RESUMO
The ATP-dependent metalloprotease FtsH12 (filamentation temperature sensitive protein H 12) has been suggested to participate in a heteromeric motor complex, driving protein translocation into the chloroplast. FtsH12 was immuno-detected in proplastids, seedlings, leaves, and roots. Expression of Myc-tagged FtsH12 under its native promotor allowed identification of FtsHi1, 2, 4, and 5, and plastidic NAD-malate dehydrogenase, five of the six interaction partners in the suggested import motor complex. Arabidopsis thaliana mutant seedlings with reduced FTSH12 abundance exhibited pale cotyledons and small, deformed chloroplasts with altered thylakoid structure. Mature plants retained these chloroplast defects, resulting in slightly variegated leaves and lower chlorophyll content. Label-free proteomics revealed strong changes in the proteome composition of FTSH12 knock-down seedlings, reflecting impaired plastid development. The composition of the translocon on the inner chloroplast membrane (TIC) protein import complex was altered, with coordinated reduction of the FtsH12-FtsHi complex subunits and accumulation of the 1 MDa TIC complex subunits TIC56, TIC214 and TIC22-III. FTSH12 overexpressor lines showed no obvious phenotype, but still displayed distinct differences in their proteome. N-terminome analyses further demonstrated normal proteolytic maturation of plastid-imported proteins irrespective of FTSH12 abundance. Together, our data suggest that FtsH12 has highest impact during seedling development; its abundance alters the plastid import machinery and impairs chloroplast development.
Assuntos
Proteases Dependentes de ATP , Proteínas de Arabidopsis , Arabidopsis , Cloroplastos , Proteínas de Membrana , Proteases Dependentes de ATP/genética , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Proteínas de Cloroplastos/genética , Proteínas de Membrana/genética , Metaloproteases/genética , MutaçãoRESUMO
FtsH (filamentation-temperature-sensitive protein H) proteases are a family of membrane-bound enzymes present in eubacteria, animals, and plants. Besides the 12 genes encoding proteolytically active members of the FtsH family in the genome of Arabidopsis, there are five genes coding for members that are assumed to be proteolytically inactive due to mutations in the protease domain; these are termed FtsHi (i for inactive). Despite their lack of proteolytic activity, these FtsHi members seem to be important for chloroplast and plant development as four out of five homozygous knockout-mutants of FtsHis are embryo-lethal. Here, we analysed the Darwinian fitness of weak homozygous (ftshi1,3,4) and heterozygous (ftshi/FTSHi2,4,5) mutants. We compared the growth and development of these mutants to their respective wild-type Arabidopsis plants under controlled laboratory conditions and in the field, and we also evaluated the photosynthetic efficiency by pulse-amplitude modulation fluorescence. Homologous genotypes were subjected to various stress conditions in a greenhouse and gene co-expression as well as phylogenetic analyses were performed. Analysis of the gene-expression network of the five FTSHi genes indicated common clusters with genes encoding FtsH12, OTP51, and methylase. Phylogenetic analyses pointed to a common evolution (and common disappearance in grasses and gymnosperms) of FtsH12 and multiple presumably proteolytically inactive FtsHi enzymes. Our data show that the FtsHi enzymes are highly important during the seedling stage and for Darwinian fitness analyses in semi-natural conditions.
