In higher plants, the chloroplast is the most important organelle for photosynthesis and for numerous essential metabolic processes in the cell. Although many genes involved in chloroplast development have been identi...In higher plants, the chloroplast is the most important organelle for photosynthesis and for numerous essential metabolic processes in the cell. Although many genes involved in chloroplast development have been identified, the mechanisms underlying such development are not fully understood. In this study, a rice(Oryza sativa) mutant exhibiting pale green color and seedling lethality was isolated from a mutant library. The mutated gene was identified as an ortholog of THA8(thylakoid assembly 8) in Arabidopsis and maize. This gene is designated as OsTHA8 hereafter. OsTHA8 showed a typical pentatricopeptide repeat(PPR) characteristic of only four PPR motifs. Inactivation of OsTHA8 led to a deficiency in chloroplast development in the rice seedling stage. OsTHA8 was expressed mainly in young leaves and leaf sheaths.The OsTHA8 protein was localized to the chloroplast. Loss of function of OsTHA8 weakened the editing efficiency of ndhB-611/737 and rps8-182 transcripts under normal conditions. Y2H and BiFC indicated that OsTHA8 facilitates RNA editing by forming an editosome with multiple organellar RNA editing factor(OsMORF8) and thioredoxin z(OsTRXz), which function in RNA editing in rice chloroplasts. Defective OsTHA8 impaired chloroplast ribosome assembly and resulted in reduced expression of PEP-dependent genes and photosynthesis-related genes. Abnormal splicing of the chloroplast gene ycf3 was detected in ostha8. These findings reveal a synergistic regulatory mechanism of chloroplast biogenesis mediated by RNA, broaden the function of the PPR family, and shed light on the RNA editing complex in rice.展开更多
Early embryogenesis is the most fundamental developmental process in biology. Screening of ethyl methanesulfonate （EMS）-mutagenized populations of Arabidopsis thaliana led to the identification of a zygote-lethal mu...Early embryogenesis is the most fundamental developmental process in biology. Screening of ethyl methanesulfonate （EMS）-mutagenized populations of Arabidopsis thaliana led to the identification of a zygote-lethal mutant embryonic factor 19 （fac19） in which embryo development was arrested at the elongated zygote to octant stage. The number of endosperm nuclei decreased significantly in fac19 embryos. Genetic analysis showed fac19 was caused by a single recessive mutation with typical mendelian segregation, suggesting equal maternal and paternal contributions of FAC19 towards zygotic embryogenesis. Positional cloning showed that FAC19 encodes a putative mitochondrial protein with 16 conserved pentatricopeptide repeat （PPR） motifs. The fac19 mutation caused a conversion from hydrophilic serine located in a previously unknown domain to hydrophobic leucine. Crosses between FAC191fac19 and the T-DNA insertion mutants in the same gene failed to complement the fac19 defects, confirming the identity of the gene. This study revealed the critical importance of a PPR protein-mediated mitochondrial function in early embryogenesis.展开更多
Functional chloroplast generation depends on the precise coordination of gene expression between the plastid and the nucleus and is essential for plant growth and development. In this study, a rice（Oryza sativa） mut...Functional chloroplast generation depends on the precise coordination of gene expression between the plastid and the nucleus and is essential for plant growth and development. In this study, a rice（Oryza sativa） mutant that exhibited albino and seedling-lethal phenotypes was isolated from a60Co-irradiated rice population. The mutant gene was identified as an ortholog of the Arabidopsis plastid transcriptionally active chromosome protein 2（p TAC2） gene, and the mutant strain was designated osptac2. Sequence and transcription analyses showed that Osp TAC2 encodes a putative chloroplast protein consisting of 10 pentratricopeptide repeat（PPR） domains and a C-terminal small Mut S-related（SMR） domain. Cytological observations via microscopy showed that the Osp TAC2-green fluorescent fusion protein is localized in the chloroplasts. Transmission electron microscopy revealed that the chloroplast of the osptac2 mutant lacks an organized thylakoid membrane. The transcript levels of all investigated PEP（plastid-encoded RNA polymerase）-dependent genes were dramatically reduced in the osptac2 mutant, whereas the transcript levels of NEP（nuclear-encoded polymerase）-dependent genes were increased. These results suggest that Osp TAC2 plays a critical role in chloroplast development and indicate that the molecular function of the Osp TAC2 gene is conserved in rice and Arabidopsis.展开更多
