灰斑古毒蛾口腔反吐物诱导沙冬青细胞Ca2+内流及H2O2积累

植物对昆虫取食活动进行成功防御的关键,取决于对昆虫口腔反吐物的激发子的快速识别。实验利用无损伤微测系统及激光共聚焦显微镜,研究了沙冬青细胞经灰斑古毒蛾口腔反吐物诱导后Ca2+流及H2O2的变化。结果发现:灰斑古毒蛾口腔反吐物诱导沙冬青细胞Ca2+内流及H2O2的积累,表明Ca2+内流及H2O2的积累是沙冬青细胞对口腔反吐物产生应答的早期响应事件;Ca2+钙通道阻断剂仅部分抑制Ca2+内流,说明Ca2+内流除经过质膜上的Ca2+通道进入细胞外,尚存在其他的内流途径;灰斑古毒蛾口腔反吐物中的某些成分与沙冬青细胞的质膜结合后,诱导质膜上形成允许Ca2+通过的孔道,而GdCl3不能抑制这类孔道的活性。胞外Ca2+螯合剂EGTA完全抑制H2O2的积累,GdCl3预处理仅部分抑制了H2O2的积累,说明灰斑古毒蛾诱导的沙冬青细胞内H2O2的积累依赖于Ca2+内流;抑制剂实验表明,H2O2的积累主要来源于质膜上NADPH氧化酶的作用。

Regurgitant from Orgyia ericae Germar induces calcium influx and accumulation of hydrogen peroxide in Ammopiptanthus mongolicus (Maxim. ex Kom.) Cheng f. cells

The battle between plants and phytophagous insects has lasted for hundreds of millions of years. Over time, plants have evolved sophisticated defence systems to counteract attacks from insects. After being attacked by herbivores, plants quickly generate herbivory-specific signals, and, through complicated networks, these signals then trigger large-scale biochemical and physiological changes. The success of plants in withstanding herbivore attacks depends on their ability to quickly recognize elicitors in insect regurgitant. When herbivorous insects feed on plants, their regurgitant inevitably comes in contact with the wounded plant tissue. Thereby, the insects provide chemical signals that might be involved in the interaction between the attacking insect and the defending plant. In general, the earliest detectable signalling events in plants defence responses include transmembrane ion fluxes and production of reactive oxygen species. In plant cells, the calcium ion is a ubiquitous intracellular second messenger involved in numerous signalling pathways. It is widely acknowledged that calcium flux across cellular membranes plays a key role in triggering and mediating defence mechanisms. Hydrogen peroxide is a common component of the defence responses of plants against herbivore attacks. The non-invasive microelectrode ion flux measurement technique, which can monitor ion/molecule-specific activities non-disruptively, has become a popular tool for studying adaptive responses of plant cells and tissues to a large number of abiotic stresses. Using a non-invasive micro-test system and a confocal laser scanning microscope, Ca²⁺ influx and variation in hydrogen peroxide production induced by the regurgitant of Orgyia ericae Germar were investigated in Ammopiptanthus mongolicus (Maxim. ex Kom.) Cheng f. cells. Experiments were conducted in the Key Laboratory of Forest Silviculture of the State Forestry Administration from June 2010 to April 2011. The results showed that Ca²⁺ influx and hydrogen peroxide accumulation were induced by the regurgitant, indicating that Ca²⁺ influx and variation in hydrogen peroxide production were early signalling events in response to the regurgitant. Ca²⁺ influx was incompletely inhibited by the Ca²⁺ channel blocker GdCl₃, demonstrating that the Ca²⁺ channel on the plasma membrane was not the only route of Ca²⁺ into the cell. Alamethicin, a voltage-gated ion channel-forming peptide mixture derived from the soil fungus Trichoderma viride Pers., induced Ca²⁺ influx, but this alamethicin-induced Ca²⁺ influx was not inhibited by the Ca²⁺ channel blocker GdCl₃. These results provided indirect evidence that one or more components of regurgitant exhibited ion channel-forming activities in the cell plasma membrane, and that the activity of those channels was not inhibited by GdCl₃. Hydrogen peroxide accumulation was completely inhibited by the extracellular calcium chelator EGTA, but GdCl₃ incompletely inhibited hydrogen peroxide production, demonstrating that Ca²⁺ influx was necessary for the production of hydrogen peroxide. Some compounds in oral secretions from O. ericae induced NADPH oxidase activity. The application of regurgitant from O. ericae resulted in immediate and rapid hydrogen peroxide accumulation. The accumulation of hydrogen peroxide in cells in response to the regurgitant of O. ericae was inhibited by the NADPH oxidase inhibitor diphenylene iodonium, demonstrating that hydrogen peroxide was primarily generated from the activation of NADPH oxidase on the plasma membrane.