Assuntos
Proteínas de Arabidopsis/genética , Arabidopsis/genética , Regulação da Expressão Gênica de Plantas , Aptidão Genética , Metaloendopeptidases/genética , Metaloproteases/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Metaloendopeptidases/metabolismo , Metaloproteases/metabolismo , Mutação , Fotossíntese , FilogeniaRESUMO
Proteases play a vital role in the removal of proteins, which become damaged due to temperature or oxidative stress. Important to this process in the cyanobacterium Synechocystis sp. PCC6803 is the family of Deg/HtrA proteases; HhoA (sll1679), HhoB (sll1427) and HtrA (slr1204). While previous studies have elucidated the structures of Deg/HtrA proteases from Escherichia coli and from the chloroplast of the higher plant Arabidopsis thaliana, no structural data have been available for any Deg/HtrA protease from cyanobacteria, the evolutionary ancestor of the chloroplast. To gain a deeper insight into the molecular mechanisms and regulation of these proteins we have solved the structure of the Synechocystis HhoA protease in complex with a co-purified peptide by X-ray crystallography. HhoA assembles into stable trimers, mediated by its protease domain and further into a cage-like hexamer by a novel interaction between the PDZ domains of opposing trimers. Each PDZ domain contains two loops for PDZ-PDZ formation: interaction clamp one and two (IC1, IC2). IC1 interacts with IC2 on the opposing PDZ domain and vice versa. Our structure shows a peptide bound to a conserved groove on the PDZ domain and the properties of this pocket suggest that it binds substrate proteins as well as the neo C-termini of cleaved substrates. In agreement with previous studies showing the proteolytic activity of HhoA to be activated by Ca2+ or Mg2+, binding of divalent metal ions to the central channel of the trimer by the L1 activation loop was observed.
Assuntos
Serina Endopeptidases/química , Synechocystis/química , Synechocystis/enzimologia , Proteínas de Bactérias , Sítios de Ligação , Cálcio/metabolismo , Cristalografia por Raios X , Magnésio/metabolismo , Domínios PDZ , Peptídeos/metabolismo , Multimerização Proteica , Serina Endopeptidases/metabolismoRESUMO
The membrane-integrated metalloprotease FtsH11 of Arabidopsis thaliana is proposed to be dual-targeted to mitochondria and chloroplasts. A bleached phenotype was observed in ftsh11 grown at long days or continuous light, pointing to disturbances in the chloroplast. Within the chloroplast, FtsH11 was found to be located exclusively in the envelope. Two chloroplast-located proteins of unknown function (Tic22-like protein and YGGT-A) showed significantly higher abundance in envelope membranes and intact chloroplasts of ftsh11 and therefore qualify as potential substrates for the FtsH11 protease. No proteomic changes were observed in the mitochondria of 6-week-old ftsh11 compared with wild type, and FtsH11 was not immunodetected in these organelles. The abundance of plastidic proteins, especially of photosynthetic proteins, was altered even during standard growth conditions in total leaves of ftsh11. At continuous light, the amount of photosystem I decreased relative to photosystem II, accompanied by a drastic change of the chloroplast morphology and a drop of non-photochemical quenching. FtsH11 is crucial for chloroplast structure and function during growth in prolonged photoperiod.
Assuntos
Proteínas de Arabidopsis/fisiologia , Arabidopsis/enzimologia , Cloroplastos/fisiologia , Metaloproteases/fisiologia , Arabidopsis/crescimento & desenvolvimento , Arabidopsis/ultraestrutura , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Clorofila/metabolismo , Cloroplastos/metabolismo , Cloroplastos/ultraestrutura , Técnicas de Inativação de Genes , Redes Reguladoras de Genes , Metaloproteases/genética , Metaloproteases/metabolismo , Fenótipo , FotoperíodoRESUMO
FtsHs are a well-characterized family of membrane bound proteases containing an AAA (ATPase associated with various cellular activities) and a Zn(2+) metalloprotease domain. FtsH proteases are found in eubacteria, animals and plants and are known to have a crucial role in housekeeping proteolysis of membrane proteins. In Arabidopsis thaliana, 12 FtsH family members are present (FtsH 1-12) and their subcellular localization is restricted to mitochondria and chloroplasts. In addition, five genes coding for proteins homologous to FtsH (FtsHi 1-5) have been detected in the genome, lacking the conserved zinc-binding motif HEXXH, which presumably renders them inactive for proteolysis. These inactive FtsHs as well as nine of the active FtsHs are thought to be localized in the chloroplast. In this article, we shortly summarize the recent findings on plastidic FtsH proteases in text and figures. We will mainly focus on FtsH 1, 2, 5 and 8 localized in the thylakoid membrane and known for their importance in photosynthesis.
