Chloroplasts are recognized as environmental sensors, capable of translating environmental fluctuations into diverse signals to communicate with the nucleus. Among the reactive oxygen species produced in chloroplasts, singlet oxygen (1O2) has been extensively studied due to its dual roles, encompassing both damage and signaling activities, and the availability of conditional mutants overproducing 1O2 in chloroplasts. In particular, investigating the Arabidopsis (Arabidopsis thaliana) mutant known as fluorescent (flu) has led to the discovery of EXECUTER1 (EX1), a plastid 1O2 sensor residing in the grana margin of the thylakoid membrane. 1O2-triggered EX1 degradation is critical for the induction of 1O2-responsive nuclear genes (SOrNGs). However, a recent study showed that EX1 relocates from chloroplasts to the nucleus upon 1O2 release, where it interacts with WRKY18 and WRKY40 (WRKY18/40) transcription factors to regulate SOrNG expression. In this study, we challenge this assertion. Our confocal microscopy analysis and subcellular fractionation assays demonstrate that EX1 does not accumulate in the nucleus. While EX1 appears in nuclear fractions, subsequent thermolysin treatment assays indicate that it adheres to the outer nuclear region rather than localizing inside the nucleus. Furthermore, luciferase complementation imaging and yeast two-hybrid assays reveal that EX1 does not interact with nuclear WRKY18/40. Consequently, our study refines the current model of 1O2 signaling by ruling out the nuclear relocation of intact EX1 as a means of communication between the chloroplast and nucleus.

中文翻译：

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EXECUTER1 和单线态氧信号转导：核活性的重新评估


叶绿体被认为是环境传感器，能够将环境波动转化为不同的信号，以便与细胞核进行通信。在叶绿体中产生的活性氧中，单线态氧 （1O2） 因其双重作用而被广泛研究，包括损伤和信号活性，以及在叶绿体中过度产生 1O2 的条件突变体的可用性。特别是，研究被称为荧光（流感）的拟南芥（拟南芥）突变体导致了 EXECUTER1 （EX1） 的发现，这是一种位于类囊体膜颗粒边缘的质体 1O2 传感器。1O2 触发的 EX1 降解对于诱导 1O2 反应性核基因 （SOrNG） 至关重要。然而，最近的一项研究表明，EX1 在 1O2 释放后从叶绿体重新定位到细胞核，在那里它与 WRKY18 和 WRKY40 （WRKY18/40） 转录因子相互作用以调节 SOrNG 表达。在这项研究中，我们挑战了这一断言。我们的共聚焦显微镜分析和亚细胞分离分析表明，EX1 不会在细胞核中积累。虽然 EX1 出现在细胞核组分中，但随后的嗜热菌蛋白酶处理测定表明它粘附在核外区域，而不是定位于细胞核内部。此外，荧光素酶互补成像和酵母双杂交测定显示 EX1 不与核 WRKY18/40 相互作用。因此，我们的研究通过排除完整 EX1 的核重定位作为叶绿体和细胞核之间通信的一种方式来改进当前的 1O2 信号模型。