1998b). A similar decrease in the [Ca2+]cyt was also observed when the cells were treated with La3+ and EGTA, suggesting that the decrease in [Ca2+]cyt may be due to an inhibition of Ca2+ influx by either blocking Ca2+-permeable channels in the plasma membrane (La3+) or decreasing Ca2+ activity in the external medium (EGTA) (Jones et al., 1998b). There is general consensus that plant cells can mobilise the intracellular Ca2+ stores to maintain the Ca2+ homeostasis in the cytosol when extracellular Ca2+ sources become limited or the Ca2+-permeable channels are blocked (Trewavas & Malho, 1997). However, the BY-2 suspension cells either do not appear to have the adequate capacity to mobilise intracellular Ca2+ stores, or Al-related blockage of Ca2+-permeable channels in these cells is ineffective. Further work is required to elucidate the mobilization of Ca2+ from intracellular sources during Al exposure. Nevertheless, since the morphology and physiology of suspension-cultured cells differ markedly from those of intact root cells, the Al-induced decrease in [Ca2+]cyt in the suspension-cultured BY-2 cells of N. tabacum may not have the ultimate relevance for a general response of plant cells in intact root tips to toxic Al.

The role of glutamate-gated Ca2+ channels Glutamate-gated Ca2+ channels were suggested to be involved in fluxes of apoplastic Ca2+ into the cytosol as part of the signalling sequence (Dennison & Spalding, 2000). Indeed, these authors have measured a large and fast (within seconds) increase in [Ca2+]cyt due to application of glutamate, facilitating the influx of Ca2+ from the apoplasm through the glutamate-gated channels. By contrast, when the source of Ca2+ for increasing [Ca2+]cyt was from internal stores, the response was much slower (Legue et al., 1997). It is interesting to note that the response to Al toxicity in intact root tips (elevated [Ca2+]cyt) is also relatively slow [measurable after about 10 min (Zhang & Rengel, 1999; Ma et al., 2002)], suggesting a major role for intracellular Ca2+ in increasing [Ca2+]cyt during Al stress. However, the role of glutamate-gated channels, if any, in increasing [Ca2+]cyt under Al exposure has yet to be ascertained.

Given relative nonselectivity of anion channels (Ward et al., 1995), a hypothesis has been put forward that exudation of glutamate may occur through anion channels activated by Al exposure (Dennison & Spalding, 2000). By implication, this increased exudation of glutamate under Al toxicity would cause an increase in influx of apoplasmic Ca2+ into the cell contributing to increased [Ca2+]cyt. However, Al-resistant genotypes exhibit much greater exudation of anions upon exposure to Al (e.g. malate in T. aestivum, Delhaize et al., 1993; Zhang et al., 2001; citrate in Z. mays, Pellet et al., 1995; Ryan et al., 1995a, 1995b; Ryan et al., 1997b; Kollmeier et al., 2001; Piñeros & Kochian, 2001), yet a much slower and smaller increase in [Ca2+]cyt (e.g. in T. aestivumZhang et al., 1998; Zhang & Rengel, 1999) compared with Al-sensitive genotypes. Further work on Al toxicity, glutamate-gated Ca2+-permeable channels and anion channels is required to resolve the apparent inconsistency about the hypothesis proposed by Dennison & Spalding (2000).

IV. The role of electrical properties of the plasma membrane in calcium–aluminium interactions

  1. Top of page
  2. Abstract
  3. I. Introduction
  4. II. Symptoms of aluminium toxicity
  5. III. Calcium – aluminium interactions
  6. IV. The role of electrical properties of the plasma membrane in calcium–aluminium interactions
  7. V. Oxidative stress
  8. VI. Callose
  9. VII. Cytoskeleton
  10. VIII. Conclusions
  11. Acknowledgements
  12. References

The surface charge of the plasma membrane as well as the transmembrane potential across the plasma membrane may influence the activity of Ca2+ channels, including hyperpolarization-activated (Kiegle et al., 2000; Very & Davies, 2000) and depolarization-activated Ca2+ channels (Thuleau et al., 1994; Piñeros & Tester, 1995, 1997; Thion et al., 1998), and may therefore alter the dynamics of cytoplasmic Ca2+ homeostasis. Exposure of plant cells to Al results in a decrease in negativity of the membrane surface charge (Ahn et al., 2001, 2002) as well as in depolarization of the transmembrane potential (e.g. Rengel, 2000; Ahn et al., 2001, 2002), which would affect the activity of these Ca2+ channels.