Assuntos
Proteínas de Arabidopsis/metabolismo , Proteínas de Cloroplastos/metabolismo , Proteínas de Membrana/metabolismo , Metaloproteases/metabolismo , Tilacoides/enzimologia , Arabidopsis/química , Arabidopsis/enzimologia , Proteínas de Cloroplastos/genética , Regulação Enzimológica da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Genes de Plantas , Proteínas de Membrana/genética , Fotossíntese , Proteólise , Transcrição GênicaRESUMO
Darwinian fitness analyses were performed, comparing single ftsh mutants with wild-type Arabidopsis thaliana plants grown under controlled laboratory conditions and in the field, by measuring plant size, survival rate, and silique and seed production. Additionally, three genotypes of ΔFtsH6 were analysed, under controlled growth conditions, with respect to both their ability to degrade the light-harvesting complex of photosystem II during senescence and light acclimation. In the field, substantial increases in variegation and reductions in growth were observed in the ΔFtsH2, ΔFtsH5 and ΔFtsH10 mutants; FtsH2 seemed particularly important for plant survival. Despite being grown in relatively cold weather, the ΔFtsH11 mutant displayed strong phenotypic deviations from wild type. Both ΔFtsH10 and ΔFtsH3 mutants exhibited less severe phenotypic changes, but were different from wild-type plants when placed in the field as young plants. When older ΔFtsH3 or ΔFtsH10 mutants were placed outdoors, no phenotypic differences from wild type were observed. Three genotypes of ΔFtsH6 displayed no phenotypic deviations from wild-type plants. Under controlled growth conditions, during senescence and light acclimation, no differences in the amount of chlorophyll or Photosystem II light-harvesting complex b3 (Lhcb3) were detected in ΔFtsH6 mutants compared with the wild type. Therefore, FtsH6 seems to be unimportant for LHCII degradation in vivo.
Assuntos
Arabidopsis/efeitos da radiação , Luz , Metaloproteases/metabolismo , Complexo de Proteína do Fotossistema II/metabolismo , Arabidopsis/enzimologia , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Clorofila/metabolismo , Cloroplastos/metabolismo , Temperatura Baixa , Genótipo , Complexos de Proteínas Captadores de Luz/genética , Complexos de Proteínas Captadores de Luz/metabolismo , Metaloproteases/genética , Fenótipo , Fotossíntese , Complexo de Proteína do Fotossistema II/genética , Folhas de Planta/genética , Folhas de Planta/metabolismo , Folhas de Planta/efeitos da radiação , Deleção de Sequência , Estresse Fisiológico , Fatores de TempoRESUMO
Illumination changes elicit modifications of thylakoid proteins and reorganization of the photosynthetic machinery. This involves, in the short term, phosphorylation of photosystem II (PSII) and light-harvesting (LHCII) proteins. PSII phosphorylation is thought to be relevant for PSII turnover, whereas LHCII phosphorylation is associated with the relocation of LHCII and the redistribution of excitation energy (state transitions) between photosystems. In the long term, imbalances in energy distribution between photosystems are counteracted by adjusting photosystem stoichiometry. In the green alga Chlamydomonas and the plant Arabidopsis, state transitions require the orthologous protein kinases STT7 and STN7, respectively. Here we show that in Arabidopsis a second protein kinase, STN8, is required for the quantitative phosphorylation of PSII core proteins. However, PSII activity under high-intensity light is affected only slightly in stn8 mutants, and D1 turnover is indistinguishable from the wild type, implying that reversible protein phosphorylation is not essential for PSII repair. Acclimation to changes in light quality is defective in stn7 but not in stn8 mutants, indicating that short-term and long-term photosynthetic adaptations are coupled. Therefore the phosphorylation of LHCII, or of an unknown substrate of STN7, is also crucial for the control of photosynthetic gene expression.