Pentatricopeptide repeat(PPR)proteins play important roles in the post-transcriptional modification of organellar RNAs in plants.However,the function of most PPR proteins remains unknown.Here,we characterized the rice...Pentatricopeptide repeat(PPR)proteins play important roles in the post-transcriptional modification of organellar RNAs in plants.However,the function of most PPR proteins remains unknown.Here,we characterized the rice(Oryza sativa L.)chlorophyll deficient 4(cde4)mutant which exhibits an albino phenotype during early leaf development,with decreased chlorophyll contents and abnormal chloroplasts at low-temperature(20℃).Positional cloning revealed that CDE4 encodes a P-type PPR protein localized in chloroplasts.In the cde4 mutant,plastid-encoded polymerase(PEP)-dependent transcript levels were significantly reduced,but transcript levels of nuclear-encoded genes were increased compared to wild-type plants at 20℃.CDE4 directly binds to the transcripts of the chloroplast genes rpl2,ndhA,and ndhB.Intron splicing of these transcripts was defective in the cde4 mutant at 20℃,but was normal at 32℃.Moreover,CDE4 interacts with the guanylate kinase VIRESCENT 2(V2);overexpression of V2 enhanced CDE4 protein stability,thereby rescuing the cde4 phenotype at 20℃.Our results suggest that CDE4 participates in plastid RNA splicing and plays an important role in rice chloroplast development under lowtemperature conditions.展开更多
Organellar biogenesis is mainly regulated by nucleus-encoded factors, which act on various steps of gene expression including RNA editing, processing, splicing, stabilization, and translation initiation. Among these r...Organellar biogenesis is mainly regulated by nucleus-encoded factors, which act on various steps of gene expression including RNA editing, processing, splicing, stabilization, and translation initiation. Among these regulatory factors, pentatricopeptide repeat （PPR） proteins form the largest family of RNA binding proteins, with hundreds of members in flowering plants. In striking contrast, the genome of the unicellular green alga Chlamydomonas reinhardtii encodes only 14 such proteins. In this study, we analyzed PPR7, the smallest and most highly expressed PPR protein in C. reinhardtii. Green fluorescent protein-based localization and gel-filtration analysis revealed that PPR7 forms a part of a high-molecular-weight ribonu- cleoprotein complex in the chloroplast stroma. RIP-chip analysis of PPRT-bound RNAs demonstrated that the protein associates with a diverse set of chloroplast transcripts in vivo, i.e. rrnS, psbH, rpoC2, rbcL, atpA, cemA-atpH, tscA, and atpl-psaJ. Furthermore, the investigation of PPR7 RNAi strains revealed that deple- tion of PPR7 results in a light-sensitive phenotype, accompanied by altered levels of its target RNAs that are compatible with the defects in their maturation or stabilization. PPR7 is thus an unusual type of small multi- functional PPR protein, which interacts, probably in conjunction with other RNA binding proteins, with numerous target RNAs to promote a variety of post-transcriptional events.展开更多