The membrane surface charge and transmembrane potential are very complex characteristics that are influenced by a myriad of ion transport and other processes (Miyasaka et al., 1989; Rengel, 1996). Among these processes, the plasma membrane H+-ATPase activity may well be the most influential.

1. H+-ATPase

At least 12 isoforms of the plasma membrane H+-ATPase are ubiquitous in higher plants, algae and fungi. They belong to the super-family of P-type ATPases, mainly cation pumps characterized by: formation of an intermediate in the phosphorylated reaction cycle; and inhibition by vanadate. H+-ATPase is composed of a single polypeptide of c. 100 kDa molecular weight (around 950 amino acid residues) (see Jahn & Palmgren, 2002).

The modulation of H+-ATPase activity, and thus H+ pumping, is involved in a wide range of fundamental cellular processes, including the formation and maintenance of an electrochemical gradient across the membrane that serves as the driving force for the secondary ion transport. The H+-ATPase activity plays a role in cell expansion, initiation of cell division, nutritional status, and is a crucial factor for the survival of plants under various environmental stresses (Serrano, 1990; Morsomme & Boutry, 2000; Axelsen & Palmgren, 2001; Palmgren, 2001; Jahn & Palmgren, 2002). Most of these processes are also affected by Al. For instance, a decrease in negativity of the plasma membrane surface charge is correlated with the decline of H+-ATPase activity in the plasma membrane vesicles of plant roots exposed to either Al toxicity (Ahn et al., 2001, 2002), cold shock (Ahn et al., 2000) or salt stress (Suhayda et al., 1990).

The H+-pumping (H+-ATPase activity) across the plasma membrane is likely to play a major role in cytoplasmic pH regulation. The H+-ATPase activity is strongly pH-dependent, with an optimal pH around 6.6. A marked stimulation of H+-ATPase occurs when cytoplasmic pH is lowered. It should be noted that there may be a link between Al toxicity and both Ca2+ and pH homeostasis in the cytoplasm (cf. Plieth et al., 1999). Also, in many cases enhanced H+-pumping activity results in apoplastic acidification (Morsomme & Boutry, 2000), which would exacerbate Al toxicity.

2. Al-related impairment of H+ fluxes across the plasma membrane

Al3+ has a very strong affinity for the plasma membrane surface (e.g. 56-fold higher than Ca2+, Akeson et al., 1989). Aluminium at 50 µm neutralized the surface charge of the plasma membrane and caused a surface potential shift from −20 to +1 mV (Ahn et al., 2001). Several responses of plant cells to Al are related to such alteration of the plasma membrane properties (Rengel, 1996; Kochian & Jones, 1997). Aluminium diminished the H+-ATPase activity in the plasma membrane vesicles prepared from Al-treated seedlings of H. vulgare (Matsumoto, 1988; Matsumoto et al., 1992), T. aestivum (Sasaki et al., 1995; Hamilton et al., 2001) and C. pepo (Ahn et al., 2001, 2002). In addition, the root zone-specific shift of the plasma membrane surface charge to less negative values in C. pepo was recently found as an early symptom of Al toxicity (Ahn et al., 2001, 2002). Aluminium also caused instantaneous plasma membrane depolarization in root cells of an Al-sensitive Z. mays cultivar, with the intensity of depolarization varying with the root growth zone (Sivaguru et al., 1999a).

In intact T. aestivum roots, small Al-related depolarization was found in the Al-sensitive Scout but not in the Al-resistant cultivar Atlas (Miyasaka et al., 1989), while no significant effect of Al on the transmembrane potential was detected in the more recent study on the same cultivars (Huang et al., 1992). However, the surface charge of plasma membrane vesicles isolated from cv. Scout was 26% more negative than that of vesicles from cv. Atlas, allowing more Al to bind to the more negative Scout vesicles (Yermiyahu et al., 1997a), causing greater Al toxicity. Such a result indicates that the importance of the membrane surface charge in Al toxicity should not be underestimated.

The membrane surface charge in the root tips of Al-sensitive T. aestivum near-isogenic line was shifted to less negative values by exposure to 2.6 µm Al, the effect accompanied by a decrease in H+-ATPase activity and H+ transport (Ahn et al. unpublished results). Such neutralization of the membrane surface charge was not observed in the Al-resistant line. It is important to note that the 2.6 µm Al inhibited root growth only in Al-sensitive T. aestivum near-isogenic line but not in the Al-resistant one (Zhang & Rengel, 1999), suggesting a link between growth inhibition and alteration of the plasma membrane properties by Al exposure. A similar link between the dose-dependent inhibition of the H+-ATPase activity by Al and the root growth was observed in root apices of C. pepo (Ahn et al., 2002).

