RESUMO
ADP-glucose pyrophosphorylase (AGPase) is a key regulatory enzyme of bacterial glycogen and plant starch synthesis as it controls carbon flux via its allosteric regulatory behavior. Unlike the bacterial enzyme that is composed of a single subunit type, the plant AGPase is a heterotetrameric enzyme (alpha2beta2) with distinct roles for each subunit type. The large subunit (LS) is involved mainly in allosteric regulation through its interaction with the catalytic small subunit (SS). The LS modulates the catalytic activity of the SS by increasing the allosteric regulatory response of the hetero-oligomeric enzyme. To identify regions of the LS involved in binding of effector molecules, a reverse genetics approach was employed. A potato (Solanum tuberosum L.) AGPase LS down-regulatory mutant (E38A) was subjected to random mutagenesis using error-prone polymerase chain reaction and screened for the capacity to form an enzyme capable of restoring glycogen production in glgC(-) Escherichia coli. Dominant mutations were identified by their capacity to restore glycogen production when the LS containing only the second site mutations was co-expressed with the wild-type SS. Sequence analysis showed that most of the mutations were decidedly nonrandom and were clustered at conserved N- and C-terminal regions. Kinetic analysis of the dominant mutant enzymes indicated that the K(m) values for cofactor and substrates were comparable with the wild-type AGPase, whereas the affinities for activator and inhibitor were altered appreciably. These AGPase variants displayed increased resistance to P(i) inhibition and/or greater sensitivity toward 3-phosphoglyceric acid activation. Further studies of Lys-197, Pro-261, and Lys-420, residues conserved in AGPase sequences, by site-directed mutagenesis suggested that the effectors 3-phosphoglyceric acid and P(i) interact at two closely located binding sites.
Assuntos
Nucleotidiltransferases/metabolismo , Solanum tuberosum/enzimologia , Sítio Alostérico , Sequência de Aminoácidos , Sequência de Bases , Primers do DNA , Glucose-1-Fosfato Adenililtransferase , Cinética , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Nucleotidiltransferases/química , Nucleotidiltransferases/genética , Nucleotidiltransferases/isolamento & purificação , Fenótipo , Estrutura Secundária de Proteína , Homologia de Sequência de AminoácidosRESUMO
During rice seed development, prolamine RNAs are localized to the surface of the prolamine storage protein bodies (PBs), organelles bounded by the endoplasmic reticulum (ER). The exact mechanism by which prolamine RNAs are enriched on this ER subdomain is not known but recent evidence indicates the directed transport and targeting of prolamine RNAs to the prolamine PBs. As such a process involves RNA signal determinants and cytoskeleton-interacting proteins that recognize these signals, we obtained an enriched cytoskeleton-PB fraction and identified a prominent RNA-binding activity, Rp120, by RNA-binding UV-cross-linking assay. Recombinant cDNA clones of Rp120 revealed that the primary sequence shared considerable structural homology to the human transcriptional coactivator p100 and possessed a modular organization, four nucleic acid-binding SN domains, a tudor domain and a coil-coil domain. Consistent with the presence of SN domains, Rp120 binds a variety of RNAs including prolamine RNA. Interaction with the latter RNA, however, was specific as binding activity was evident only to the prolamine 3' UTR and not to the 5' UTR or coding sequences. Rp120 is also able to interact with other proteins as its sedimentation behavior in sucrose density gradient suggests an association with the cytoskeleton. The presence of a tudor domain, suggested to have a role in RNA processing or transport, together with the SN and coiled-coil domains are consistent with the view that Rp120 may be involved in RNA sorting in rice endosperm.
Assuntos
Citoesqueleto/metabolismo , Oryza/química , Proteínas de Ligação a RNA/isolamento & purificação , Sementes/química , Sequência de Aminoácidos , Sítios de Ligação , Immunoblotting , Dados de Sequência Molecular , Peso Molecular , Ligação Proteica , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Sementes/crescimento & desenvolvimento , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Frações Subcelulares/química , Distribuição TecidualRESUMO
Plant productivity and yield are dependent on source-sink relationships, i.e. the capacity of source leaves to fix CO2 and the capacity of developing sink tissues and organs to assimilate and convert this fixed carbon into dry matter. Studies from our laboratories as well as others have demonstrated that rice productivity and yield are mainly sink-limited during its development because of limited capacity to utilize the initial photosynthetic product (triose phosphate). This limitation in triose phosphate utilization, evident at both the vegetative and reproductive stages of rice development, may be associated with limited capacity for carbohydrate synthesis in rice leaves (which are poor accumulators of starch) or feedback due to limited sink strength of developing seeds. Strategies in improving triose phosphate utilization by enhancing starch production in leaves and developing seeds by the expression of engineered genes for ADP glucose pyrophosphorylase, a key regulatory enzyme of starch biosynthesis, are discussed.
