RESUMO
Algae biotechnology holds immense promise for revolutionizing the bioeconomy through the sustainable and scalable production of various bioproducts. However, their development has been hindered by the lack of advanced genetic tools. This study introduces a synthetic biology approach to develop such tools, focusing on the construction and testing of synthetic promoters. By analyzing conserved DNA motifs within the promoter regions of highly expressed genes across six different algal species, we identified cis-regulatory elements (CREs) associated with high transcriptional activity. Combining the algorithms POWRS, STREME, and PhyloGibbs, we predicted 1511 CREs and inserted them into a minimal synthetic promoter sequence in 1, 2, or 3 copies, resulting in 4533 distinct synthetic promoters. These promoters were evaluated in vivo for their capacity to drive the expression of a transgene in a high-throughput manner through next-generation sequencing post antibiotic selection and fluorescence-activated cell sorting. To validate our approach, we sequenced hundreds of transgenic lines showing high levels of GFP expression. Further, we individually tested 14 identified promoters, revealing substantial increases in GFP expressionâup to nine times higher than the baseline synthetic promoter, with five matching or even surpassing the performance of the native AR1 promoter. As a result of this study, we identified a catalog of CREs that can now be used to build superior synthetic algal promoters. More importantly, here we present a validated pipeline to generate building blocks for innovative synthetic genetic tools applicable to any algal species with a sequenced genome and transcriptome data set.
Assuntos
Biologia Computacional , Regiões Promotoras Genéticas , Biologia Sintética , Regiões Promotoras Genéticas/genética , Biologia Computacional/métodos , Biologia Sintética/métodos , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Sequenciamento de Nucleotídeos em Larga Escala/métodos , AlgoritmosRESUMO
Expression of the genes of the photosystem II (PSII) core polypeptides D1 and D2, of three proteins of the oxygen evolving complex of PSII and of the light harvesting chlorophyll a/b binding proteins (LHCP) has been compared in wild-type (wt) and in the y-1 mutant of Chlamydomonas reinhardtii. Since wt, but not y-1 cells produce a fully developed photosynthetic system in the dark, comparison of the two has allowed us to distinguish the direct effect of light from the influence of plastid development on gene expression. The PSII core polypeptides and LHCP are nearly undetectable in dark-grown y-1 cells but they accumulate progressively during light induced greening. The levels of these proteins in wt are the same in the light and the dark. The amounts of the proteins of the oxygen evolving complex do not change appreciably in the light or in the dark for both wt and y-1. Steady state levels of chloroplast mRNA encoding the core PSII polypeptides remain nearly constant in the light or the dark and are not affected by the developmental stage of the plastid. Levels of nuclear encoded mRNAs for the oxygen evolving proteins and of LHCP increase during light growth in wt and y-1. In contrast to wt, synthesis of LHCP proteins is not detectable in y-1 cells in the dark but starts immediately after transfer to light, indicating that LHCP synthesis is controlled by a light-induced factor or process. While the rates of synthesis of D1 and D2 are immediately enhanced by light in wt, this increase occurs only after a lag in y-1 and thus must be dependent on an early light-induced event in the plastid. These results show that the biosynthesis of PSII is affected by light directly, by the stage of plastid development, and by the interaction of light and events associated with plastid development.