Chloroplast biogenesis requires the coordinated expression of chloroplast and nuclear genes.Here, we show that EMB1270, a plastid-localized pentatricopeptide repeat(PPR) protein, is required for chloroplast biogenesis...Chloroplast biogenesis requires the coordinated expression of chloroplast and nuclear genes.Here, we show that EMB1270, a plastid-localized pentatricopeptide repeat(PPR) protein, is required for chloroplast biogenesis in Arabidopsis thaliana.Knockout of EMB1270 led to embryo arrest,whereas a mild knockdown mutant of EMB1270 displayed a virescent phenotype. Almost no photosynthetic proteins accumulated in the albino emb1270 knockout mutant. By contrast, in the emb1270 knockdown mutant, the levels of ClpP1 and photosystem I(PSI) subunits were significantly reduced, whereas the levels of photosystem II(PSII) subunits were normal. Furthermore, the splicing efficiencies of the clpP1.2,ycf3.1, ndhA, and ndhB plastid introns were dramatically reduced in both emb1270 mutants. RNA immunoprecipitation revealed that EMB1270 associated with these introns in vivo. In an RNA electrophoretic mobility shift assay(REMSA), a truncated EMB1270 protein containing the 11 Nterminal PPR motifs bound to the predicted sequences of the clpP1.2, ycf3.1, and ndhA introns.In addition, EMB1270 specifically interacted with CRM Family Member 2(CFM2). Given that CFM2 is known to be required for splicing the same plastid RNAs, our results suggest that EMB1270 associates with CFM2 to facilitate the splicing of specific group II introns in Arabidopsis.展开更多
细胞质雄性不育(cytoplasmic male sterility,CMS)是植物杂种优势利用的基础,大量研究表明CMS是线粒体基因与细胞核基因互作的结果。蛋白质是基因表达的产物,也是基因功能的执行者,研究线粒体蛋白质有利于探索CMS产生机理。本文综述了CM...细胞质雄性不育(cytoplasmic male sterility,CMS)是植物杂种优势利用的基础,大量研究表明CMS是线粒体基因与细胞核基因互作的结果。蛋白质是基因表达的产物,也是基因功能的执行者,研究线粒体蛋白质有利于探索CMS产生机理。本文综述了CMS相关的线粒体蛋白质的研究进展,并探讨了PPR(penta- tricopeptide repeats)基因的结构特征、生物学功能及对CMS相关基因的表达调控。展开更多
The hotABA-deficiency suppressor2 （has2） mutation increases drought tolerance and the ABA sensitivity of stomata closure and seed germination. Here we report that the HAS2 locus encodes the MITOCHONDRIAL EDITING FAC...The hotABA-deficiency suppressor2 （has2） mutation increases drought tolerance and the ABA sensitivity of stomata closure and seed germination. Here we report that the HAS2 locus encodes the MITOCHONDRIAL EDITING FACTOR11 （MEF11）, also known as LOVASTATIN INSENSITIVE1. has21mef11 mutants exhibited phenotypes very similar to the ABA-hypersensitive mutant, hail-1 pp2ca-1 hab 1-1 abil-2, which is impaired in four genes encoding type 2C protein phosphatases （PP2C） that act as upstream negative regulators of the ABA signaling cascade. Like pp2c, mef11 plants were more resistant to progressive water stress and seed germination was more sensitive to paclobutrazol （a gibberellin biosynthesis inhibitor） as well as mannitol and NaCI, compared with the wild-type plants. Phenotypic alterations in mef11 were associated with the lack of editing of transcripts for the mitochondrial cytochrome c maturation FN2 （cCmFN2） gene, which encodes a cytochrome c-heme lyase subunit involved in cytochrome c biogenesis. Although the abundance of electron transfer chain complexes was not affected, their dysfunction could be deduced from increased respiration and altered production of hydrogen peroxide and nitric oxide in reef11 seeds. As minor defects in mitochondrial respiration affect ABA signaling, this suggests an essential role for ABA in mitochondrial retrograde regulation.展开更多
三角状五肽重复(pentatricopeptide repeat,PPR)蛋白通常包含2~27个串联重复的结构域,每个结构域含有35个氨基酸残基。PPR蛋白是一类广泛存在于真核生物体内的RNA结合蛋白,对单链RNA具有序列特异性识别模式,与RNA的转录、剪切、编辑、...三角状五肽重复(pentatricopeptide repeat,PPR)蛋白通常包含2~27个串联重复的结构域,每个结构域含有35个氨基酸残基。PPR蛋白是一类广泛存在于真核生物体内的RNA结合蛋白,对单链RNA具有序列特异性识别模式,与RNA的转录、剪切、编辑、稳定性及翻译等过程密切相关。迄今为止,在哺乳动物中仅发现7个PPR蛋白,它们均存在于线粒体中,包括线粒体RNA聚合酶(mitochondrial RNA polymerase,POLRMT)、富含亮氨酸的三角状五肽重复结构蛋白(leucinerich pentatricopeptide repeat-containing protein,LRPPRC)蛋白、线粒体核糖体小亚基蛋白27(mitochondrial ribosomal protein of the small subunit 27,MRPS27)、线粒体核糖核酸酶P蛋白3(mitochondrial RNase P protein 3,MRPP3)以及五肽重复结构域蛋白(pentaricopeptide repeat domain protein,PTCD)1~3。哺乳动物PPR蛋白家族的成员在线粒体转录、RNA代谢和翻译中具有多种调控作用,对于线粒体功能和细胞健康至关重要。本文简要概述了哺乳动物细胞中PPR蛋白家族7个成员的结构和亚细胞定位,探讨了PPR蛋白在线粒体基因表达调控中的作用,并展望了PPR蛋白对人类线粒体相关疾病的应用前景。展开更多
基金supported by the Natural Science Foundation of Hebei Province (C2021208014)the Key R&D Program of Hebei Province (22326312D, 21326332D)。
