Aluminum Rapidly Depolymerizes Cortical Microtubules and Depolarizes the Plasma Membrane: Evidence that these Responses are Mediated by a Glutamate Receptor

Abstract

;Efforts to understand how plants respond to aluminum have focused on describing the symptoms of toxicity and elucidating mechanisms of tolerance; however, little is known about the signal transduction steps that initiate the plant's response. Here, we image cortical microtubules and quantify plasma-membrane potential in living, root cells of intact Arabidopsis seedlings. We show that aluminum depolymerizes microtubules and depolarizes the membrane, and that these responses are prevented by calcium channel blockade. Calcium influx might involve glutamate receptors, which in animals are ligand-gated cation channels and are present in the Arabidopsis genome. We show that glutamate depolymerizes microtubules and depolarizes the plasma membrane. These responses, and also the inhibition of root elongation, occur within the first few min of treatment, but are evoked more rapidly by glutamate than by aluminum. Microtubule depolymerization and membrane depolarization, induced by either glutamate or aluminum, are blocked by a specific antagonist of ionotropic glutamate receptors, 2-amino-5-phosphonopentanoate; whereas an antagonist of an aluminum-gated anion channel blocks the two responses to aluminum but not to glutamate. For growth, microtubule integrity, and membrane potential, responses to combined glutamate and aluminum were not greater than to glutamate alone. We propose that signaling in response to aluminum is initiated by efflux of a glutamatelike ligand through an anion channel and the binding of this ligand to a glutamate receptor.

Fluorescence Resonance Energy Transfer-Sensitized Emission of Yellow Cameleon 3.60 Reveals Root Zone-Specific Calcium Signatures in Arabidopsis in Response to Aluminum and Other Trivalent Cations

“…Bar = 100 mm. Sivaguru et al (2003) to block Al 3+ -induced microtubule disruption and membrane depolarization. Interestingly, 1 mM AP-5 on its own was able to induce rapid [Ca 2+ ] cyt transients resembling that of roots treated with Glu alone (Fig.…”

“…These results indicate that roots have a mechanism to prevent Ca 2+ influx into the cytosol despite high Ca 2+ in the external medium, which is altered by Al 3+ application. It was previously reported that the impact of Al 3+ on Arabidopsis root growth and the cytoskeleton could be mediated in part by interactions with Glu signaling pathways (Sivaguru et al, 2003). We asked whether imaging of [Ca 2+ ] cyt using sensitized FRET of YC3.60 could reveal additional insights into the nature of this interaction.…”

“…We also tested whether the Al 3+ -induced biphasic [Ca 2+ ] cyt signature in the root transition zone was affected by the anion channel blocker 5-nitro-2-(3#-phenylpropyl-amino) benzoate (NPPB), since this compound was also shown to prevent Al 3+ -triggered cellular changes in Arabidopsis roots (Sivaguru et al, 2003). NPPB alone did not evoke any [Ca 2+ ] cyt changes in the root transition zone (Fig.…”

“…For long-term Al 3+ exposure growth assays, we followed the protocol of Sivaguru et al (2003), as this proved to be the most effective method for long-term application of Al 3+ on Arabidopsis seedlings growing on agar plates. Six-day-old seedlings expressing YC3.60 of the wild type, alr104, and als3-1 were transplanted onto agar plates perfused with a range of Al 3+ concentrations.…”

“…Six-day-old seedlings expressing YC3.60 of the wild type, alr104, and als3-1 were transplanted onto agar plates perfused with a range of Al 3+ concentrations. The position of the root tip was marked on the back of the petri dish at 0 and 24 h after transplanting, and the increment between marks was measured (Sivaguru et al, 2003). The data were analyzed using ANOVA and Duncan's multiple comparison test.…”