Assuntos
Chlamydomonas/genética , Clorofila/genética , Genes , Fotossíntese , Proteínas de Plantas/genética , Clorofila/biossíntese , Cloroplastos/análise , Eletroforese em Gel de Poliacrilamida , Regulação da Expressão Gênica , Complexos de Proteínas Captadores de Luz , Mutação , Hibridização de Ácido Nucleico , Biossíntese Peptídica , Peptídeos/genética , Complexo de Proteínas do Centro de Reação Fotossintética , Complexo de Proteína do Fotossistema II , Proteínas de Plantas/biossíntese , Biossíntese de Proteínas , Proteínas/genética , RNA Mensageiro/análise , RNA Mensageiro/genéticaRESUMO
The 5' untranslated region of the chloroplast psbA mRNA, encoding the D1 protein, is processed in Chlamydomonas reinhardtii. Processing occurs just upstream of a consensus Shine-Dalgarno sequence and results in the removal of 54 nucleotides from the 5' terminus, including a stem-loop element identified previously as an important structure for D1 expression. Examination of this processing event in C. reinhardtii strains containing mutations within the chloroplast or nuclear genomes that block psbA translation reveals a correlation between processing and ribosome association. Mutations within the 5' untranslated region of the psbA mRNA that disrupt the Shine-Dalgarno sequence, acting as a ribosome binding site, preclude translation and prevent mRNA processing. Similarly, nuclear mutations that specifically affect synthesis of the D1 protein specifically affect processing of the psbA mRNA. In vitro, loss of the stem-loop element does not prohibit the binding of a message-specific protein complex required for translational activation of psbA upon illumination. These results are consistent with a hierarchical maturation pathway for chloroplast messages, mediated by nuclear-encoded factors, that integrates mRNA processing, message stability, ribosome association, and translation.
Assuntos
Regiões 5' não Traduzidas/genética , Regiões 5' não Traduzidas/metabolismo , Chlamydomonas reinhardtii/genética , Chlamydomonas reinhardtii/metabolismo , Complexo de Proteínas do Centro de Reação Fotossintética/genética , Animais , Sequência de Bases , Sítios de Ligação/genética , Núcleo Celular/genética , Núcleo Celular/metabolismo , Chlamydomonas reinhardtii/efeitos da radiação , Cloroplastos/genética , Cloroplastos/metabolismo , Primers do DNA/genética , Luz , Dados de Sequência Molecular , Mutação , Conformação de Ácido Nucleico , Complexo de Proteínas do Centro de Reação Fotossintética/metabolismo , Complexo de Proteínas do Centro de Reação Fotossintética/efeitos da radiação , Complexo de Proteína do Fotossistema II , Processamento Pós-Transcricional do RNA , RNA Mensageiro/química , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA de Plantas/química , RNA de Plantas/genética , RNA de Plantas/metabolismo , RNA de Protozoário/química , RNA de Protozoário/genética , RNA de Protozoário/metabolismo , Ribossomos/metabolismoRESUMO
Translational regulation is a key modulator of gene expression in chloroplasts of higher plants and algae. Genetic analysis has shown that translation of chloroplast mRNAs requires nuclear-encoded factors that interact with chloroplastic mRNAs in a message-specific manner. Using site-specific mutations of the chloroplastic psbA mRNA, we show that RNA elements contained within the 5' untranslated region of the mRNA are required for translation. One of these elements is a Shine-Dalgarno consensus sequence, which is necessary for ribosome association and psbA translation. A second element required for high levels of psbA translation is located adjacent to and upstream of the Shine-Dalgarno sequence, and maps to the location on the RNA previously identified as the site of message-specific protein binding. This second element appears to act as a translational attenuator that must be overcome to activate translation. Mutations that affect the secondary structure of these RNA elements greatly reduce the level of psbA translation, suggesting that secondary structure of these RNA elements plays a role in psbA translation. These data suggest a mechanism for translational activation of the chloroplast psbA mRNA in which an RNA element containing the ribosome-binding site is bound by message-specific RNA binding proteins allowing for increased ribosome association and translation initiation. These elements may be involved in the light-regulated translation of the psbA mRNA.
Assuntos
Chlamydomonas reinhardtii/genética , Conformação de Ácido Nucleico , Complexo de Proteínas do Centro de Reação Fotossintética/biossíntese , Biossíntese de Proteínas , RNA Mensageiro/genética , Animais , Sequência de Bases , Chlamydomonas reinhardtii/metabolismo , Chlamydomonas reinhardtii/efeitos da radiação , Análise Mutacional de DNA , Escuridão , Regulação da Expressão Gênica/efeitos da radiação , Luz , Dados de Sequência Molecular , Complexo de Proteínas do Centro de Reação Fotossintética/genética , Complexo de Proteína do Fotossistema II , RNA Mensageiro/metabolismo , Sequências Reguladoras de Ácido Nucleico/genéticaRESUMO
A set of nuclear mutants of C. reinhardtii were identified that specifically lack translation of the chloroplast-encoded psbA mRNA, which encodes the photosystem II reaction center polypeptide D1. Two of these mutants are deficient in the 47-kD member (RB47) of the psbA RNA-binding complex, which has previously been identified both genetically and biochemically as a putative translational activator of the chloroplast psbA mRNA. RB47 is a member of the poly(A)-binding protein family, and binds with high affinity and specificity to the 5' untranslated region of the psbA mRNA. The results presented here confirm RB47's role as a message-specific translational activator in the chloroplast, and bring together genetic and biochemical data to form a cohesive model for light- activated translational regulation in the chloroplast.
