Rearrangements of microtubule cytoskeleton in stomatal closure of <Emphasis Type="Italic">Arabidopsis</Emphasis> induced by nitric oxide

NO (nitric oxide), known as a key signal molecule in plant, plays important roles in regulation of stomatal movement. In this study, microtubule dynamics and its possible mechanism in the NO signal pathway were investigated. The results were as follows: (i) In vivo stomatal aperture assays revealed that both vinblastine (microtubule-disrupting drug) and SNP (exogenous NO donor) prevented stomatal opening in the light, and vinblastine even could enhance the inhibitory effect of SNP, whereas taxol (a microtubule-stabilizing agent) was able to reduce this effect; (ii) microtubules in the opening Arabidopsis guard cells expressing GFP:α-tubulin-6 (AtGFP:α-tubulin-6) were organized in parallel, straight and dense bundles, radiating from the ventral side to the dorsal side, and most of them were localized perpendicularly to the ventral wall; (iii) under the same environmental conditions, treated with SNP for 30 min, the radial arrays of microtubules in guard cells began to break down, twisted partially and became oblique or exhibited a random pattern; (iv) furthermore, the involvement of cytosolic Ca²⁺ in this event was tested. Stomatal aperture assays revealed that BAPTA-AM (a chelator of Ca²⁺) greatly suppressed the effect of NO on stomatal closure; however, it did not show the same function on stomatal closure induced by vinblastine. When BAPTA-AM was added to the SNP-pretreated solution, the SNP-induced disordered microtubulue cytoskeleton in guard cells underwent rearrangement in a time-dependent manner. After 30 min of treatment with BAPTA-AM, the cortical microtubules resumed the original radial distribution, almost the same as the control. All this indicates that NO may promote rearrangement of microtubule cytoskeleton via elevation of Ca²⁺]_cyt (free Ca²⁺ concentration in the cytoplasm), finally leading to stomatal closure. These authors contributed equally to this work. Supported by the New Star Plan of Science & Technology of Beijin, China (Grant No. 2003B34) and National Natural Science Foundation of China (Grant Nos. 30600318 and 30400228)