Assuntos
Aclimatação/fisiologia , Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Fotossíntese/fisiologia , Complexo de Proteína do Fotossistema II/metabolismo , Proteínas Quinases/metabolismo , Arabidopsis/enzimologia , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Complexos de Proteínas Captadores de Luz/metabolismo , Mutação/genética , Fosforilação , Complexo de Proteína do Fotossistema II/genética , Proteínas Quinases/genética , Proteínas Serina-Treonina QuinasesRESUMO
The expression of genes in higher plant chloroplasts includes a complex transcriptional regulation which can be explained only in part with the action of the actually known components of the transcriptional machinery. This suggests the existence of still unknown important regulatory factors which influence chloroplast transcription. In order to test if such factors could exist we performed in silico analyses of Arabidopsis genes encoding putative transcription factors looking for putative N-terminal chloroplast transit peptides in the amino acid sequences. Our results suggest that 48 (and maybe up to 100) transcription factors of eukaryotic origin are likely to be imported into plastids. None of them has been described yet. This set of transcription factors highly expands the actually known regulation capacity of the chloroplast transcription machinery and provides a possible explanation for the complex initiation patterns of chloroplast transcripts. As consequence of a massive import of eukaryotic transcription factors a comprehensive reconstruction of the ancient prokaryotic gene expression machinery must be assumed resulting in a novel compatible combination of eukaryotic and prokaryotic protein components. In turn, the opposite process has been induced in the nucleus by the integration of prokaryotic components of the plastid ancestor via its loss of genes during endosymbiosis. Thus, a mutual exchange of regulatory factors, i.e. transcription factors occurred which resulted in the unique signalling network of today's plants. An evolutionary model of how this could have emerged during endosymbiosis in a timely coordinated manner is proposed.
Assuntos
Biologia Computacional , Plastídeos/genética , Fatores de Transcrição/genética , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Núcleo Celular/genética , Cloroplastos/fisiologia , Mapeamento Cromossômico , Cromossomos de Plantas/genética , Células Eucarióticas , Evolução Molecular , Regulação da Expressão Gênica de Plantas , Genoma de Planta , Fatores de Transcrição/metabolismo , Transcrição Gênica/genéticaRESUMO
Proteins are the main machinery for all living processes in a cell; they provide structural elements, regulate biochemical reactions as enzymes, and are the interface to the outside as receptors and transporters. Like any other machinery proteins have to be assembled correctly and need maintenance after damage, e.g., caused by changes in environmental conditions, genetic mutations, and limitations in the availability of cofactors. Proteases and chaperones help in repair, assembly, and folding of damaged and misfolded protein complexes cost-effective, with low energy investment compared with neo-synthesis. Despite their importance for viability, the specific biological role of most proteases in vivo is largely unknown. Deg/HtrA proteases, a family of serine-type ATP-independent proteases, have been shown in higher plants to be involved in the degradation of the Photosystem II reaction center protein D1. The objective of this review is to highlight the structure and function of their cyanobacterial orthologs. Homology modeling was used to find specific features of the SynDeg/HtrA proteases of Synechocystis sp. PCC 6803. Based on the available data concerning their location and their physiological substrates we conclude that these Deg proteases not only have important housekeeping and chaperone functions within the cell, but also are needed for remodeling the cell exterior.
RESUMO
The long-term response (LTR) to light-quality gradients improves performance and survival of plants in dense stands. It involves redox-controlled transcriptional regulation of the plastome-encoded genes psaAB (encoding the P700 apoproteins of photosystem I) and psbA (encoding the D1 protein of photosystem II) and requires the action of plastid-localized kinases. To study the potential impact of phosphorylation events on plastid gene expression during the LTR, we analyzed mustard seedlings acclimated to light sources favoring either photosystem I or photosystem II. Primer extension analyses of psaA transcripts indicate that the redox regulation occurs at the principal bacterial promoters, suggesting that the plastid encoded RNA polymerase (PEP) is the target for redox signals. Chloroplast protein fractions containing PEP and other DNA-binding proteins were purified from mustard via heparin-Sepharose chromatography. The biochemical properties of these fractions were analyzed with special emphasis on promoter recognition and specificity, phosphorylation state, and kinase activity. The results demonstrate that the LTR involves the action of small DNA-binding proteins; three of them exhibit specific changes in the phosphorylation state. Auto-phosphorylation assays, in addition, exhibit large differences in the activity of endogenous kinase activities. Chloroplast run-on transcription experiments with the kinase inhibitor H7 and the reductant DTT indicate that phosphorylation events are essential for the mediation of redox signals toward psaA and psbA transcription initiation, but require the synergistic action of a thiol redox signal. The data support the idea that redox signals from the thylakoid membrane are linked to gene expression via phosphorylation events; however, this mediation appears to require a complex network of interacting proteins rather than a simple phosphorelay.
