油菜素内酯对植物细胞钙离子分布的影响

【目的】明确油菜素内酯对Ca²⁺分布的影响，分析油菜素内酯对影响钙稳态的编码Ca²⁺通道和Ca²⁺-ATPase相关基因表达量的变化，明确油菜素内酯对钙稳态的影响。【方法】采用焦锑酸钙沉淀法对油菜素内酯（brassinosteroid，BR）处理后Ca²⁺的分布进行细胞化学定位；利用real-time PCR技术对调控细胞内Ca²⁺水平的位于细胞质膜、液泡膜和内质网上的编码Ca²⁺-ATPase的基因及位于细胞质膜、液泡膜和溶酶体上的编码Ca²⁺通道基因的表达量进行分析。【结果】在未经BR处理的拟南芥细胞中，Ca²⁺主要分布在细胞壁、细胞间隙和液泡中，细胞质和叶绿体中仅有少量Ca²⁺的分布；在1 µmol·L^-1 BR处理3 h后，Ca²⁺呈聚集状分布在液泡膜和细胞质膜附近，同时细胞质和叶绿体上的Ca²⁺分布增多；BR处理6 h后，细胞质和叶绿体中Ca²⁺分布继续增加，细胞壁中Ca²⁺分布有所减少；BR处理9 h后，细胞质和叶绿体中Ca²⁺分布减少，细胞间隙和液泡中Ca²⁺分布有所增加，但细胞壁中Ca²⁺分布明显减少，说明BR具有移除细胞壁中Ca²⁺的作用。CNGC2和CNGC12是细胞质膜上编码Ca²⁺通道的基因，在1 µmol·L^-1BR处理3 h后，CNGC2和CNGC12的表达量均明显下降；处理6 h后，CNGC2和CNGC12的表达量有所恢复；处理9 h后，CNGC2和CNGC12的表达量明显增加。TPC1和TPC2分别是液泡和溶酶体上钙离子通道相关基因，TPC1和TPC2的表达量在1 µmol·L^-1 BR处理3 h后也表现为明显下降，但TPC1的表达量在BR处理6 h后的表达量明显高于未用BR处理的对照，而TPC2的表达量直到BR处理9 h后才明显升高。可见，BR可阻滞细胞质中Ca²⁺浓度的快速上升，液泡膜上编码Ca²⁺通道基因的表达恢复早于细胞质膜和溶酶体上的Ca²⁺通道基因，说明液泡中Ca²⁺大量进入细胞质的时间早于胞外钙库和溶酶体等胞内细胞器。ACA8和ACA10是定位在细胞质膜上Ca²⁺-ATPase基因，1 µmol·L^-1 BR处理3和6 h后，ACA8和ACA10的表达量没有明显的变化；BR处理9 h后，ACA8和ACA10的表达量明显增加；ACA4和ACA11是液泡膜上编码Ca²⁺-ATPase的基因，BR处理后，ACA4和ACA11的表达量变化与质膜上的ACA8和ACA10的表达变化类似。ACA2是内质网上编码Ca²⁺-ATPase的基因，ACA2的表达量同样在BR处理9 h后出现了表达量的最高峰。可见，BR处理9 h后，Ca²⁺-ATPase表达量增加，把细胞质中高浓度的Ca²⁺泵入细胞间隙、液泡和内质网等胞外和胞内钙库中，调控细胞质中的Ca²⁺稳态。【结论】BR对第二信使Ca²⁺具有调控作用，并可通过对钙稳态调控系统的调控传递信号。

Effect of Brassinolide on Calcium Ion Distribution of Plant Cell

【Objective】The objective of this study is to analyze the effect of Brassinosteroid (BR) on Ca²⁺distribution of plant cell, analyze the influence of BR on the expression level of the genes coding Ca²⁺ channel and Ca²⁺-ATPase, and clarify the effect of BR on calcium homeostasis in Arabidopsis thaliana. 【Method】The effect of BR on Ca²⁺ location in plant cell was studied using the potassium pyruantimonatc technique. The expression level of genes which coded Ca²⁺-ATPase (located in the membrane, vacuole, endoplasmic reticulum) and Ca²⁺ channel (located in membrane, vacuole, lysosome) was studied by Real-time PCR technique.【Result】Under normal condition, Ca²⁺ mainly distributed in cell walls, intercellular space, vacuole, and there were only a few Ca²⁺ distributed in the chloroplast and cytoplasm in plant cells. After 1 µmol·L^-1 BR treatment for 3 hours, Ca²⁺ gathered nearby vacuole membrane and cell membrane, meanwhile, the distribution of Ca²⁺ in the cytoplasm and chloroplast was increased. After BR treated for 6 hours, the Ca²⁺ distribution in the cytoplasm and chloroplast was continue increased, the Ca²⁺ distribution in the cell walls was reduced; after BR treated for 9 hours, the Ca²⁺ distribution in the cytoplasm and chloroplast was decreased, but increased in the intercellular space and vacuole, while the Ca²⁺ distribution in the cell walls was obviously reduced. It suggested that BR had function to release Ca²⁺ from cell wall. CNGC2 and CNGC12 are genes which encoded Ca²⁺ channel in the cell membrane. After 1 µmol·L^-1 BR treated for 3 hours, the expression level of CNGC2 and CNGC12 were all obviously decreased, this indicated that BR could block the extracellular Ca²⁺ transfer into cytoplasm. After 1 µmol·L^-1 BR treated for 6 hours, the expression levelsof CNGC2 and CNGC12 recovered；after 1 µmol·L^-1 BR treated for 9 hours, the expression levels of CNGC2 and CNGC12 were obviously increased. TPC1 and TPC2 are genes encoding Ca²⁺ channel in the vacuole and lysosome, respectively. The expression levels of TPC1 and TPC2 were significantly decreased after 1 µmol·L^-1 BR treated for 3 hours, but the TPC1 expression level was obviously higher than that without BR treated; the expression level of TPC2 was also significantly increased after 1 µmol·L^-1 BR treated for 9 hours. The results showed that BR could block the fast rising of the cytoplasm Ca²⁺ Concentrations, the expression level of the genes coding vacuole Ca²⁺ channel recovered earlier than that of the Ca²⁺ channel genes in the cell membrane and lysosome. ACA8 and ACA10 are genes encoding Ca²⁺-ATPase located in the cell membrane. After 1 µmol·L^-1 BR treated for 3 and 6 hours, the expression level of ACA8 and ACA10 had no significant change. After BR treated for 9 hours, the expression level of ACA8 and ACA10 was significantly increased. ACA4 and ACA11 were genes encoding Ca²⁺-ATPase located in the vacuole membrane, the change of ACA4 and ACA11 expression level was similar to the change of ACA8 and ACA10. ACA2 was one gene encoding Ca²⁺-ATPase located in the endoplasmic reticulum, the expression level of ACA2 also appeared the highest peak after 1 µmol·L^-1 BR treated for 9 hours. The results indicated that the expression level of Ca²⁺-ATPase genes increased after BR treated for 9 hours, and high concentrations cytoplasm Ca²⁺ was infused into extracellular and intracellular calcium store including intercellular space, vacuole, endoplasmic reticulum, etc. So BR could regulate the cytoplasm calcium homeostasis.【Conclusion】The second messenger Ca²⁺ was regulated by BR, and the signaling of BR was conducted through regulating the calcium homeostasis regulation system.