Reports published so far have dealt with the effects of Al exposure on H+-ATPase activity, but the likely effects on the amount of actual H+-ATPase protein have not been tested. However, the most recent immunofluorescence study using the Z. mays plasma membrane H+-ATPase antibody coupled with confocal laser scanning microscopy showed that the amount of H+-ATPase protein in the apical (2–3 mm) and basal (4–5 mm) cells in roots of Al-sensitive T. aestivum near-isogenic line decreased after 4 h of Al treatment (Ahn et al. unpublished results). Indeed, it was proposed that Al could interact with proteins by either competing with other cations for negatively charged binding sites, or inducing conformational changes (Haug, 1984), which could influence ion–protein interactions.

While Al-caused neutralisation of membrane surface charge (Sivaguru et al., 1999a; Rengel, 2000; Ahn et al., 2001, 2002) and the transmembrane potential (Lindberg et al., 1991), with such depolarization potentially enhancing influx of apoplasmic Ca2+ through depolarization-activated channels (Thuleau et al., 1994; Thion et al., 1998), there has been only one study so far (Rengel, 2000) in which Al-caused depolarization of the transmembrane potential and an increase in [Ca2+]cyt were measured simultaneously in the same cells. While there is a possibility that changes in [Ca2+]cyt are relatively minor in terms of overall electrical charge difference across the plasma membrane as well as in the cation activity in the cytoplasm, it is possible that depolarization occurred as a consequence of relative accumulation of positive charge in the form of Ca2+ (cf. Dennison & Spalding, 2000). Comparative measurements of Ca2+ fluxes through various types of Ca2+-permeable channels in relation to the changes in the plasma membrane potential are required to clarify this issue.

V. Oxidative stress

  1. Top of page
  2. Abstract
  3. I. Introduction
  4. II. Symptoms of aluminium toxicity
  5. III. Calcium – aluminium interactions
  6. IV. The role of electrical properties of the plasma membrane in calcium–aluminium interactions
  7. V. Oxidative stress
  8. VI. Callose
  9. VII. Cytoskeleton
  10. VIII. Conclusions
  11. Acknowledgements
  12. References

Under physiological conditions, plants produce significant amounts of superoxide and hydrogen peroxide (e.g. in the electron transport that occurs during photosynthetic reactions or during ATP generation in mitochondria, in the process of β-oxidation of fatty acids in glyoxysomes, etc.) (Bowler et al., 1992). These oxygen species can form hydroxyl radicals that cause lipid peroxidation, protein denaturation, DNA mutation, photosynthesis inhibition, etc. (Bowler et al., 1992; Foyer et al., 1994).

Aluminium induces the expression of the oxidative stress gene in A. thaliana (Richards et al., 1998). Aluminium toxicity and/or Ca deficiency cause peroxidation of membrane lipids and inhibition of root elongation due to stress-related production of highly toxic oxygen free radicals (Cakmak & Horst, 1991). However, lipid peroxidation is observed only after prolonged Al treatment (24 h or more), indicating that it is a consequence of some other primary effect of Al on the membrane structure and function.

Oxidative stress causes an increase in [Ca2+]cyt of animal (Klyubin et al., 1996) as well plant cells (Price et al., 1994; McAinsh et al., 1996; Jones et al., 1998b; Zhang et al., 1998; Cessna & Low, 2001), probably by enhancing the activity of hyperpolarization-activated Ca2+ channels in the plasma membrane (Pei et al., 2000; Murata et al., 2001; Klusener et al., 2002; Lecourieux et al., 2002) and/or Ca2+-permeable nonselective cation channels (Demidchik et al., 2003), thus facilitating the flux of apoplasmic Ca2+ into the cell. Aluminium also causes an increase in [Ca2+]cyt (e.g. Nichol & Oliveira, 1995; Jones et al., 1998a; Zhang et al., 1998; Zhang & Rengel, 1999; Rengel, 2000; Ma et al., 2002) and can induce oxidative stress due to disruption of mitochondria function (Yamamoto et al., 2002). While oxidative stress activates (Pei et al., 2000; Murata et al., 2001; Klusener et al., 2002; Lecourieux et al., 2002), Al toxicity blocks hyperpolarization-activated Ca2+ channels (Kiegle et al., 2000; Very & Davies, 2000) in the plasma membrane, which may influence the relative contribution of extracellular vs intracellular Ca2+ sources to increasing [Ca2+]cyt due to Al stress. Hence, the interaction between the two stresses (Al toxicity and oxidative stress) in influencing [Ca2+]cyt is unclear and warrants further work.

