Electrochemical Monitoring of Cell Wall-Regulated Transient Extracellular Oxidative Burst from Single Plant Cells
- 2015年第21卷 
植物细胞活性氧爆发在植物的抗病以及信号转导中起着非常重要的作用，植物内活性氧产生及代谢受到复杂而精确的机制调控，从而维持正常的活性氧水平以发挥其生理功能. 然而，在单细胞水平开展活性氧爆发实时监测及其调控机制研究一直受到很大的挑战. 本文以碳纤维微盘电极（CFMDE）为基底电极，利用Nafion的模板效应，采用电化学沉积法制得纳米铂颗粒修饰电极（NPt/Nafion/ CFMDE）；同时采用基于聚二甲基硅氧烷（PDMS）的软光刻技术，制备了一种高效固定植物悬浮细胞的琼脂糖阵列微孔芯片. 使用NPt/Nafion/CFMDE实时监测了单个拟南芥原生质体活性氧爆发，并证明电化学监测活性氧的主要成分为过氧化氢. 在此基础上，采用浅层培养法培养原生质体再生植物细胞壁. 电化学监测结果表明，与单个原生质体相比，植物细胞在受到刺激时释放的过氧化氢量显著降低；然而当采用过氧化物酶抑制剂抑制植物细胞壁上过氧化物酶活性后，植物细胞释放过氧化氢量显著回升. 研究结果表明细胞壁在活性氧爆发过程具有很好的调控功能，可望促进植物细胞活性氧爆发及其调控机制的研究.Oxidative burst by rapid, transient generation of reactive oxygen species (ROS) is a fundamental process for plant disease defense and signal transduction. ROS can be protective or toxic depending on their concentrations, and critical balance between ROS production and scavenging is regulated by complex and perfect mechanisms in plant cells. However, the process of rapid and transient ROS burst from plant cells and their regulatory mechanisms are far from being completely understood, owing to the methodological obstacles in real-time monitoring such a fast process. In this work, we showed the application of microelectrode electrochemistry with high spatiotemporal resolution to monitor the kinetics of ROS burst from single plant cell and to investigate the regulatory functions of cell wall. An agarose chip with numerous microwell arrays was fabricated to efficiently trap individual plant cells for electrochemical detection. The results revealed that the oxidative burst from single Arabidopsis thaliana cells is characterized by numerous transient spikes with fast kinetics. Further experiments indicated that intact plant cells released much less H2O2 molecules with faster kinetics to the extracellular environment compared with single protoplasts, while the ROS level could be recovered when the activity of cell wall peroxidase (POD) was inhibited. The results provide evidence that cell wall participates in regulating transient oxidative burst transient by a mechanism involving cell wall POD machinery at single cell level. We think that the results presented here would facilitate better understanding on ROS burst and their regulatory mechanisms.