Assuntos
Chlamydomonas reinhardtii/genética , Chlamydomonas reinhardtii/metabolismo , Complexo de Proteínas do Centro de Reação Fotossintética/genética , RNA Mensageiro/genética , Proteínas de Ligação a RNA/metabolismo , Animais , Núcleo Celular/metabolismo , Chlamydomonas reinhardtii/efeitos da radiação , Cloroplastos/metabolismo , Cloroplastos/efeitos da radiação , Expressão Gênica , Luz , Modelos Biológicos , Mutagênese Insercional , Mutação , Complexo de Proteína do Fotossistema II , Proteínas de Ligação a Poli(A) , Biossíntese de Proteínas , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , RNA Mensageiro/metabolismo , RNA de Protozoário/genética , RNA de Protozoário/metabolismoRESUMO
We have established schedules of expression during maize leaf development in light and darkness for the messenger RNAs (mRNAs) and polypeptides for ribulose 1,5-bisphosphate carboxylase (RuBPCase) subunits, phosphoenolpyruvate carboxylase (PEPCase), and the light-harvesting chlorophyll a/b-binding protein (LHCP). Levels of mRNAs were measured by hybridization with cloned probes, and proteins were measured by immunodetection on protein gel blots. The initial synthesis in leaves of all four mRNAs follows a light-independent schedule; illumination influences only the level to which each mRNA accumulates. The synthesis of RuBPCase small and large subunits and of PEPCase polypeptides also follows a light-independent schedule which is modified quantitatively by light. However, the accumulation of LHCP polypeptides absolutely requires illumination. The accumulation of each protein closely follows the accumulation of its mRNA during growth in light. Higher ratios of PEPCase and RuBPCase protein to mRNA occur during dark growth.
Assuntos
Clonagem Molecular , Genes , Desenvolvimento Vegetal , Proteínas de Plantas/genética , DNA/isolamento & purificação , Escuridão , Luz , Fosfoenolpiruvato Carboxilase/genética , Plantas/genética , RNA Mensageiro/genética , Ribulose-Bifosfato Carboxilase/genética , Zea mays/genética , Zea mays/crescimento & desenvolvimentoRESUMO
Translation of key proteins in the chloroplast is regulated by light. Genetic and biochemical studies in the unicellular alga Chlamydomonas reinhardtii suggest that light may regulate translation by modulating the binding of activator proteins to the 5' untranslated region of chloroplast messenger RNAs. In vitro binding of the activator proteins to psbA messenger RNA and in vivo translation of psbA messenger RNA is regulated by the redox state of these proteins, suggesting that the light stimulus is transduced by the photosynthesis-generated redox potential.
Assuntos
Chlamydomonas reinhardtii/genética , Luz , Biossíntese de Proteínas , RNA de Cloroplastos/genética , RNA Mensageiro/genética , Proteínas de Ligação a RNA/metabolismo , Animais , Chlamydomonas reinhardtii/efeitos dos fármacos , Chlamydomonas reinhardtii/metabolismo , Ácido Ditionitrobenzoico/farmacologia , Ditiotreitol/farmacologia , Etilmaleimida/farmacologia , Mercaptoetanol/farmacologia , Oxirredução , Complexo de Proteínas do Centro de Reação Fotossintética/genética , Complexo de Proteína do Fotossistema II , Biossíntese de Proteínas/efeitos dos fármacos , RNA de Cloroplastos/metabolismo , RNA Mensageiro/metabolismo , Tiorredoxinas/metabolismo , Tiorredoxinas/farmacologiaRESUMO
Light-regulated translation of chloroplast messenger RNAs (mRNAs) requires trans-acting factors that interact with the 5' untranslated region (UTR) of these mRNAs. Chloroplast polyadenylate-binding protein (cPABP) specifically binds to the 5'-UTR of the psbA mRNA and is essential for translation of this mRNA. A protein disulfide isomerase that is localized to the chloroplast and copurifies with cPABP was shown to modulate the binding of cPABP to the 5'-UTR of the psbA mRNA by reversibly changing the redox status of cPABP through redox potential or adenosine 5'-diphosphate-dependent phosphorylation. This mechanism allows for a simple reversible switch regulating gene expression in the chloroplast.