Aluminium caused the inhibition of H2O2-elicited IP3 production (Jones & Kochian, 1995), thus potentially inhibiting IP3-related activation of vacuolar Ca2+ release channels (cf. Alexandre & Lassales, 1992; Canut et al., 1993; Allen & Sanders, 1995), and thus diminishing the contribution from vacuolar Ca2+ to increasing [Ca2+]cyt (Fig. 4). Further work is required to elucidate the role of oxidative stress and IP3 in eliciting disturbance in [Ca2+]cyt as a consequence of exposure to Al.

VI. Callose

  1. Top of page
  2. Abstract
  3. I. Introduction
  4. II. Symptoms of aluminium toxicity
  5. III. Calcium – aluminium interactions
  6. IV. The role of electrical properties of the plasma membrane in calcium–aluminium interactions
  7. V. Oxidative stress
  8. VI. Callose
  9. VII. Cytoskeleton
  10. VIII. Conclusions
  11. Acknowledgements
  12. References

Al-induced callose formation has often been associated with the inhibition of root growth in several monocot plants, including T. aestivum and Z. mays (see Horst, 1995). For instance, increased Al accumulation caused growth inhibition and callose formation in a narrow zone (apical 2-mm segment) of T. aestivum (Rincón & Gonzales, 1992; Tice et al., 1992; Samuels et al., 1997) and in a 1-mm segment of Z. mays root tips (Sivaguru & Horst, 1998). In C. pepo, however, the maximum Al-induced callose accumulation did not coincide with the pattern of Al-induced growth inhibition and Al accumulation (Ahn et al., 2002), potentially reflecting the intrinsic differences between monocot and dicot plants and their differential response to Al.

The positive correlation between Al-induced increase in [Ca2+]cyt and inhibition of root growth (Zhang & Rengel, 1999) is consistent with observations that: Al-elicited callose formation in root tips is directly related to inhibition of root growth in T. aestivum (Zhang et al., 1994) and Triticum turgidum (Frantzios et al., 2001); and the Al-induced callose synthesis is inversely correlated with Al resistance in Z. mays (Horst et al., 1997). Therefore, an Al-induced increase in [Ca2+]cyt serves as a trigger to elicit callose synthesis by activating 1,3-β-glucan synthase (Kauss, 1987; Kauss & Jeblick, 1991). Direct simultaneous measurements of [Ca2+]cyt and formation of callose in T. aestivum root tips exposed to Al would be required to support that hypothesis. The work along these lines is in progress in the first author's laboratory.

A link between increased [Ca2+]cyt levels upon exposure to Al and increased biosynthesis of callose may be applicable to various experimental systems (e.g. Zhang et al., 1994; Wissemeier & Horst, 1995; Ahn et al., 2001, 2002). Al-treated log-phase SL cells of N. tabacum also showed an increase in both [Ca2+]cyt and callose accumulation (Rengel, 2000). However, prolonged exposure to Al did not result in a consistent and a large increase in callose content, indicating that callose synthesis started when [Ca2+]cyt activity increased above a certain threshold, but cells might have a finite capacity to accumulate callose. In the study where a decrease in [Ca2+]cyt was found in the BY-2 cells of N. tabacum (Jones et al., 1998b), callose was unfortunately not measured. We would hypothesize that a decrease in [Ca2+]cyt would prevent any callose accumulation.

There was co-localization of callose accumulation, depolarization of the plasma membrane and an increase in [Ca2+]cyt in the Al-treated suspension cells of N. tabacum (Rengel, 2000). Given the spotty co-localization, there is a possibility that all these phenomena can occur at the plasmodesmata. Indeed, the plasmodesmata have been implicated in the Al toxicity syndrome as a place of Al-induced lesion (Sivaguru et al., 2000).