Assuntos
Oxirredução , Fotossíntese/fisiologia , Complexo de Proteína do Fotossistema I/metabolismo , Aclimatação , Proteínas de Ligação a DNA/metabolismo , Luz , Fosforilação , Transcrição GênicaRESUMO
Flowering plants control energy allocation to their photosystems in response to light quality changes. This includes the phosphorylation and migration of light-harvesting complex II (LHCII) proteins (state transitions or short-term response) as well as long-term alterations in thylakoid composition (long-term response or LTR). Both responses require the thylakoid protein kinase STN7. Here, we show that the signaling pathways triggering state transitions and LTR diverge at, or immediately downstream from, STN7. Both responses require STN7 activity that can be regulated according to the plastoquinone pool redox state. However, LTR signaling does not involve LHCII phosphorylation or any other state transition step. State transitions appear to play a prominent role in flowering plants, and the ability to perform state transitions becomes critical for photosynthesis in Arabidopsis thaliana mutants that are impaired in thylakoid electron transport but retain a functional LTR. Our data imply that STN7-dependent phosphorylation of an as yet unknown thylakoid protein triggers LTR signaling events, whereby an involvement of the TSP9 protein in the signaling pathway could be excluded. The LTR signaling events then ultimately regulate in chloroplasts the expression of photosynthesis-related genes on the transcript level, whereas expression of nuclear-encoded proteins is regulated at multiple levels, as indicated by transcript and protein profiling in LTR mutants.
Assuntos
Proteínas de Arabidopsis/fisiologia , Arabidopsis/enzimologia , Fotossíntese/fisiologia , Proteínas Quinases/fisiologia , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Transporte de Elétrons/fisiologia , Regulação da Expressão Gênica de Plantas/genética , Regulação da Expressão Gênica de Plantas/efeitos da radiação , Immunoblotting , Luz , Espectrometria de Massas , Modelos Biológicos , Análise de Sequência com Séries de Oligonucleotídeos , Fosforilação/fisiologia , Fotossíntese/genética , Proteínas Quinases/genética , Proteínas Quinases/metabolismo , Proteínas Serina-Treonina Quinases , Transdução de Sinais/genética , Transdução de Sinais/efeitos da radiação , Tilacoides/enzimologia , Tilacoides/genética , Tilacoides/metabolismoRESUMO
The long-term response (LTR) of higher plants to varying light qualities increases the photosynthetic yield; however, the benefit of this improvement for physiology and survival of plants is largely unknown, and its functional relation to other light acclimation responses has never been investigated. To unravel positive effects of the LTR we acclimated Arabidopsis thaliana for several days to light sources, which preferentially excite photosystem I (PSI) or photosystem II (PSII). After acclimation, plants revealed characteristic differences in chlorophyll fluorescence, thylakoid membrane stacking, phosphorylation state of PSII subunits and photosynthetic yield of PSII and PSI. These LTR-induced changes in the structure, function and efficiency of the photosynthetic machinery are true effects by light quality acclimation, which could not be induced by light intensity variations in the low light range. In addition, high light stress experiments indicated that the LTR is not involved in photoinhibition; however, it lowers non-photochemical quenching (NPQ) by directing more absorbed light energy into photochemical work. NPQ in turn is not essential for the LTR, since npq mutants performed a normal acclimation. We quantified the beneficial potential of the LTR by comparing wild-type plants with the LTR-deficient mutant stn7. The mutant exhibited a decreased effective quantum yield and produced only half of seeds when grown under fluctuating light quality conditions. Thus, the LTR represents a distinct acclimation response in addition to other already known responses that clearly improves plant physiology under low light conditions resulting in a pronounced positive effect on plant fitness.
