A possible mechanism and sequence of events that lead to the Al3+-induced [Ca2+]_cyt transients and inhibition of root growth

Abstract

Fluorescence resonance energy transfer (FRET)- sensitized emission imaging of Arabidopsis thaliana roots expressing the yellow cameleon 3.60 calcium (Ca²⁺) reporter showed that the concentration of calcium in the cytosol ([Ca²⁺]_cyt) increased upon aluminum ion (Al³⁺) treatment in root cells from the transition zone within seconds. The Al³⁺-induced [Ca²⁺]_cyt transients were biphasic and were modified by Ca²⁺ channel blockers and by an antagonist of neuronal glutamate receptors, 2-amino-5-phosphonopentanoate (AP-5), and by the anion channel blocker, 5-nitro-2-(3’-phenylpropyl-amino) benzoate (NPPB). The [Ca²⁺]_cyt transients were not uniquely associated with Al³⁺ toxicity mechanisms since lanthanum (La³⁺) and gadolinium (Gd³⁺) also elicited [Ca²⁺]_cyt transients that were similar to those induced by Al³⁺. Here a testable model that describes a possible mechanism and sequence of events that lead to the Al³⁺-induced [Ca²⁺]_cyt transients and inhibition of root growth is proposed. This model can be applied to study also the signal-response coupling of the trivalent ions La³⁺ and Gd³⁺.

Aluminum (Al) is a naturally occurring component of soil particles and is the third most abundant element in the earth's crust.1 In acidic soils, Al dissolves in the soil solution and different ionic Al species form.2,3 The most toxic Al species in acidic soils is ionic Al, Al³⁺.4 Al³⁺ toxicity stems from its interference with a plethora of cellular processes that control plant growth and development.3,5–7

The interactions between calcium (Ca²⁺) and Al³⁺ are well documented in the literature. One of the toxic effects of Al³⁺ on plant growth and development has been ascribed to the disruption of Ca²⁺ homeostasis by Al³⁺.8,9 The fact that Al³⁺ inhibits Ca²⁺ uptake by roots,10 blocks voltage-regulated Ca²⁺ channels,11,12 and affects the concentration of Ca²⁺ in the cytosol ([Ca²⁺]_cyt)13–18 support this view. Ca²⁺ alleviates Al³⁺ toxicity19–22 perhaps by inhibiting Al³⁺ accumulation in the roots and cells.23,24

Rincón-Zachary et al.18 using fluorescence resonance energy transfer (FRET)-sensitized emission to image Arabidopsis thaliana roots expressing the yellow cameleon 3.60 Ca²⁺ reporter demonstrated increases in the concentration of free Ca²⁺ in the cytosol ([Ca²⁺]_cyt) within seconds of Al³⁺ application. Al³⁺ induced distinct [Ca²⁺]_cyt signatures in cells from the different developmental root regions-meristem, elongation and maturation zones. The [Ca²⁺]_cyt signature in the transition zone, which is the most Al-sensitive root region,25 was biphasic and was modified by treatments that chelate external Ca²⁺ (EGTA), block Ca²⁺ entry through the plasma membrane (verapamil), by an antagonist of neuronal glutamate receptors, 2-amino-5-phosphonopentanoate (AP-5), and by the anion channel blocker, 5-nitro-2-(3′-phenylpropyl-amino) benzoate (NPPB). All of these agents affected the first peak of the Al³⁺-induced [Ca²⁺]_cyt signature by reducing its magnitude or abolishing it. These results support the notion that Al³⁺ interacts with different types of plasma membrane Ca²⁺ channels, causing them to open. Al³⁺-induced [Ca²⁺]_cyt transients were also observed in the Arabidopsis Al-resistant and Al-sensitive mutants alr104 and als3, respectively. In addition, the trivalent ions lanthanum (La³⁺) and gadolinium (Gd³⁺) evoked [Ca²⁺]_cyt signatures in the transition zone of the wild-type Arabidopsis and of the alr104 and als3 roots similar to those elicited by Al³⁺. Hence the authors concluded that the observed [Ca²⁺]_cyt transients were not uniquely associated with Al³⁺ toxicity mechanisms. Al³⁺, La³⁺ and Gd³⁺ appear to elicit the same Ca²⁺ signaling pathway.