文摘In higher plants, the chloroplast is the most important organelle for photosynthesis and for numerous essential metabolic processes in the cell. Although many genes involved in chloroplast development have been identified, the mechanisms underlying such development are not fully understood. In this study, a rice(Oryza sativa) mutant exhibiting pale green color and seedling lethality was isolated from a mutant library. The mutated gene was identified as an ortholog of THA8(thylakoid assembly 8) in Arabidopsis and maize. This gene is designated as OsTHA8 hereafter. OsTHA8 showed a typical pentatricopeptide repeat(PPR) characteristic of only four PPR motifs. Inactivation of OsTHA8 led to a deficiency in chloroplast development in the rice seedling stage. OsTHA8 was expressed mainly in young leaves and leaf sheaths.The OsTHA8 protein was localized to the chloroplast. Loss of function of OsTHA8 weakened the editing efficiency of ndhB-611/737 and rps8-182 transcripts under normal conditions. Y2H and BiFC indicated that OsTHA8 facilitates RNA editing by forming an editosome with multiple organellar RNA editing factor(OsMORF8) and thioredoxin z(OsTRXz), which function in RNA editing in rice chloroplasts. Defective OsTHA8 impaired chloroplast ribosome assembly and resulted in reduced expression of PEP-dependent genes and photosynthesis-related genes. Abnormal splicing of the chloroplast gene ycf3 was detected in ostha8. These findings reveal a synergistic regulatory mechanism of chloroplast biogenesis mediated by RNA, broaden the function of the PPR family, and shed light on the RNA editing complex in rice.
基金supported by the National Natural Science Foundation of China (30625018 and 30821007)CAS/SAFEA International Partnership Program for Creative Research Teams (20090491019)CAS 100-Talent Projects of China
文摘Early embryogenesis is the most fundamental developmental process in biology. Screening of ethyl methanesulfonate （EMS）-mutagenized populations of Arabidopsis thaliana led to the identification of a zygote-lethal mutant embryonic factor 19 （fac19） in which embryo development was arrested at the elongated zygote to octant stage. The number of endosperm nuclei decreased significantly in fac19 embryos. Genetic analysis showed fac19 was caused by a single recessive mutation with typical mendelian segregation, suggesting equal maternal and paternal contributions of FAC19 towards zygotic embryogenesis. Positional cloning showed that FAC19 encodes a putative mitochondrial protein with 16 conserved pentatricopeptide repeat （PPR） motifs. The fac19 mutation caused a conversion from hydrophilic serine located in a previously unknown domain to hydrophobic leucine. Crosses between FAC191fac19 and the T-DNA insertion mutants in the same gene failed to complement the fac19 defects, confirming the identity of the gene. This study revealed the critical importance of a PPR protein-mediated mitochondrial function in early embryogenesis.
基金supported by grants from the National Natural Science Foundation of China (31271800 and 31571742)the Zhejiang Provincial Natural Science Foundation of China (Z3110509)+1 种基金the National High-tech R&D Program of China (863 Program) (2012AA10A302-6)the Transgenic Project (2014ZX08010-002)
文摘Functional chloroplast generation depends on the precise coordination of gene expression between the plastid and the nucleus and is essential for plant growth and development. In this study, a rice（Oryza sativa） mutant that exhibited albino and seedling-lethal phenotypes was isolated from a60Co-irradiated rice population. The mutant gene was identified as an ortholog of the Arabidopsis plastid transcriptionally active chromosome protein 2（p TAC2） gene, and the mutant strain was designated osptac2. Sequence and transcription analyses showed that Osp TAC2 encodes a putative chloroplast protein consisting of 10 pentratricopeptide repeat（PPR） domains and a C-terminal small Mut S-related（SMR） domain. Cytological observations via microscopy showed that the Osp TAC2-green fluorescent fusion protein is localized in the chloroplasts. Transmission electron microscopy revealed that the chloroplast of the osptac2 mutant lacks an organized thylakoid membrane. The transcript levels of all investigated PEP（plastid-encoded RNA polymerase）-dependent genes were dramatically reduced in the osptac2 mutant, whereas the transcript levels of NEP（nuclear-encoded polymerase）-dependent genes were increased. These results suggest that Osp TAC2 plays a critical role in chloroplast development and indicate that the molecular function of the Osp TAC2 gene is conserved in rice and Arabidopsis.
