Oxidative post-translational modifications of specific chloroplast proteins contribute to the initiation of retrograde signaling.The Arabidopsis thaliana EXECUTER1(EX1)protein,a chloroplast-localized singlet oxygen(^(...Oxidative post-translational modifications of specific chloroplast proteins contribute to the initiation of retrograde signaling.The Arabidopsis thaliana EXECUTER1(EX1)protein,a chloroplast-localized singlet oxygen(^(1)O_(2))sensor,undergoes tryptophan(Trp)643 oxidation by^(1)O_(2),a chloroplast-derived and light-dependent reactive oxygen species.The indole side chain of Trp is vulnerable to^(1)O_(2),leading to the generation of oxidized Trp variants and priming EX1 for degradation by a membrane-bound FtsH protease.The perception of^(1)O_(2)via Trp643 oxidation and subsequent EX1 proteolysis facilitate chloroplast-to-nucleus retrograde signaling.In this study,we discovered that the EX1-like protein EX2 also undergoes^(1)O_(2)-dependent Trp530 oxidation and FtsH-dependent turnover,which attenuates^(1)O_(2)signaling by decelerating EX1-Trp643 oxidation and subsequent EX1 degradation.Consistent with this finding,the loss of EX2 function reinforces EX1-dependent retrograde signaling by accelerating EX1-Trp643 oxidation and subsequent EX1 proteolysis,whereas overexpression of EX2 produces molecular phenotypes opposite to those observed in the loss-of-function mutants of EX2.Intriguingly,phylogenetic analysis suggests that EX2 may have emerged evolutionarily to attenuate the sensitivity of EX1 toward^(1)O_(2).Collectively,these results suggest that EX2 functions as a negative regulator of the EX1 signalosome through its own^(1)O_(2)-dependent oxidation,providing a new mechanistic insight into the regulation of EX1-mediated^(1)O_(2)signaling.展开更多
Chloroplasts performing oxygenic photosynthesis frequently overproduce reactive oxygen species(Ros)under stress conditions,with singlet oxygen(O2)being particularly harmful due to its high reactivity and short lifespa...Chloroplasts performing oxygenic photosynthesis frequently overproduce reactive oxygen species(Ros)under stress conditions,with singlet oxygen(O2)being particularly harmful due to its high reactivity and short lifespan.The nuclear-encoded chloroplast protein EXECUTER1(EX1)identifies elevated'O2 levels through Trp643 oxidation and undergoes proteolysis,a process essential for activating'02-induced EX1-mediated chloroplast-to-nucleus retrograde signaling('O2-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-mu-tagenized flu mutant seeds of Arabidopsis thaliana harboring FLAG-fused EX1 driven by its native promoter(referred to as fluEx1).Like flu,fluExi plants conditionally produce'o2 in chloroplasts in response to a dark-to-light shift.In the fluEx1 sof1,all'02-induced stress responses are largely suppressed,despite'02 levels being similar to those in the fluExi,soF1 encodes the chloroplast outer-envelope-anchored preprotein import receptor TOc33.While Toc33 loss does not impact EX1 import,abundance,localization,and 102-induced proteolysis in the chloroplast,it blocks'o2-induced chloroplast-to-nucleus retrograde signaling.TOC33 interacts with the UVR domain of EX1(EX1-UVR)in the chloroplast envelope,enabling 102-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 chloro-plast envelope,activating'O2 signaling and inducing stress responses.Our findings indicate that soF1/TOC33 mediates 102-EX1 signaling from the chloroplast to the nucleus and that the EX1-UVR domain can substitute for full-length EX1 in this signaling pathway.展开更多
基金This research was supported by the Strategic Priority Research Program from the Chinese Academy of Sciences(grant no.XDB27040102)the 100-Talent Program of the Chinese Academy of Sciences and the National Natural Science Foundation of China(NSFC)(grant no.31871397)to C.K.Support from a President's International Fellowship Initiative(PIFI)postdoctoral fellowship from the Chinese Academy of Sciences(no.2019PB0066)to V.D.is also acknowledged.
文摘Oxidative post-translational modifications of specific chloroplast proteins contribute to the initiation of retrograde signaling.The Arabidopsis thaliana EXECUTER1(EX1)protein,a chloroplast-localized singlet oxygen(^(1)O_(2))sensor,undergoes tryptophan(Trp)643 oxidation by^(1)O_(2),a chloroplast-derived and light-dependent reactive oxygen species.The indole side chain of Trp is vulnerable to^(1)O_(2),leading to the generation of oxidized Trp variants and priming EX1 for degradation by a membrane-bound FtsH protease.The perception of^(1)O_(2)via Trp643 oxidation and subsequent EX1 proteolysis facilitate chloroplast-to-nucleus retrograde signaling.In this study,we discovered that the EX1-like protein EX2 also undergoes^(1)O_(2)-dependent Trp530 oxidation and FtsH-dependent turnover,which attenuates^(1)O_(2)signaling by decelerating EX1-Trp643 oxidation and subsequent EX1 degradation.Consistent with this finding,the loss of EX2 function reinforces EX1-dependent retrograde signaling by accelerating EX1-Trp643 oxidation and subsequent EX1 proteolysis,whereas overexpression of EX2 produces molecular phenotypes opposite to those observed in the loss-of-function mutants of EX2.Intriguingly,phylogenetic analysis suggests that EX2 may have emerged evolutionarily to attenuate the sensitivity of EX1 toward^(1)O_(2).Collectively,these results suggest that EX2 functions as a negative regulator of the EX1 signalosome through its own^(1)O_(2)-dependent oxidation,providing a new mechanistic insight into the regulation of EX1-mediated^(1)O_(2)signaling.
基金supported by the National Key Research and Development Program of China(grant no.2023YFF1000203-2)the National Natural Science Foundation of China(NSFC)(grant no.32170284)the open funds of the State Key Laboratory of Plant Environmental Resilience(SKLPERKF2418)to L.W.,and NSFC(grant no.32370249)to C.K.
文摘Chloroplasts performing oxygenic photosynthesis frequently overproduce reactive oxygen species(Ros)under stress conditions,with singlet oxygen(O2)being particularly harmful due to its high reactivity and short lifespan.The nuclear-encoded chloroplast protein EXECUTER1(EX1)identifies elevated'O2 levels through Trp643 oxidation and undergoes proteolysis,a process essential for activating'02-induced EX1-mediated chloroplast-to-nucleus retrograde signaling('O2-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-mu-tagenized flu mutant seeds of Arabidopsis thaliana harboring FLAG-fused EX1 driven by its native promoter(referred to as fluEx1).Like flu,fluExi plants conditionally produce'o2 in chloroplasts in response to a dark-to-light shift.In the fluEx1 sof1,all'02-induced stress responses are largely suppressed,despite'02 levels being similar to those in the fluExi,soF1 encodes the chloroplast outer-envelope-anchored preprotein import receptor TOc33.While Toc33 loss does not impact EX1 import,abundance,localization,and 102-induced proteolysis in the chloroplast,it blocks'o2-induced chloroplast-to-nucleus retrograde signaling.TOC33 interacts with the UVR domain of EX1(EX1-UVR)in the chloroplast envelope,enabling 102-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 chloro-plast envelope,activating'O2 signaling and inducing stress responses.Our findings indicate that soF1/TOC33 mediates 102-EX1 signaling from the chloroplast to the nucleus and that the EX1-UVR domain can substitute for full-length EX1 in this signaling pathway.