I would like to propose a testable model that describes the possible sequence of events during Ca²⁺ signaling triggered by trivalent ions using Al³⁺ as a prototype (Fig. 1). (1) Al³⁺ causes Ca²⁺ channels in cells of the root transition zone to open allowing Ca²⁺ influx into the cells. (2) [Ca²⁺]_cyt rises producing the first peak of the biphasic [Ca²⁺]_cyt signature. (3) Increased [Ca²⁺]_cyt activates internal Ca²⁺ channels located in membranes of internal Ca²⁺ stores such as the vacuole, ER, mitochondria or plastids producing the second peak of the [Ca²⁺]_cyt signature. Ca²⁺-induced Ca²⁺ release from internal stores has been described in plant cells.26 (4) Al³⁺ may permeate plasma membrane Ca²⁺ and non-selective cation channels and interact with internal Ca²⁺ channels allowing Ca²⁺ to be released into the cytosol, contributing to the rise in [Ca²⁺]_cyt. In this context, supporting data come from unpublished results (Leblanc J and Rincón-Zachary M) that show Al³⁺ transport across plasma membrane (PM) vesicles isolated from 5 mm wheat (Triticum aestivum) root tips by aqueous two-phase partitioning27 (Fig. 2). In this experiment isolated PM vesicles were loaded with the fluorescent histochemical aluminum indicator morin (2′, 3′, 4′, 5, 7-pentahydroxyflavone) for 30 min at room temperature and then centrifuged at 100,000 xg for 15 min at 4°C and the pellet was washed twice to remove excess morin. The PM vesicles (25 µg protein mL⁻¹) were then incubated in a 2 mL buffer (250 mM sucrose, 50 mM K₂SO⁴, 1 mM DTT, 5 mM MES-Tris [pH 7.0]) containing different concentrations of Al³⁺ for 10 min at room temperature. Al³⁺uptake by the PM vesicles was monitored by fluorometry (excitation at 420 nm; emission at 475 nm). The results show that PM vesicles isolated from the Al-sensitive wheat cultivar Scout 66 root tips are more permeable to Al³⁺ than those isolated from the Al-tolerant cultivar Atlas 66 (Fig. 2A). In this experiment, the relationship between the rate of Al³⁺ uptake and the Al³⁺ concentration in the solution was linear for both Scout 66 (Y = 0.114X + 0.741, R² = 0.99) and Atlas 66 (Y = 0.108X + 0.193, R² = 0.98) PM vesicles. In addition, Leblanc28 showed that compounds known to block Ca²⁺ channels inhibited Al³⁺ uptake by plasma membrane vesicles (Fig. 2B; Leblanc J and Rincón-Zachary M, unpublished data). La³⁺, verapamil and nifedipine were very effective in inhibiting Al³⁺ uptake by plasma membrane vesicles: 5 µM La³⁺ and 1 mM nifedipine caused 67% and 73% inhibition, respectively, and 1 mM verapamil completely abolished the Al³⁺ uptake by the vesicles. Thus, it is feasible that Al³⁺ permeates non-selective cation channels or/and Ca²⁺ channels. (5) Lastly, the overall [Ca²⁺]_cyt elevation could set off mechanisms that inhibit root growth (e.g., callose synthesis and its deposition in the cell wall, disruption of the cytoskeleton organization, formation of reactive oxygen species, etc.). Testing these hypotheses is underway.

Figures and Tables

Figure 1 A model that describes a possible mechanism and sequence of events that lead to the [Ca²⁺]_cyt transients and inhibition of root growth. (1) Al³⁺ interacts with Ca²⁺ channels in the plasma membrane of root cells in the root transition zone. The Ca²⁺channels open and external Ca²⁺ enters the cytosol. (2) [Ca²⁺]_cyt rises producing the first peak of the biphasic [Ca²⁺]_cyt signature. (3) Increased [Ca²⁺]_cyt activates internal Ca²⁺ channels located in membranes of internal Ca²⁺ stores (e.g., tonoplast, ER, mitochondria or plastids) producing the second peak of the [Ca²⁺]_cyt signature. (4) Al³⁺ permeates the PM through Ca²⁺- and non-selective cation channels. (5) Al³⁺ opens internal Ca²⁺ channels in the tonoplast, ER, mitochondria or plastids and as a result more Ca²⁺ is released into the cytosol. (6) The overall [Ca²⁺]_cyt elevation stimulates mechanisms that inhibit root growth.

Figure 2 Al³⁺ uptake by PM vesicles isolated from 5 mm root tips of both the Al-sensitive cultivar Scout 66 and the Al-tolerant cultivar Atlas 66. (A) Rate of Al³⁺ uptake by PM vesicles incubated in increasing concentrations of Al³⁺. The PM vesicles from the Al sensitive cultivar Scout 66 were more permeable to Al³⁺ than those of the Al-tolerant cultivar Atlas 66. The values are means ± SD. Rates of Al³⁺ uptake are expressed in Fluorescence Intensity Units (FIU) mg⁻¹ protein min⁻¹. (B) Effect of Ca²⁺ channel blockers on the rate of Al³⁺ uptake by PM vesicles s percent of the control. All Ca²⁺ channel blockers tested inhibited the rate Al³⁺ uptake by the PM vesicles in both cultivars. The accumulation of Al³⁺ in the PM vesicles was monitored by measuring the fluorescence emitted by the Al-morin complex as described in the text. Both experiments were repeated three times in triplicate (n = 9). The PM vesicles were pooled from multiple independent membrane isolations in order to obtain enough membrane protein for the assays.

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