油菜素内酯对植物细胞钙离子分布的影响

Effect of Brassinolide on Calcium Ion Distribution of Plant Cell

【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1μmol·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1μmol·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1μmol·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,1μmol·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。

[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,＆amp;nbsp;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-1BR 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 levels of 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.