Chloroplast transit peptides(CTPs) can be used to transport non-chloroplastic proteins into the chloroplasts. Here, we studied the CTPs of three rice(Oryza sativa L.) chloroplast-localized proteins and found that thei...Chloroplast transit peptides(CTPs) can be used to transport non-chloroplastic proteins into the chloroplasts. Here, we studied the CTPs of three rice(Oryza sativa L.) chloroplast-localized proteins and found that their CTPs could be used to transport non-chloroplast-localized proteins into the chloroplasts. Fusion proteins lacking the CTP remained located in the cytoplasm. Furthermore, we constructed green fluorescent protein fusion vectors with the three CTPs and three non-chloroplast-localized proteins, Ghd10, MULTI-FLORET SPIKELET1(MFS1), and SHORTENED UPPERMOST INTERNODE 1(SUI1). After transforming these constructs into rice protoplasts, the fusion proteins all localized in the chloroplasts. Collectively, our results showed that these CTPs can transport non-chloroplast-localized proteins into the chloroplasts, and more importantly, these CTPs can be applied to engineer chloroplast metabolism.展开更多
Lesion mutants can be valuable tools to reveal the interactions between genetic factors and environmental signals and to improve grain production.Here we identified a rice(Oryza sativa)mutant,lesion spotleaf1(lsl1),wh...Lesion mutants can be valuable tools to reveal the interactions between genetic factors and environmental signals and to improve grain production.Here we identified a rice(Oryza sativa)mutant,lesion spotleaf1(lsl1),which produces necrotic leaf lesions throughout its life cycle.LSL1 encodes a protein of unknown function and belongs to a grass-specific clade.The lesion phenotype of the lsl1 mutant was sharply induced by shading,and its detached leaves incubated in 6-benzylamino purine similarly formed lesions in the dark.In addition,the lsl1 mutant exhibited reactive oxygen species accumulation and cell death.The terminal deoxynucleotidyl transferase d UTP nick end-labeling(TUNEL)and comet assays revealed that the lsl1 mutant contained severe DNA damage,resulting in reduced grain yield and quality.RNA sequencing,gene expression,and protein activity analyses indicate that LSL1 is required for chloroplast function.Furthermore,LSL1 interacts with Psa D and PAP10 to form a regulatory module that functions in chlorophyll synthesis and chloroplast development to maintain redox balance.Our results reveal that LSL1 maintains chloroplast structure,redox homeostasis,and DNA stability,and plays important roles in the interaction between genetic factors and environmental signals and in regulating grain size and quality.展开更多
Most chloroplast proteins （cp proteins） are nucleus-encoded, synthesized on cytosolic ribosomes as precursor proteins containing a presequence （cTP）, and post-translationally imported via the Tic/Toc complex into ...Most chloroplast proteins （cp proteins） are nucleus-encoded, synthesized on cytosolic ribosomes as precursor proteins containing a presequence （cTP）, and post-translationally imported via the Tic/Toc complex into the organelle, where the cTP is removed. Only a few unambiguous instances of cp proteins that do not require cTPs （non-canonical cp proteins） have been reported so far. However, the survey of data from large-scale proteomic studies presented here suggests that the fraction of such proteins in the total cp proteome might be as large as -30%. To explore this discrepancy, we chose a representative set of 28 putative non-canonical cp proteins, and used in vitro import and Red Fluorescent Protein （RFP）-fusion assays to determine their sub-cellular destinations. Four proteins, including embryo defective 1211, glycolate oxidase 2, protein disulfide isomerase-like protein （PDII）, and a putative glutathione S-transferase, could be unambiguously assigned to the chloroplast. Several others （＇potential cp proteins＇） were found to be imported into chloroplasts in vitro, but failed to localize to the organelle when RFP was fused to their C-terminal ends. Extrapolations suggest that the fraction of cp proteins that enter the inner compartments of the organelle, although they lack a cTP, might be as large as 11.4% of the total cp proteome. Our data also support the idea that cytosolic proteins that associate with the cp outer membrane might account for false positive cp proteins obtained in earlier studies.展开更多
基金Acknowledgements We thank the RIKEN BRC in Japan for provision of all full-length cDNA in this study. National Natural Science Foundation of China （grants numbers 30530100 and 90408010）, the State Key Program of Basic Research of China （grant numbers 2007CB947600 and 2007CB108800）, and Hi-Tech Research and Development Program of China （grant number 2006AA02Z313） supported this project.
