蚕豆叶保卫细胞和根边缘细胞对2种桉叶挥发物的响应及信号调节

Response and signal regulation of guard cells and root border cells in Vicia faba L.to two Eucalyptus volatiles

【目的】研究在蓝桉(Eucalyptus globulus)和巨桉(E.grandis)2种桉树的挥发油及其共有成分α-蒎烯和桉油精作用下,蚕豆(Vicia faba L.)根边缘细胞和叶保卫细胞的生理变化及保卫细胞内信号调节,探讨2种桉树的化感作用机制。【方法】运用显微技术、细胞化学技术和qRT-PCR技术,测定不同挥发油及其共有成分α-蒎烯和桉油精浓度(2、4、6、8和10μL)处理下蚕豆根边缘细胞和叶保卫细胞的活性以及气孔开度的变化及其保卫细胞内信号分子ROS、NO和Ca^(2+)含量变化。【结果】随着2种桉叶挥发油、α-蒎烯和桉油精处理剂量增加,蚕豆根边缘细胞和叶保卫细胞的活性逐渐降低。叶片NADPH氧化酶基因上调,叶保卫细胞内ROS、NO、Ca^(2+)等信号分子水平升高,细胞核出现多种类型的畸变,TUNEL检测表明叶保卫细胞发生凋亡,当挥发物分别与泛Caspase抑制剂Z-VAD-FMK、Ca^(2+)通道阻断剂(LaCl_(3))、活性氧清除剂抗坏血酸(AsA)和硝酸还原酶抑制剂(NaN3)共同作用于叶保卫细胞时,挥发物引起叶保卫细胞死亡率下降。ROS、NO和Ca^(2+)原位观察显示,与挥发油处理组相比,挥发物与AsA共处理组的NO和Ca^(2+)水平较低,NaN_(3)共处理组ROS和Ca^(2+)水平较低,LaCl3共处理组ROS和NO水平差异不明显。表明ROS与NO串扰,导致叶保卫细胞内Ca^(2+)浓度升高,从而触发叶保卫细胞出现Ca^(2+)依赖性和Caspase依赖性细胞凋亡;此外,蚕豆叶气孔开度随桉叶挥发物处理剂量增加而降低,在微丝聚合抑制剂细胞松弛素B(CB)。NADPH氧化酶抑制剂二联苯碘(DPI)、活性氧清除剂抗坏血酸(AsA)与挥发物共处理时,桉叶挥发物引起的气孔开度减小现象得到缓解,表明叶保卫细胞内ROS爆发,诱发微丝聚合引起气孔关闭。【结论】蓝桉和巨桉通过挥发途径释放化感物质诱发氧化损伤,破坏了根系保护屏障--根边缘细胞结构和功能,以及叶保卫细胞的结构和功能,进而干扰植物吸收作用和光合作用过程,导致受体植物生长受阻。

【Objective】To explore the allelopathy mechanism of Eucalyptus globulus and E.grandis,the effects of two volatile oils from Eucalyptus leaf and their two common components,alpha-pinene and eucalyptol,the physiological changes of root border cells and leaf guard cells of Vicia faba L.and the signal regulation in guard cells were studied to explore the allelopathic mechanism of the two eucalyptus species.【Method】The activity of root border cells and leaf guard cells and stomatal aperture and the regulation of ROS,NO and Ca^(2+)signal molecules in guard cells by different volatile oils and their common componentsα-Concentration of pinene and cineole(2,4,6,8 and 10μL)were studied by using microscopy technology,cytochemistry mothed and qRT-PCR.【Result】With the increase of the treatment dose of volatile oil,α-pinene and eucalyptol,the activities of root border cells and leaf guard cells decreased gradually.The NADPH oxidase gene in leaves was up-regulated,the levels of intracellular signaling molecules such as ROS,NO,Ca^(2+)were increased and various nuclear aberrations appeared in guard cells.TUNEL results showed that the guard cells died in the form of apoptosis.The guard cells mortality was decreased when the volatiles were combined with Z-VAD-FMK(a pan-Caspase inhibitor),LaCl_(3)(a specific calcium-channel blocker),ascorbic acid(AsA,a reactive oxygen scavenger)and NaN_3(a nitrate reductase inhibitor).In situ observation of ROS,NO and Ca^(2+)showed that compared with volatile oil treatment group,the levels of NO and Ca^(2+)in volatiles and AsA co-treated group were lower,and the levels of ROS and Ca^(2+)in volatiles and NaN_(3) co-treated group were lower,and there was no significant difference in ROS and no levels in volatiles and LaCl_(3) co-treated group.The results showed that the crosstalk between ROS and NO led to the increase of Ca^(2+)concentration in guard cells,which triggered Ca^(2+)-dependent and Caspase-dependent apoptosis.In addition,the stomatal aperture decreased with the increase of the treatment dose of Eucalyptus volatiles.The reduction of stomatal aperture caused by Eucalyptus volatiles was alleviated when microfilament polymerization inhibitor Cytochalasin B(CB),NADPH oxidase inhibitor diphenylene iodonium(DPI),active oxygen scavenger ascorbic acid(ASA)were co-treated with volatiles,indicating that ROS burst in guard cells induced microfilament polymerization and caused stomatal closure.【Conclusion】The allelochemicals released by E.globulus and E.grandis through the volatile pathway induced oxidative damage,which damaged the structure and function of guard cells and root border cells with root protective barrier function,as well as absorption and photosynthesis processes,and receptor growth was inhibited.