| Biological sciences & biotechnology |
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| Effect of oxalic acid on Ca2+ concentration and signaling pathways in plants. |
| Li Yang1, Zhao Yuan1, Xu Youping2, Wang Jipeng1, Cai Xinzhong1* |
| (1. Institute of Biotechnology, College of Agriculture and Biotechnology, Zhejiang University, Hangzhou 310058, China; 2. Centre of Analysis and Measurement, Zhejiang University, Hangzhou 310058, China) |
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Abstract Sclerotinia sclerotiorum (Lib.) de Bary is one of the most important necrotrophic fungal pathogens. It has a very wide host range, reportedly infecting over 400 species of plants worldwide. S. sclerotiorum causes white mould (stem rot) disease of many important crops, especially oil crops such as rape, soybean, peanut and sunflower. However, so far, the molecular mechanism of pathogenicity of this pathogen has not been fully understood. It is clear that cell wall degrading enzymes (CWDEs) and oxalic acid (OA) are the important pathogenicity factors of S. sclerotiorum. During infection, the pathogen secretes CWDEs to degrade components of host plant cell wall and thus facilitates its infection. OA is of concern as another essential pathogenicity factor. The known roles of OA in pathogenicity include lowering extracellular pH value and thus increasing activity of CWDEs, altering redox status to regulate accumulation of reactive oxygen species (ROS) and programmed cell death (PCD), and having direct toxicity to plant cells. Additionally, OA is thought to be able to chelate calcium in cell wall and thus enhances its degradation by some CWDEs secreted by the pathogen. However, the effect of OA on Ca2+ concentration of the whole cells and Ca2+ signaling pathways remains unclear, which is examined in this study. The dye Fluo4 interacts with Ca2+ inside living cells and can be detected with the green fluorescence by confocal laserscanning microscope. Employing Fluo4 as an indicator of Ca2+ concentration inside cells, the effect of OA on Ca2+ concentration was analyzed. Infiltration of 1 mmol/L OA only weakly lowered the Ca2+ concentration inside leaf cells of Arabidopsis. However, 10 mmol/L OA rapidly and dramatically reduced it. The fluorescent signal was almost undetectable since 10 min post this treatment. This result reveals that the effect of OA on Ca2+ concentration inside plant cells is dependent on their concentrations. Additionally, the effect of OA on gene expression of Ca2+ signaling pathways was examined using realtime quantitative reverse transcriptasepolymerase chain reaction (qRTPCR). The genes under investigation included CRT gene family, which play roles in regulation of Ca2+ homeostasis and signaling and disease resistance, two CDPKs, which are Ca2+ sensors, and CAMTA3, a calmodulinbinding transcriptional factor involved in regulation of plant disease resistance. Expression results showed that the tested genes responded differently to OA treatment. Generally speaking, the expression of two CDPKs and CAMTA3 altered more strongly but in contrast direction in response to OA in comparison with CRT family genes. In addition, these genes were expressed differently in response to OA of different concentrations. The expression level of these genes was much higher in response to OA of higher concentration. In response to 1 mmol/L OA, the expression of three CRT genes and CDPK1 was continuously upregulated by about 10 times for CRT3 and 130 times for CDPK1 at 24 h post treatment (hpt), and the expression of CAMTA3 gene was continuously downregulated to be almost undetectable at 24 hpt, while the expression of CDPK2 gene was significantly upregulated at 6 hpt and then slightly reduced at 24 hpt. In summary, the data of this study reveal that 10 mmol/L OA, which is typically secreted by S. sclerotiorum during infection, rapidly and dramatically reduces Ca2+ concentration inside plant cells. OA may target Ca2+ signaling pathway at some key components such as CRTs, CDPKs and CAMTA3 during plant and S. sclerotiorum interactions.
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Published: 20 March 2014
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草酸对植物体内Ca2+浓度及信号传导途径的影响
从影响植物体内Ca2+浓度及其信号通路的角度分析了草酸的致病作用分子机制.采用Fluo4染色和荧光检测方法，在激光共聚焦显微镜下观察分析了草酸对拟南芥植株体内Ca2+浓度的影响.结果表明：1 mmol/L草酸处理后拟南芥体内Ca2+浓度变化不大；但10 mmol/L草酸处理迅速、强烈地降低了拟南芥体内Ca2+浓度，处理10 min后几乎完全检测不到Ca2+荧光信号.同时，利用实时定量反转录聚合酶链反应技术分析了草酸对3个与Ca2+浓度稳定和信号传导相关的CRT基因以及3个Ca2+信号途径重要基因CDPK1、CDPK2和CAMTA3的表达动态的影响.结果显示：除了处理6 h后CRT3的基因表达外，0.1和1 mmol/L草酸处理对上述基因表达的影响总体趋势一致，但1 mmol/L草酸处理的影响程度更显著；草酸处理对不同基因的表达影响也有显著区别：1 mmol/L草酸处理后CRT基因家族与CDPK1基因表达持续上调，CDPK1基因表达上调倍数显著大于CRT基因；CAMTA3基因表达持续下调，到处理后24 h，表达几乎被完全抑制；而CDPK2基因表达则在处理后6 h显著上调，到处理后24 h略微下降.这些结果说明草酸可通过改变Ca2+浓度及Ca2+信号通路对植物与核盘菌互作起重要的调控作用.
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