基金supported by the National Natural Science Foundation of China(Grant Nos.31601284 and 31661143006)the Transgenic Plant Research and Commercialization Project of the Ministry of Agriculture of China(Grant No.2016ZX08001003-002)+1 种基金Zhejiang Province Outstanding Youth Fund(Grant No.LR16C130001)the Collaborative Innovation Project of the Chinese Academy of Agricultural Sciences(Grant No.Y2016XT05)
文摘Chloroplast transit peptides(CTPs) can be used to transport non-chloroplastic proteins into the chloroplasts. Here, we studied the CTPs of three rice(Oryza sativa L.) chloroplast-localized proteins and found that their CTPs could be used to transport non-chloroplast-localized proteins into the chloroplasts. Fusion proteins lacking the CTP remained located in the cytoplasm. Furthermore, we constructed green fluorescent protein fusion vectors with the three CTPs and three non-chloroplast-localized proteins, Ghd10, MULTI-FLORET SPIKELET1(MFS1), and SHORTENED UPPERMOST INTERNODE 1(SUI1). After transforming these constructs into rice protoplasts, the fusion proteins all localized in the chloroplasts. Collectively, our results showed that these CTPs can transport non-chloroplast-localized proteins into the chloroplasts, and more importantly, these CTPs can be applied to engineer chloroplast metabolism.
基金Supported by National 863 Project of China (2002AA227011)Natural Science Foundation of Hubei Province (2003ABAI18)Natural Science Foundation of Shandong Province (ZR2010HQ054)~~
基金the National Natural Science Foundation of China(32188102,32071993,31971872,31861143006,U2004204)the Zhejiang Provincial“Ten Thousand Talent Program”Project(2018R52025,2019R52031)+3 种基金the Hainan Yazhou Bay Seed Laboratory(a project of B21HJ0220)the Key Research and Development Program of Zhejiang Province(2021C02056)the Nanfan Special Project,CAAS(ZDXM06)the Central Public-Interest Scientific Institution Basal Research Fund(CPSIBRF-CNRRI-202111,CPSIBRF-CNRRI202110)。
文摘Lesion mutants can be valuable tools to reveal the interactions between genetic factors and environmental signals and to improve grain production.Here we identified a rice(Oryza sativa)mutant,lesion spotleaf1(lsl1),which produces necrotic leaf lesions throughout its life cycle.LSL1 encodes a protein of unknown function and belongs to a grass-specific clade.The lesion phenotype of the lsl1 mutant was sharply induced by shading,and its detached leaves incubated in 6-benzylamino purine similarly formed lesions in the dark.In addition,the lsl1 mutant exhibited reactive oxygen species accumulation and cell death.The terminal deoxynucleotidyl transferase d UTP nick end-labeling(TUNEL)and comet assays revealed that the lsl1 mutant contained severe DNA damage,resulting in reduced grain yield and quality.RNA sequencing,gene expression,and protein activity analyses indicate that LSL1 is required for chloroplast function.Furthermore,LSL1 interacts with Psa D and PAP10 to form a regulatory module that functions in chlorophyll synthesis and chloroplast development to maintain redox balance.Our results reveal that LSL1 maintains chloroplast structure,redox homeostasis,and DNA stability,and plays important roles in the interaction between genetic factors and environmental signals and in regulating grain size and quality.
文摘Most chloroplast proteins （cp proteins） are nucleus-encoded, synthesized on cytosolic ribosomes as precursor proteins containing a presequence （cTP）, and post-translationally imported via the Tic/Toc complex into the organelle, where the cTP is removed. Only a few unambiguous instances of cp proteins that do not require cTPs （non-canonical cp proteins） have been reported so far. However, the survey of data from large-scale proteomic studies presented here suggests that the fraction of such proteins in the total cp proteome might be as large as -30%. To explore this discrepancy, we chose a representative set of 28 putative non-canonical cp proteins, and used in vitro import and Red Fluorescent Protein （RFP）-fusion assays to determine their sub-cellular destinations. Four proteins, including embryo defective 1211, glycolate oxidase 2, protein disulfide isomerase-like protein （PDII）, and a putative glutathione S-transferase, could be unambiguously assigned to the chloroplast. Several others （＇potential cp proteins＇） were found to be imported into chloroplasts in vitro, but failed to localize to the organelle when RFP was fused to their C-terminal ends. Extrapolations suggest that the fraction of cp proteins that enter the inner compartments of the organelle, although they lack a cTP, might be as large as 11.4% of the total cp proteome. Our data also support the idea that cytosolic proteins that associate with the cp outer membrane might account for false positive cp proteins obtained in earlier studies.