Assuntos
Chlamydomonas reinhardtii/genética , Cloroplastos/genética , Regulação da Expressão Gênica , Biossíntese de Proteínas , Isomerases de Dissulfetos de Proteínas/metabolismo , Proteínas de Ligação a RNA/metabolismo , Difosfato de Adenosina/metabolismo , Sequência de Aminoácidos , Animais , Sítios de Ligação , Catálise , Chlamydomonas reinhardtii/enzimologia , Chlamydomonas reinhardtii/metabolismo , Cloroplastos/metabolismo , Clonagem Molecular , Ditiotreitol/farmacologia , Dissulfeto de Glutationa/farmacologia , Dados de Sequência Molecular , Oxirredução , Fosforilação , Complexo de Proteínas do Centro de Reação Fotossintética/genética , Complexo de Proteína do Fotossistema II , Isomerases de Dissulfetos de Proteínas/química , Isomerases de Dissulfetos de Proteínas/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Homologia de Sequência de AminoácidosRESUMO
Translational regulation has been identified as one of the key steps in chloroplast-encoded gene expression. Genetic and biochemical analysis with Chlamydomonas reinhardtii has implicated nucleus-encoded factors that interact specifically with the 5' untranslated region of chloroplast mRNAs to mediate light-activated translation. F35 is a nuclear mutation in C. reinhardtii that specifically affects translation of the psbA mRNA (encoding D1, a core polypeptide of photosystem II), causing a photosynthetic deficiency in the mutant strain. The F35 mutant has reduced ribosome association of the psbA mRNA as a result of decreased translation initiation. This reduction in ribosome association correlates with a decrease in the stability of the mRNA. Binding activity of the psbA specific protein complex to the 5' untranslated region of the mRNA is diminished in F35 cells, and two members of this binding complex (RB47 and RB55) are reduced compared with the wild type. These data suggest that alteration of members of the psbA mRNA binding complex in F35 cells results in a reduction in psbA mRNA-protein complex formation, thereby causing a decrease in translation initiation of this mRNA.
Assuntos
Núcleo Celular/metabolismo , Chlamydomonas reinhardtii/genética , Chlamydomonas reinhardtii/metabolismo , Cloroplastos/metabolismo , Iniciação Traducional da Cadeia Peptídica , Complexo de Proteínas do Centro de Reação Fotossintética/biossíntese , Biossíntese de Proteínas/genética , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/metabolismo , Acetatos/metabolismo , Animais , Proteínas de Membrana/biossíntese , Proteínas de Membrana/isolamento & purificação , Mutação , Complexo de Proteínas do Centro de Reação Fotossintética/genética , Complexo de Proteína do Fotossistema II , Polirribossomos/metabolismoRESUMO
Chloroplast development and function relies both on structural and on regulatory factors encoded within the nucleus. Recent work has lead to the identification of several nuclear encoded genes that participate in a wide array of chloroplast functions. Characterization of these genes has increased our understanding of the signalling between these two compartments. Accumulating evidence shows that a variety of molecular mechanisms are used for intercompartmental communication and for regulating co-ordinated chloroplast protein expression.
