Differences between apoplastic and cytosolic reactive oxygen species production in Arabidopsis during pattern-triggered immunity
Differences between apoplastic and cytosolic reactive oxygen species production in Arabidopsis during pattern-triggered immunity
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DOI：
10.1101/2021.04.20.440614
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发表时间：
2021-04
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影响因子：
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通讯作者：
D. Arnaud;M. Deeks;N. Smirnoff
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作者：
D. Arnaud;M. Deeks;N. Smirnoff
尽管人们对植物-微生物相互作用过程中的活性氧 (ROS) 信号传导越来越感兴趣，但主要出于技术原因，关于 PAMP 触发免疫过程中调节细胞内过氧化氢 (H2O2) 信号传导的分子机制的信息却很少。在这里，我们使用了灵敏的荧光测定方法和 H2O2 传感器 roGFP2-Orp1，揭示了拟南芥对病原体相关分子模式 (PAMP) 感知的细胞质 H2O2 和硫醇氧化还原动力学的调节的意想不到的特征。延长的 PAMP 诱导的胞质 roGFP2-Orp1 氧化与通过鲁米诺氧化测量的质外体中的瞬时氧化爆发不同。药理学和遗传分析表明，PAMP 诱导的细胞质中 H2O2 的延长在很大程度上独立于 NADPH 氧化酶和质外体过氧化物酶。相比之下，NADPH 氧化酶突变体 rbohF 对 H2O2 和 PAMP 的 roGFP2-Orp1 氧化高度敏感，表明抗氧化能力较低。与之前的报道不同，rbohF 突变体（而不是 rbohD）在 PAMP 触发的气孔关闭和保卫细胞中基于荧光素的探针测量的 ROS 产生方面受到损害，导致气孔对细菌的防御缺陷。然而，气孔关闭与保卫细胞中 roGFP2-Orp1 氧化的增加无关。有趣的是，RBOHF 也参与了 PAMP 诱导的质外体碱化。总而言之，我们的结果为质外体和细胞质 ROS 动力学之间的相互作用提供了新的见解，并强调了 RBOHF 在植物免疫中的重要性。意义陈述 植物通过检测病原体相关分子模式（PAMP）来发起对病原体的防御反应。一种反应是 NADPH 氧化酶和细胞壁过氧化物酶在细胞壁（质外体）中快速瞬时爆发活性氧（ROS，例如超氧化物和过氧化氢）。使用基因编码的过氧化氢传感器 roGFP2-Orp1，我们发现，与短暂的质外体 ROS 爆发相反，在 PAMP 感知时，胞质溶胶中也会长时间产生过氧化氢，这与 NADPH 氧化酶和细胞壁过氧化物酶无关。我们的结果表明，在 PAMP 触发的免疫过程中，质外体 ROS 而不是细胞内过氧化氢是触发气孔关闭的信号。此外，我们重新讨论了 NADPH 氧化酶 D 和 F 在气孔免疫中的相对贡献。
Despite an ever-increasing interest in reactive oxygen species (ROS) signalling during plant-microbe interactions, very little information exists, mainly for technical reasons, on the molecular mechanisms regulating intracellular hydrogen peroxide (H2O2) signalling during PAMP-triggered immunity. Here, we used a sensitive fluorimetry method and the H2O2 sensor roGFP2-Orp1, which revealed unsuspected features on the regulation of cytoplasmic H2O2 and thiol redox dynamics upon pathogen-associated molecular patterns (PAMPs) perception by Arabidopsis thaliana. Extended PAMP-induced cytosolic roGFP2-Orp1 oxidation was distinct from the transient oxidative burst in the apoplast measured by luminol oxidation. Pharmacological and genetic analyses indicate that the prolonged PAMP-induced H2O2 increase in the cytoplasm was largely independent on NADPH oxidases and apoplastic peroxidases. By contrast, the NADPH oxidase mutant rbohF was hyper-sensitive to roGFP2-Orp1 oxidation by H2O2 and PAMP indicating a lower antioxidant capacity. Unlike previous reports, the rbohF mutant, but not rbohD, was impaired in PAMP-triggered stomatal closure and ROS production measured by a fluorescein-based probe in guard cells resulting in defects in stomatal defences against bacteria. However, stomatal closure was not correlated with an increase in roGFP2-Orp1 oxidation in guard cells. Interestingly, RBOHF also participated in PAMP-induced apoplastic alkalinisation. Altogether, our results provide novel insights on the interplay between apoplastic and cytosolic ROS dynamics and highlight the importance of RBOHF in plant immunity. Significance statement Plants mount defence responses to pathogens by detecting pathogen-associated molecular patterns (PAMPs). One response is a rapid and transient burst of reactive oxygen species (ROS, e.g. superoxide and hydrogen peroxide) in the cell wall (apoplast) produced by NADPH oxidases and cell wall peroxidases. Using a genetically-encoded hydrogen peroxide sensor roGFP2-Orp1, we found that, in contrast to the transient apoplastic ROS burst, there is also prolonged hydrogen peroxide production in the cytosol upon PAMP perception which is independent of NADPH oxidase and cell wall peroxidases. Our results suggest that apoplastic ROS rather than intracellular hydrogen peroxide is a signal triggering stomatal closure during PAMP-triggered immunity. Additionally, we re-address the relative contribution of the NADPH oxidases D and F in stomatal immunity.