CAJ | 학술논문

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 Fluo-4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.
종영향식물체내 Ca2+농도급기신호통로적각도분석료초산적치병작용분자궤제.채용 Fluo-4염색화형광검측방법，재격광공취초현미경하관찰분석료초산대의남개식주체내 Ca2+농도적영향.결과표명：1 mmol/L 초산처리후의남개체내 Ca2+농도변화불대；단10 mmol/L 초산처리신속、강렬지강저료의남개체내 Ca2+농도，처리10 min 후궤호완전검측불도 Ca2+형광신호.동시，이용실시정량반전록취합매련반응기술분석료초산대3개여 Ca2+농도은정화신호전도상관적 CRT 기인이급3개 Ca2+신호도경중요기인 CDPK1、CDPK2화 CAMTA3적표체동태적영향.결과현시：제료처리6 h 후 CRT3적기인표체외，0.1화1 mmol/L 초산처리대상술기인표체적영향총체추세일치，단1 mmol/L 초산처리적영향정도경현저；초산처리대불동기인적표체영향야유현저구별：1 mmol/L 초산처리후 CRT 기인가족여 CDPK1기인표체지속상조，CDPK1기인표체상조배수현저대우CRT 기인；CAMTA3기인표체지속하조，도처리후24 h，표체궤호피완전억제；이 CDPK2기인표체칙재처리후6 h 현저상조，도처리후24 h 략미하강.저사결과설명초산가통과개변 Ca2+농도급 Ca2+신호통로대식물여핵반균호작기중요적조공작용.
Summary 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. <br> The dye Fluo-4 interacts with Ca2+ inside living cells and can be detected with the green fluorescence by confocal laser-scanning microscope.Employing Fluo-4 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. <br> Additionally,the effect of OA on gene expression of Ca2+ signaling pathways was examined using real-time quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR).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 calmodulin-binding 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 up-regulated 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 down-regulated to be almost undetectable at 24 hpt,while the expression of CDPK2 gene was significantly up-regulated at 6 hpt and then slightly reduced at 24 hpt. <br> 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.