Assuntos
Núcleo Celular/fisiologia , Cloroplastos/fisiologia , Fenômenos Fisiológicos Vegetais , Transdução de Sinais , Cloroplastos/genética , Regulação da Expressão Gênica de Plantas , Plantas/genética , Plastídeos/genética , Biossíntese de Proteínas , RNA Mensageiro/genéticaRESUMO
By transformation with a cloned wild-type oee1 gene, which codes for the oxygen-evolving enhancer 1 (OEE1)protein, we have constructed a strain of Chlamydomonas reinhardtii containing multiple copies of this gene. A transformant (R1-K-50) containing four to five copies of the oee1 gene accumulated oee1 mRNA in approximately threefold excess of the wild type. The OEE1 protein accumulated in proportion to the oee1-mRNA levels in these cells. These data indicate that no apparant feedback mechanism is operating to reduce either transcription or translation of the introduced oee1 genes as a means to regulate OEE1-protein accumulation. The OEE1 protein in R1-K-50 was all of mature size, indicating that the transit peptide had been completely removed, and that all of the protein was located within the thylakoid lumen. Photosystem II reaction-center proteins D1 and D2 accumulated to wild-type levels, but not greater, in these cells, while there was no effect on accumulation of any of the PSII peripheral proteins such as OEE2 or LHCII. The OEE1 protein which accumulated in excess of wild-type levels was not bound to the thylakoid membranes, indicating that a limited number of binding sites for OEE1 exist on the thylakoid membranes. No difference in photosynthetic oxygen evolution was observed between wild-type and Rl-K-50 strains. These data show that whatever mechanisms are used to determine stoichiometry within the PSII complex they are not perturbed by overexpression of the OEE1 protein.
RESUMO
Genetic analysis has revealed a set of nuclear-encoded factors that regulate chloroplast mRNA translation by interacting with the 5' leaders of chloroplastic mRNAs. We have identified and isolated proteins that bind specifically to the 5' leader of the chloroplastic psbA mRNA, encoding the photosystem II reaction center protein D1. Binding of these proteins protects a 36 base RNA fragment containing a stem-loop located upstream of the ribosome binding site. Binding of these proteins to the psbA mRNA correlates with the level of translation of psbA mRNA observed in light- and dark-grown wild type cells and in a mutant that lacks D1 synthesis in the dark. The accumulation of at least one of these psbA mRNA-binding proteins is dependent upon chloroplast development, while its mRNA-binding activity appears to be light modulated in developed chloroplasts. These nuclear encoded proteins are prime candidates for regulators of chloroplast protein synthesis and may play an important role in coordinating nuclear-chloroplast gene expression as well as provide a mechanism for regulating chloroplast gene expression during development in higher plants.
Assuntos
Chlamydomonas reinhardtii/genética , Cloroplastos , Luz , Complexo de Proteínas do Centro de Reação Fotossintética/genética , Biossíntese de Proteínas/efeitos da radiação , RNA Mensageiro/genética , Proteínas de Ligação a RNA/genética , Animais , Autorradiografia , Sequência de Bases , Sítios de Ligação , Western Blotting , Cromatografia de Afinidade , Eletroforese em Gel Bidimensional , Camundongos , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Complexo de Proteína do Fotossistema II , RNARESUMO
We have developed a stable nuclear transformation system for the unicellular green alga Chlamydomonas reinhardtii. Transformation was accomplished by introducing the cloned C. reinhardtii oxygen-evolving enhancer protein 1 (OEE1) gene into C. reinhardtii cells by bombardment with DNA-coated tungsten particles. The recipient strain was an OEE1-deficient, nonphotosynthetic, acetate-requiring mutant, which recovered photosynthetic competence after transformation, and was therefore able to grow in the absence of acetate. Analysis of several transformants indicates that transformation has proceeded via second-site integration of the cloned gene, leaving the endogenous mutant gene intact. In genetic crosses of transformants with wild type, both mutant and wild-type phenotypes were recovered, showing that the photosynthetic competence of transformants was due not to reversion of the original locus but rather to expression of the introduced gene. We suggest that the success of the present system is largely due to using a homologous C. reinhardtii gene, leading to stable maintenance and expression of the gene. Transformation with heterologous genes may be problematic because of poor expression due to an unusual codon bias in C. reinhardtii.