基金This work was supported by the Natural Science Foundation of Zhejiang province(LR20C13002)the special support plan for high level talents in Zhejiang Province(2019R52032)Agricultural Sciences and Technologies Innovation Program of the Chinese Academy of Agricultural Sciences.
文摘Pentatricopeptide repeat(PPR)proteins play important roles in the post-transcriptional modification of organellar RNAs in plants.However,the function of most PPR proteins remains unknown.Here,we characterized the rice(Oryza sativa L.)chlorophyll deficient 4(cde4)mutant which exhibits an albino phenotype during early leaf development,with decreased chlorophyll contents and abnormal chloroplasts at low-temperature(20℃).Positional cloning revealed that CDE4 encodes a P-type PPR protein localized in chloroplasts.In the cde4 mutant,plastid-encoded polymerase(PEP)-dependent transcript levels were significantly reduced,but transcript levels of nuclear-encoded genes were increased compared to wild-type plants at 20℃.CDE4 directly binds to the transcripts of the chloroplast genes rpl2,ndhA,and ndhB.Intron splicing of these transcripts was defective in the cde4 mutant at 20℃,but was normal at 32℃.Moreover,CDE4 interacts with the guanylate kinase VIRESCENT 2(V2);overexpression of V2 enhanced CDE4 protein stability,thereby rescuing the cde4 phenotype at 20℃.Our results suggest that CDE4 participates in plastid RNA splicing and plays an important role in rice chloroplast development under lowtemperature conditions.
文摘Organellar biogenesis is mainly regulated by nucleus-encoded factors, which act on various steps of gene expression including RNA editing, processing, splicing, stabilization, and translation initiation. Among these regulatory factors, pentatricopeptide repeat （PPR） proteins form the largest family of RNA binding proteins, with hundreds of members in flowering plants. In striking contrast, the genome of the unicellular green alga Chlamydomonas reinhardtii encodes only 14 such proteins. In this study, we analyzed PPR7, the smallest and most highly expressed PPR protein in C. reinhardtii. Green fluorescent protein-based localization and gel-filtration analysis revealed that PPR7 forms a part of a high-molecular-weight ribonu- cleoprotein complex in the chloroplast stroma. RIP-chip analysis of PPRT-bound RNAs demonstrated that the protein associates with a diverse set of chloroplast transcripts in vivo, i.e. rrnS, psbH, rpoC2, rbcL, atpA, cemA-atpH, tscA, and atpl-psaJ. Furthermore, the investigation of PPR7 RNAi strains revealed that deple- tion of PPR7 results in a light-sensitive phenotype, accompanied by altered levels of its target RNAs that are compatible with the defects in their maturation or stabilization. PPR7 is thus an unusual type of small multi- functional PPR protein, which interacts, probably in conjunction with other RNA binding proteins, with numerous target RNAs to promote a variety of post-transcriptional events.
