【作者】 孙清鹏；

【导师】 王小菁； 孙大业；

【作者基本信息】 华南师范大学 ， 植物学， 2003， 博士

【摘要】 Ca²⁺是细胞响应多种刺激反应的重要第二信使。植物受到生物和非生物胁迫以及激素刺激时，胞质游离钙离子浓度([Ca²⁺]_i)升高。茉莉酸（JA）信号也可能通过触发[Ca²⁺]_i升高而调节细胞的生理生化反应。前人有关JA与Ca²⁺关系的研究有两种观点，一种认为JA可以诱导胞内游离钙离子浓度的升高，且钙离子是来源于胞内钙库中的钙离子的释放而非胞外钙离子的流入；另一种观点认为JA不能诱导胞内游离钙离子浓度的变化。 尽管JA与胞质游离钙离子有关，但直接测定细胞[Ca²⁺]_i的变化还鲜见报道。我们将Ca²⁺敏感的酯化荧光探针Fluo-3／AM低温下装载到10 d的拟南芥叶细胞后，利用激光共聚焦显微镜直接测定了JA刺激对细胞中[Ca²⁺]_i变化的影响。结果表明JA处理后1 min内就可诱导[Ca²⁺]_i增加，并于5 min时达到最大。在所试验的范围内，JA的最适浓度为100 μ mol／L。 为探究JA诱导的钙动员的来源、钙调素（CaM）的影响以及Ca²⁺／GaM在JA信号通路中的作用，本文研究了不可过膜的质膜钙通道抑制剂（nifedipine、verpamil）、IP₃受体拮抗剂（heparin）等对JA诱导的[Ca²⁺]_i升高的影响；同时还利用上述拮抗剂、钙调素（CaM）拮抗剂（W-5、W-7）、TMB-8及不同浓度CaCl₂处理10 d的拟南芥幼苗，检测了药物处理对茉莉酸反应基因（JR1或VSP）表达的影响。结果表明： 1)不可过膜的质膜钙通道抑制剂尼群地平（nifedipine）可以抑制JA诱导的[Ca²⁺]i升高，且药剂浓度和[Ca²⁺]i之间存在剂量关系。250 μ mol／L尼群地平对[Ca²⁺]_i升高的抑制作用最强；而另一个抑制剂异博定（verapamil）对JA诱导的[Ca²⁺]i升高则没有显著的影响。IP₃受体拮抗剂肝素（heparin）预处理既可降低胞质的基态荧光强度，也可降低JA诱导的胞质钙离子荧光强度的增加。拟南芥叶细胞经不同浓度肝素预处理后，再用100 μ mol／L JA处理时，其胞内钙离子荧光强度仅升高至与不做任何处理的对照的水平。 2)尼群地平可抑制JA诱导的茉莉酸反应基因JR1的表达且具有浓 ca2+/caM在拟南芥茉莉酸信号通路中的作用度依赖性，而异博定对尸了的表达则没有显著影响。在实验所使用的浓度范围内，肝素和TMB一8均可促进莉酸反应基因尸1的表达。3)钙调素拮抗剂W一7可以抑制尸1及拭夕尸的表达，而对CaM亲和性较低的W一7同系物W一5在同样浓度下对了刀了及只夕尸的表达则没有明显的影响。4)外源10 mmol/L及50 mmol/L CaC12处理也可部分诱导尸1的表达。说明外源Ca卜参与了JA的信号转导。 从以上实验可以推知:JA可以诱导拟南芥叶细胞【CaZ，]主升高，胞外钙和胞内钙库共同参与了JA诱导的钙动员和信号转导。尼群地平敏感的质膜钙通道对JA诱导的JR基因起正调节作用，使基因表达上调，而IP3敏感的胞内钙库中的Ca卜则对其起负调节作用。Ca卜可能通过钙离子结合蛋白CaM或其相关蛋白参与调节JA反应基因的表达。对拟南芥JA信号传导过程中可能存在精细的 Ca2+调控机制进行了讨论。更多还原

【Abstract】 In plant Ca2+ ion acts as an important secondary messenger responding to a variety of environmental stimuli. The increase of cytoplasmic free Ca2+ ([ Ca2+]i) is involved in the signaling network triggered by some stimuli such as biotic and abiotic stress and phytohormones as well. It is possible that exogenous jasmonic acid (JA) can trigger the physiological and biochemical reactions in the cell by the increase of [ Ca2*]i. It has been reported that JA could induce the raising of [ Ca2+]i due to the release of Ca2+ from intracellular store rather than the influx of extracellular Ca2+ from apoplast. Another evidence, however, showed that JA has no effect on the [Ca2+], change.Although there is a relationship between JA stimulation and [Ca2+]i increase, very few evidence has been obtained with a direct measurement of [ Ca2+]i after JA treatment. In the present study, the [Ca2+]i was directly detected by means of fluorescence method in the leaf cell of 10-d -old seedlings in Arabidopsis. After the application of exogenous JA in different concentrations, the Ca2+ fluorescence in the leaf pre-loaded with Fluo-3/AM was measured by confocal laser scanning microscopy (CLSM). The results showed that fluorescence was observed within 1 min after JA treatment and reached a peak at 5 min. The optimun concentration of JA was 100 μmol/L.To evaluate the role of apoplast Ca2+ and calmodulin (CaM) in JA signaling, pharmacological effectors of Ca2+ influx across the plasma membrane, antagonist of IPs receptor and antagonist of CaM were used for the investigation. Moreover, the expression of JA response gene, JR1 and VSP, was also investigated. The main results were as follows:1) With the pretreatment of nifedipine, a nonpermeable L-type channel blocker, the fluorescence of [Ca2+]i induced by JA was inhibited in a dose dependent manner in the leaves and 250μmol/L nifedipine was most effective. Verapamil, another nonpermeable L-type channel blocker, had no significant effect on JA triggered increase of [Ca2+]i. Both basal level and JA-induced increase of [Ca2+]i were reduced upon pretreatment with heparin, an antagonist of IP3 receptor, and the level of [Ca2+]i only reachedthe level of the control which not treated with heparin and JA.2) After JA treatment, the JA response genes JR1 and VSP increased their expression markedly in 10d-old seedings. Nifedipine inhibited the JA-induced JR1 expression in a dose dependent manner, while verapamil had no effect on it. In the range of concentration used in the experiment, heparin and TMB-8, antother antagonist of IP3 receptor, had the positive effect on JA-triggered JR1 expression and the gene expression were up-regulated.3) W-7, an antagonist of CaM, inhibited the expression of JR1 and VSP, but W-5, a less potent analog of W-7, was found to be incapable of inhibiting the expression of JR1 and VSP under the same concentrations as W-7.4) The application of exogenous Ca2+ ion could mimic the JA partially to induce the gene expression of JR1 and support another evidence that apoplastic Ca2+ was involved in the JA response.The data reported here demonstrate that JA can trigger the increase of cytoplasmic free Ca2+ concentration. Apoplast Ca2+ and the intracellular Ca2+ store contributed to the rising of [Ca2*]j in JA signaling pathway. Nifedipine sensitive plasma membrane Ca2+ channel is a positive regulator of JA-induced [Ca2+]i increase and the gene expression of JR1, but the Ca2+ mobilized from IP3 sensitive calcium stores is a negative regulator of JR1 expression. Ca2+ exerts its functions through activate the CaM or CaM related proteins. Fine processes involved in modulation of the JA-triggered Ca2+ signaling network was discussed.更多还原