Assuntos
Proteínas de Algas , Chlamydomonas/genética , Proteínas de Plantas/genética , Transformação Genética , Sequência de Bases , Clonagem Molecular , Códon/genética , Cruzamentos Genéticos , Escherichia coli/genética , Genes , Marcadores Genéticos , Dados de Sequência Molecular , Mutação , Fotossíntese , Plasmídeos , Mapeamento por RestriçãoRESUMO
Light-regulated translation of chloroplastic mRNAs in the green alga Chlamydomonas reinhardtii requires nuclear encoded factors that interact with the 5'-untranslated region (5'-UTR) of specific mRNAs to enhance their translation. We have previously identified and characterized a set of proteins that bind specifically to the 5'-UTR of the chloroplastic psbA mRNA. Accumulation of these proteins is similar in dark- and light-grown cells, whereas their binding activity is enhanced during growth in the light. We have identified a serine/threonine protein phosphotransferase, associated with the psbA mRNA-binding complex, that utilizes the beta-phosphate of ADP to phosphorylate and inactivate psbA mRNA-binding in vitro. The inactivation of mRNA-binding in vitro is initiated at high ADP levels, levels that are attained in vivo only in dark-grown chloroplasts. These data suggest that the translation of psbA mRNA is attenuated by phosphorylation of the mRNA-binding protein complex in response to a rise in the stromal concentration of ADP upon transfer of cells to dark.
Assuntos
Proteínas de Bactérias/metabolismo , Chlamydomonas reinhardtii/metabolismo , Cloroplastos/metabolismo , Complexo de Proteínas do Centro de Reação Fotossintética/metabolismo , Proteínas de Plantas/metabolismo , Biossíntese de Proteínas/efeitos da radiação , RNA Mensageiro/metabolismo , Difosfato de Adenosina/metabolismo , Trifosfato de Adenosina/antagonistas & inibidores , Trifosfato de Adenosina/metabolismo , Animais , Chlamydomonas reinhardtii/genética , Chlamydomonas reinhardtii/crescimento & desenvolvimento , Íntrons , Luz , Fosforilação , Complexo de Proteína do Fotossistema II , Proteínas Quinases/metabolismo , Proteínas de Ligação a RNA/metabolismoRESUMO
In vitro culture of pericarp segments from fruit of Citrus sinensis (L.) Osbeck cv Valencia was used to determine the temporal sequence in development of chloroplasts from chromoplasts during regreening of the epicarp. Regreening of chromoplasts closely resembled greening of etioplasts, except that regreening proceeded much more slowly. Chlorophyll, the light-harvesting chlorophyll a/b binding protein of photosystem II, the chlorophyll a binding protein of reaction center P-700 of photosystem I, thylakoid membranes, and adenosine triphosphate synthetase were all detected at very low levels in degreened epicarp. All of these increased in parallel during regreening of the epicarp. Ribulose 1,5-bisphosphate carboxylase (RuBPCase) levels were high in degreened epicarp and declined for the first 10 days of culture before reaccumulating in the regreening segments. Light was necessary for the accumulation of all of the chloroplastic components. A lack of exogenous nitrogen did not prevent the accumulation of any chloroplastic component except Ru-BPCase, although accumulation of the other components was reduced. Sucrose at 150 millimolar in media lacking nitrogen markedly inhibited the accumulation of chlorophyll and light-harvesting chlorophyll a/b-protein.
RESUMO
Yellow leaves of chlorophyll-deficient seedlings and white leaves of carotenoid-deficient seedlings contain no detectable light-harvesting chlorophyll a/b binding proteins (LHCP). Chlorophyll-deficient leaves contain plastids which are arrested in development prior to chloroplast formation [Mascia, P.N. and Robertson, D.S. (1978) Planta (Berl.) 143, 207-211] while carotenoid-deficient leaves contain plastids which are arrested in development at a rudimentary stage [Bachmann, M. D., Robertson, D.S., Bowen, C.C., and Anderson, I.C. (1967) J. Ultrastruc. Res. 21, 41-60]. Chlorophyll-deficient leaves have normal levels of nuclear-encoded LHCP mRNA while carotenoid-deficient leaves contain only trace amounts of LHCP mRNA. Similar results were obtained with carotenoid deficiencies caused by nuclear gene mutations and by treatment with the herbicide norflurazon which blocks carotenoid biosynthesis. We conclude that events at early stages of plastid development influence the accumulation of a nuclear-encoded mRNA.