基金This study was supported by the National Natural Science Foundation of China(31100180,31470337,31570231)Shanghai Natural Science Foundation(18ZR1428200)+1 种基金The Fund of Innovation Program of Shanghai Municipal Education Commission(2021-01-07-00-02-E00117)Shanghai Engineering Research Center of Plant Germplasm Resources(17DZ2252700)。
文摘Chloroplast biogenesis requires the coordinated expression of chloroplast and nuclear genes.Here, we show that EMB1270, a plastid-localized pentatricopeptide repeat(PPR) protein, is required for chloroplast biogenesis in Arabidopsis thaliana.Knockout of EMB1270 led to embryo arrest,whereas a mild knockdown mutant of EMB1270 displayed a virescent phenotype. Almost no photosynthetic proteins accumulated in the albino emb1270 knockout mutant. By contrast, in the emb1270 knockdown mutant, the levels of ClpP1 and photosystem I(PSI) subunits were significantly reduced, whereas the levels of photosystem II(PSII) subunits were normal. Furthermore, the splicing efficiencies of the clpP1.2,ycf3.1, ndhA, and ndhB plastid introns were dramatically reduced in both emb1270 mutants. RNA immunoprecipitation revealed that EMB1270 associated with these introns in vivo. In an RNA electrophoretic mobility shift assay(REMSA), a truncated EMB1270 protein containing the 11 Nterminal PPR motifs bound to the predicted sequences of the clpP1.2, ycf3.1, and ndhA introns.In addition, EMB1270 specifically interacted with CRM Family Member 2(CFM2). Given that CFM2 is known to be required for splicing the same plastid RNAs, our results suggest that EMB1270 associates with CFM2 to facilitate the splicing of specific group II introns in Arabidopsis.
文摘细胞质雄性不育(cytoplasmic male sterility,CMS)是植物杂种优势利用的基础,大量研究表明CMS是线粒体基因与细胞核基因互作的结果。蛋白质是基因表达的产物,也是基因功能的执行者,研究线粒体蛋白质有利于探索CMS产生机理。本文综述了CMS相关的线粒体蛋白质的研究进展,并探讨了PPR(penta- tricopeptide repeats)基因的结构特征、生物学功能及对CMS相关基因的表达调控。
文摘The hotABA-deficiency suppressor2 （has2） mutation increases drought tolerance and the ABA sensitivity of stomata closure and seed germination. Here we report that the HAS2 locus encodes the MITOCHONDRIAL EDITING FACTOR11 （MEF11）, also known as LOVASTATIN INSENSITIVE1. has21mef11 mutants exhibited phenotypes very similar to the ABA-hypersensitive mutant, hail-1 pp2ca-1 hab 1-1 abil-2, which is impaired in four genes encoding type 2C protein phosphatases （PP2C） that act as upstream negative regulators of the ABA signaling cascade. Like pp2c, mef11 plants were more resistant to progressive water stress and seed germination was more sensitive to paclobutrazol （a gibberellin biosynthesis inhibitor） as well as mannitol and NaCI, compared with the wild-type plants. Phenotypic alterations in mef11 were associated with the lack of editing of transcripts for the mitochondrial cytochrome c maturation FN2 （cCmFN2） gene, which encodes a cytochrome c-heme lyase subunit involved in cytochrome c biogenesis. Although the abundance of electron transfer chain complexes was not affected, their dysfunction could be deduced from increased respiration and altered production of hydrogen peroxide and nitric oxide in reef11 seeds. As minor defects in mitochondrial respiration affect ABA signaling, this suggests an essential role for ABA in mitochondrial retrograde regulation.
文摘三角状五肽重复(pentatricopeptide repeat,PPR)蛋白通常包含2~27个串联重复的结构域,每个结构域含有35个氨基酸残基。PPR蛋白是一类广泛存在于真核生物体内的RNA结合蛋白,对单链RNA具有序列特异性识别模式,与RNA的转录、剪切、编辑、稳定性及翻译等过程密切相关。迄今为止,在哺乳动物中仅发现7个PPR蛋白,它们均存在于线粒体中,包括线粒体RNA聚合酶(mitochondrial RNA polymerase,POLRMT)、富含亮氨酸的三角状五肽重复结构蛋白(leucinerich pentatricopeptide repeat-containing protein,LRPPRC)蛋白、线粒体核糖体小亚基蛋白27(mitochondrial ribosomal protein of the small subunit 27,MRPS27)、线粒体核糖核酸酶P蛋白3(mitochondrial RNase P protein 3,MRPP3)以及五肽重复结构域蛋白(pentaricopeptide repeat domain protein,PTCD)1~3。哺乳动物PPR蛋白家族的成员在线粒体转录、RNA代谢和翻译中具有多种调控作用,对于线粒体功能和细胞健康至关重要。本文简要概述了哺乳动物细胞中PPR蛋白家族7个成员的结构和亚细胞定位,探讨了PPR蛋白在线粒体基因表达调控中的作用,并展望了PPR蛋白对人类线粒体相关疾病的应用前景。
