单线态氧与叶绿体逆行信号

Singlet oxygen (1O2), the excitation stage of the ground-state molecular oxygen, is a fundamental reactive oxygen species (ROS) with important functions in plant growth, development, and stress responses. In plant cells, 1O2 is mainly generated in the chloroplast due to photosensitizing activity of tetrapyrroles. Moreover, 1O2 can be generated in non-photosynthetic tissues when plants suffer environmental stresses. Although 1O2 was initially considered as a cytotoxin—causing merely photooxidative damages, more recent work suggests that 1O2 also acts as a signal that either triggers a programmed cell death response or promotes acclimation. The 1O2 signaling pathway is distinct and operates independently of other ROS signaling cascades. In Arabidopsis, EXECUTER1 (EX1) protein has been identified as a crucial signaling component that perceives and relays 1O2 signals to the nucleus, thereby initiating extensive transcriptional reprogramming. Additionally, oxidative products of carotenoids, such as β-cyclocitral, are also recognized as 1O2-derived signaling molecules. Through specific chloroplast-to-nucleus signaling and cross talk with hormone signaling networks—including jasmonic acid (JA) and salicylic acid (SA)—1O2 helps finely coordinate plant growth, defense responses, and cell fate decisions under fluctuating environmental conditions. This review aims to summarize current knowledge on 1O2 generation and signaling, 1O2-induced chloroplast changes under diverse stress conditions, and cross talk between 1O2 and phytohormone signaling.

Chloroplasts are fundamental plant organelles in green plants and algae. As an endosymbiosis-originated organelle, most chloroplast proteins are encoded by nuclear genes, translated in the cytosol, and imported into the chloroplast. Here, we present a protocol for the purification of Arabidopsis chloroplasts and in vitro translation of the desired protein. We also describe steps for import system preparation, import reaction, and Western blot. This approach eliminates centrifugation, thermolysis, and radioisotope labeling steps and reduces sample preparation time and cost by approximately 50%. For complete details on the use and execution of this protocol, please refer to Zhao et al.1

Chloroplasts performing oxygenic photosynthesis frequently overproduce reactive oxygen species (ROS) under stress conditions, with singlet oxygen (1O2) being particularly harmful due to its high reactivity and short lifespan. The nuclear-encoded chloroplast protein EXECUTER1 (EX1) identifies elevated 1O2 levels through Trp643 oxidation and undergoes proteolysis, a process essential for activating 1O2-induced EX1-mediated chloroplast-to-nucleus retrograde signaling (1O2-EX1 signaling). However, the association between EX1 proteolysis and subsequent nuclear transcriptome alterations remains unclear. In this study, we isolated SOF1 (suppressor of flu 1) through a forward genetic screen using ethyl methanesulfonate-mutagenized flu mutant seeds of Arabidopsis thaliana harboring FLAG-fused EX1 driven by its native promoter (referred to as fluEX1). Like flu, fluEX1 plants conditionally produce 1O2 in chloroplasts in response to a dark-to-light shift. In the fluEX1sof1, all 1O2-induced stress responses are largely suppressed, despite 1O2 levels being similar to those in the fluEX1. SOF1 encodes the chloroplast outer-envelope-anchored preprotein import receptor TOC33. While TOC33 loss does not impact EX1 import, abundance, localization, and 1O2-induced proteolysis in the chloroplast, it blocks 1O2-induced chloroplast-to-nucleus retrograde signaling. TOC33 interacts with the UVR domain of EX1 (EX1-UVR) in the chloroplast envelope, enabling 1O2-induced decrease of the chloroplast EX1-UVR and increased nuclear EX1-UVR. Moreover, ectopic expression of EX1-UVR outside of the chloroplast overcomes the restrictive barrier imposed by the chloroplast envelope, activating 1O2 signaling and inducing stress responses. Our findings indicate that SOF1/TOC33 mediates 1O2-EX1 signaling from the chloroplast to the nucleus and that the EX1-UVR domain can substitute for full-length EX1 in this signaling pathway.

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 2-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.