Assuntos
Carotenoides/deficiência , Clorofila/biossíntese , Fotossíntese , Proteínas de Plantas/biossíntese , Zea mays/metabolismo , Clorofila/deficiência , Clorofila/genética , Regulação da Expressão Gênica , Luz , Complexos de Proteínas Captadores de Luz , Mutação , Complexo de Proteínas do Centro de Reação Fotossintética , Proteínas de Plantas/genética , Biossíntese de Proteínas , RNA Mensageiro/metabolismo , Zea mays/genéticaRESUMO
The appearance of photosynthetic proteins was directly measured in developing maize (Zea mays L.) leaves. Third leaves of 10-14-d-old seedlings were dissected into six successive sections from the basal meristem to the tip of the leaf. The membrane and soluble proteins were separated by polyacrylamide gel electrophoresis and then transferred onto cyanogen bromide paper. After transfer of membrane proteins the paper was reacted with antisera raised against the light-harvesting chlorophyll a/b protein of photosystem II, the chlorophyll a-binding protein of reaction center P-700 of photosystem I and the α-subunit of chloroplast-coupling factor 1. The blots of soluble proteins were reacted with antisera raised against the electron-transport proteins plastocyanin and Fe-NADP reductase (EC 1.6.7.1), the carbon-fixing enzymes ribulose-1,5-bisphosphate carboxylase (EC 4.1.1.39) and phosphoenolpyruvate carboxylase (EC 4.1.1.31), as well as pyruvate orthophosphate dikinase (EC 2.7.9.1). The schedule of appearance of proteins shows that the light-harvesting and ATP-generating proteins are present in the most immature segments at the leaf base and accumulate rapidly as the cells mature. The carbon-reducing enzymes, however, appear only in tissue that has differentiated into mesophyll and bundle-sheath cells.
RESUMO
To study the interaction of the nuclear and chloroplast genomes in the biogenesis of the photosynthetic apparatus, nuclear mutants of Chlamydomonas reinhardtii deficient in photosystem II (PSII) activity were analyzed. Two independently-isolated, allelic nuclear mutants show a pleiotropic reduction in a set of functionally related PSII polypeptides. Immunoblot analysis reveals that the two mutants, nac-1-18 and nac-1-11, accumulate reduced amounts of the chloroplast-encoded polypeptides P5 and P6 and are completely deficient in polypeptides D1 and D2. Polypeptides of the oxygen-evolving and light-harvesting complexes associated with PSII, however, are present at wild-type levels. Analysis of mRNAs encoding PSII polypeptides from these mutants indicates that all messages are present, although some species, including the D2 message, are significantly elevated. When mutant cells are pulse-labeled for 10 min with [C]acetate, a greatly reduced amount of labeled D2 protein is observed, while all other PSII polypeptides are synthesized normally. These data indicate that the mutations present in nac-1-18 and nac-1-11 affect a nuclear gene whose product specifically controls the translation and/or degradation of the chloroplast-encoded D2 polypeptide.
RESUMO
In Chlamydomonas reinhardtii the oxygen evolving enhancer protein 1 (OEE1), which is part of the oxygen evolving complex of photosystem II (PS II), is coded for by a single nuclear gene (psb1). The nuclear mutant FuD44 specifically lacks the OEE1 polypeptide and is completely deficient in photosynthetic oxygen evolution. In this mutant a 5 kb DNA insertion into the 5' region of the psb1 gene results in the complete absence of OEE1 mRNA and protein. A revertant, FuD44-R 2, which is capable of 30% of the photosynthetic oxygen evolution of wild-type cells, has lost 4 kb of the 5 kb DNA insert, and accumulates both OEE1 mRNA and protein, although at levels somewhat less than those of wild-type cells. Absence of the OEE1 protein in the FuD44 mutant does not affect the accumulation of other nuclear encoded PS II peripheral polypeptides. OEE1 absence does, however, result in a more rapid turnover of the chloroplast encoded PS II core polypeptides, thus resulting in a substantial deficiency of PS II core polypeptides in FuD44 cells. These PS II core proteins again accumulate in revertant FuD44-R2 cells.