Assuntos
Aclimatação , Arabidopsis/efeitos da radiação , Fotossíntese/efeitos da radiação , Complexo de Proteína do Fotossistema I/fisiologia , Complexo de Proteína do Fotossistema II/fisiologia , Arabidopsis/genética , Arabidopsis/fisiologia , Cloroplastos/fisiologia , Cloroplastos/ultraestrutura , Fertilidade , Fluorescência , Mutação , Complexo de Proteína do Fotossistema I/análise , Complexo de Proteína do Fotossistema II/análise , Tempo de Reação , Plântula , Tilacoides/ultraestruturaRESUMO
Chloroplasts contain 3000-4000 different proteins but only a small subset of them is encoded in the plastid genome while the majority is encoded in the nucleus. Expression of these genes therefore requires a high degree of co-ordination between nucleus and chloroplast. This is achieved by a bilateral information exchange between both compartments including nucleus-to-plastid (anterograde) and plastid-to-nucleus (retrograde) signals. The latter represent a functional feedback control which couples the expression of nuclear encoded plastid proteins to the actual functional state of the organelle. The efficiency of photosynthesis is a very important parameter in this context since it is influenced by many environmental conditions and therefore represents a sensor for the residing environment. Components of the photosynthetic electron transport chain exhibit significant changes in their reduction/oxidation (redox) state depending on the photosynthetic electron flow and therefore serve as signalling parameters which report environmental influences on photosynthesis. Such redox signals control chloroplast and nuclear gene expression events and play an important role in the co-ordination of both genetic compartments. It is discussed here which photosynthetic parameters are known to control nuclear gene expression, how these signals are transduced toward the nucleus, and how they interact with other plastid retrograde signals and cytosolic light perception systems.
Assuntos
Regulação da Expressão Gênica de Plantas/fisiologia , Fotossíntese/fisiologia , Proteínas de Plantas/biossíntese , Adaptação Fisiológica , Núcleo Celular/metabolismo , Luz , Oxirredução , Transdução de SinaisRESUMO
Excitation imbalances between photosystem I and II generate redox signals in the thylakoid membrane of higher plants which induce acclimatory changes in the structure of the photosynthetic apparatus. They affect the accumulation of reaction center and light-harvesting proteins as well as chlorophylls a and b. In Arabidopsis thaliana the re-adjustment of photosystem stoichiometry is mainly mediated by changes in the number of photosystem I complexes, which are accompanied by corresponding changes in transcripts for plastid reaction center genes. Because chloroplast protein complexes contain also many nuclear encoded components we analyzed the impact of such photosynthetic redox signals on nuclear genes. Light shift experiments combined with application of the electron transport inhibitor 3-(3',4'-dichlorophenyl)-1,1'-dimethyl urea have been performed to induce defined redox signals in the thylakoid membrane. Using DNA macroarrays we assessed the impact of such redox signals on the expression of nuclear genes for chloroplast proteins. In addition, studies on mutants with lesions in cytosolic photoreceptors or in chloroplast-to-nucleus communication indicate that the defective components in the mutants are not essential for the perception and/or transduction of light-induced redox signals. A stable redox state of glutathione suggest that neither glutathione itself nor reactive oxygen species are involved in the observed regulation events pointing to the thylakoid membrane as the main origin of the regulatory pathways. Our data indicate a distinct role of photosynthetic redox signals in the cellular network regulating plant gene expression. These redox signals appear to act independently and/or above of cytosolic photoreceptor or known chloroplast-to-nucleus communication avenues.