VII. Cytoskeleton

  1. Top of page
  2. Abstract
  3. I. Introduction
  4. II. Symptoms of aluminium toxicity
  5. III. Calcium – aluminium interactions
  6. IV. The role of electrical properties of the plasma membrane in calcium–aluminium interactions
  7. V. Oxidative stress
  8. VI. Callose
  9. VII. Cytoskeleton
  10. VIII. Conclusions
  11. Acknowledgements
  12. References

Aluminium alters the cytoskeleton system (Blancaflor et al., 1998; Grabski et al., 1998; Horst et al., 1999; Sivaguru et al., 1999a, 1999b; Frantzios et al., 2000, 2001; Alessa & Oliveira, 2001; Schwarzerova et al., 2002) by acting either directly on the cytoskeleton elements or indirectly through Ca2+-related signalling cascades (e.g. increased [Ca2+]cyt depolymerizes microtubules and microfilaments in Z. mays cells, Bokros et al., 1996). In Glycine max root cells, Al-induced an increase in rigidity of actin network (Grabski & Schindler, 1995). A similar change in the actin network in the G. max cells can also be induced by increased [Ca2+]cyt (Grabski et al., 1994). These observations are in line with Al increasing [Ca2+]cyt in intact root tip and root hair cells (e.g. Jones et al., 1998a; Zhang et al., 1998; Zhang & Rengel, 1999; Ma et al., 2002).

Because the depolarization-activated Ca2+-permeable channels are modulated by microtubules in D. carota (Thion et al., 1996) and A. thaliana cells (Thion et al., 1998), disruption of the microtubules increases the fluxes of Ca2+ through depolarization-activated Ca2+ channels (Thion et al., 1996). Therefore, the Al-caused disruption of microtubules (Grabski & Schindler, 1995; Horst et al., 1999; Sivaguru et al., 1999a, 1999b) may enhance the activity of depolarization-activated Ca2+ channels, resulting in increased [Ca2+]cyt (cf. Zhang et al., 1998; Zhang & Rengel, 1999; Ma et al., 2002) (cf. Fig. 4). It should also be borne in mind that Al causes depolarization of the plasma membrane (Lindberg et al., 1991; Sivaguru et al., 1999a; Rengel, 2000; Ahn et al., 2001, 2002), thus potentially enhancing depolarization-activated Ca2+ channels directly (see Ca2+-permeable channels in section Calcium–aluminium interactions).

VIII. Conclusions

  1. Top of page
  2. Abstract
  3. I. Introduction
  4. II. Symptoms of aluminium toxicity
  5. III. Calcium – aluminium interactions
  6. IV. The role of electrical properties of the plasma membrane in calcium–aluminium interactions
  7. V. Oxidative stress
  8. VI. Callose
  9. VII. Cytoskeleton
  10. VIII. Conclusions
  11. Acknowledgements
  12. References

There is mounting evidence that disruption of cytoplasmic Ca2+ homeostasis plays a decisive role in the earliest stages of Al toxicity, with an Al-related increase in cytosolic Ca2+ impairing the capacity of cells to regulate metabolic processes. The source of Ca2+ for increasing cytosolic Ca2+ during the Al stress is both extracellular (Al depolarizes the plasma membrane, thus potentially enhancing the flux of Ca2+ through depolarization-activated channels that are only partially inhibited by Al in the absence of membrane depolarization) and intracellular (increased [Ca2+]cyt stimulates Ca2+-release channels in the tonoplast and the endoplasmic reticulum). Nothing is known about the role of Ca2+-permeable nonselective cation channels as well as Ca2+ exchangers in the Al toxicity syndrome.

Further work is eagerly awaited on: using caged Ca2+ probes with or without Al exposure to ascertain the link between cytosolic Ca2+ and Al toxicity; measurements of Ca2+ fluxes through the depolarization-activated Ca2+ channels and Ca2+-permeable nonselective cation channels during Al exposure; elucidating the interaction between IP3 and Al with respect to effects on dynamics of [Ca2+]cyt; simultaneously measuring callose accumulation, cell elongation and dynamics of [Ca2+]cyt in intact root cells exposed to Al; and devising analytical techniques to allow measurements of Al effects in intact root cells within seconds of Al exposure.

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  1. Top of page
  2. Abstract
  3. I. Introduction
  4. II. Symptoms of aluminium toxicity
  5. III. Calcium – aluminium interactions
  6. IV. The role of electrical properties of the plasma membrane in calcium–aluminium interactions
  7. V. Oxidative stress
  8. VI. Callose
  9. VII. Cytoskeleton
  10. VIII. Conclusions
  11. Acknowledgements
  12. References
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