Nitric oxide suppresses stomatal opening by inhibiting inward-rectifying K<Stack><Subscript>in</Subscript><Superscript>+</Superscript></Stack> channels in <Emphasis Type="Italic">Arabidopsis</Emphasis> guard cells

We explore nitric oxide （NO） effect on K^＋in, channels in Arabidopsis guard cells. We observed NO inhibited K^＋in, currents when Ca^2＋ chelator EGTA （Ethylene glycol-bis（2-aminoethylether）-N,N,N′,N;tetraacetic acid） was not added in the pipette solution; K^＋in currents were not sensitive to NO when cytosolic Ca^2＋ was chelated by EGTA. NO inhibited the Arabidopsis stomatal opening, but when EGTA was added in the bath solution, inhibition effect of NO on stomatal opening vanished. Thus, it implies that NO elevates cytosolic Ca^2＋ by activating plasma membrane Ca^2＋ channels firstly, then inactivates K^＋in, chartnels, resulting in stomatal opening suppressed subsequently.     

Rearrangements of microtubule cytoskeleton in stomatal closure of Arabidopsis 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 Arabi- dopsis 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 be- came 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 sup- pressed 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 cy- toplasm), finally leading to stomatal closure.     

NO诱导血管平滑肌细胞凋亡中Ca~(2 )的作用

探讨了ＮＯ诱导血管平滑肌细胞凋亡与细胞内游离Ｃａ２＋之间的关系．通过粘附式细胞仪和Ｃａ２＋ 荧光探针Ｆｌｕｏ－３／ＡＭ,检测分析了ＮＯ在供体ＳＮＡＰ的作用下,血管平滑肌细胞中游离Ｃａ２＋浓度的变化; 又通过ＳＮＡＰ与维拉帕米、ＥＧＴＡ、肝素钠、普鲁卡因共同孵育的方法,观测了Ｃａ２＋浓度变化在细胞凋亡中 的作用．得出ＳＮＡＰ能使细胞中游离Ｃａ２＋浓度升高,而胞外Ｃａ２＋内流在其中起主要作用;并且阻断胞外 Ｃａ２＋内流能够抑制ＳＮＡＰ所诱导的血管平滑肌细胞的凋亡．提示了胞内Ｃａ２＋浓度升高可能是ＳＮＡＰ诱导血管 平滑肌细胞凋亡的一条途径．     

Localization of NOS-like protein in guard cells of Vicia faba L. and its possible function

Using the immuno-fluorescence and immuno-gold electron microscope technology, localization of ni- tric oxide synthase (NOS)-like proteins was determined in guard cells of Vicia faba L. NOS is mainly localized in nucleus, cytoplasm, chloroplast, mitochondria and the cell wall of guard cells. Scorch and exogenous JA can enhance the level of nitric oxide (NO) and increase NOS activity in both leaf and epidermis, and the changing pattern of NOS activity was consistent with the change of NO. NOS in- hibitor, L-NAME, inhibited JA-induced NO generation. From the results, we presumed that NO genera- tion from NOS pathway is the main pathway in the stress and JA responses. The pharmacological ex- periment showed that increasing the Ca2 at a suitable concentration promoted leaf NOS activity and the NO level, indicating that NOS activity together with the distribution of NO is Ca2 -dependent. NOS and NO are possibly involved in the regulation of stomatal movement thus playing an important role in plant stress responses.     

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 stomatsl aperture assays revealed that both vinblastine （microtubule-disrupUng drug） and SNP （exogenous NO donor） prevented stomatsl opening in the light, and vinblastine even could enhance the inhibitory effect of SNP, whereas tsxol （a microtubule-stsbilizing agent） was able to reduce this effect; （ii） microtubules in the opening Arabi-dopsis guard cells expressing GFP：a-tubulin-6 （AtGFP：a-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 be- came oblique or exhibited a random pattern; （iv） furthermore, the involvement of cytosolic Ca^2＋ in this event was tested. Stomatal aperture assays revealed that BAPTA-AM （a chelator of Ca^2＋） 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 [Ca^2＋]cyt （free Ca^2＋ concentration in the cytoplasm）, finally leading to stomatsl closure.