Assuntos
Oryza/crescimento & desenvolvimento , Oryza/metabolismo , Amido/biossíntese , Arabidopsis/metabolismo , Biomassa , Mutação , Fotossíntese/fisiologia , Folhas de Planta/crescimento & desenvolvimento , Folhas de Planta/metabolismo , ReproduçãoRESUMO
Three ADP-glucose pyrophosphorylase clones were isolated from the cotyledon cDNA library of the oil plant, Perilla frutescens, and their intracellular localization investigated. Two of three cDNAs (PfagpS1 and PfagpS2) were homologous to the catalytic small subunit of AGPases found in other plants, while the third clone (PfagpL) was highly similar to the large subunit type. Transcripts for PfagpS1 and PfagpS2 were observed in both photosynthetic and non-photosynthetic tissue, showing the highest expression in the stem, while PfagpL transcripts were abundantly expressed in stem and cotyledon. To evaluate the subcellular localization of PfagpS2 and PfagpL as well as the maize BT2, N-terminus-GFP DNA fusion were constructed and transformed into tobacco plants. Immunoblot analysis showed that the expressed PfagpS2- and PfagpL-GFP fusions were targeted to the plastid in the heterologous tobacco system whereas the BT2-GFP remained intact, suggesting a cytoplasmic location. These intracellular assignments were confirmed by direct confocal microscopic examination. GFP signals were localized to the cytoplasm as well as in the nucleus in BT2-GFP plants, and to the plastids in PfagpS2- and PfagpL-GFP plants. Our results indicate that Perilla cotyledons contain multiple AGPase subunits, of which at least two isoforms and very likely the third, are plastidial in nature.
Assuntos
Regulação Enzimológica da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Lamiaceae/enzimologia , Nucleotidiltransferases/genética , Sequência de Aminoácidos , Sequência de Bases , Clonagem Molecular , DNA de Plantas , Expressão Gênica , Glucose-1-Fosfato Adenililtransferase , Proteínas de Fluorescência Verde , Immunoblotting , Líquido Intracelular/metabolismo , Lamiaceae/genética , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Dados de Sequência Molecular , Nucleotidiltransferases/metabolismo , Óleos de Plantas , RNA de Plantas , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Homologia de Sequência de Aminoácidos , Frações Subcelulares , Transcrição Gênica , Ácido alfa-LinolênicoRESUMO
ADP-glucose pyrophosphorylase (AGPase), a key regulatory enzyme in higher plant starch biosynthesis, is composed of a pair of large and small subunits (alpha(2)beta(2)). Current evidence suggests that the large subunit has primarily a regulatory function, while the small subunit has both regulatory and catalytic roles. To define the structure-function relationship of the large subunit (LS), the LS of potato AGPase was subjected to chemical mutagenesis and coexpressed with the wild-type (WT) small subunit (SS) cDNA in an AGPase defective Escherichia coli strain. An LS mutant (M143) was isolated, which accumulated very low levels of glycogen compared to the WT recombinant AGPase, but maintained normal catalytic activity when assayed under saturating conditions. Sequence analysis revealed that M143 has a single amino acid change, V463I, which lies adjacent to the C-terminus. This single mutation had no effect on the Km for ATP and Mg(2+), which were similar to the WT enzyme. The K(m) for glucose 1-P, however, was sixfold higher than the WT enzyme. These results suggest that the LS plays a role in binding glucose 1-P through its interaction with the SS.
Assuntos
Nucleotidiltransferases/metabolismo , Sequência de Aminoácidos , Catálise , Eletroforese em Gel de Poliacrilamida , Glucose-1-Fosfato Adenililtransferase , Mutagênese , Nucleotidiltransferases/química , Nucleotidiltransferases/genética , Nucleotidiltransferases/isolamento & purificação , Ligação Proteica , Homologia de Sequência de AminoácidosRESUMO
ADP-glucose pyrophosphorylase (AGPase) is the allosterically regulated gateway for carbon entry into transient and storage starch in plants as well as glycogen in bacteria. This enzyme plays a key role in the modulation of photosynthetic efficiency in source tissues and directly determines the level of storage starch in sink tissues, thus influencing overall crop yield potential. AGPase is a tetrameric enzyme; in higher plants it consists of two regulatory large subunits (LS) and two catalytic small subunits (SS), while in cyanobacteria and prokaryotes the enzyme is homotetrameric. The potato SS gene in pML10 was mutated by hydroxylamine and mutants were screened for elevated homotetrameric activity by iodine vapor staining. This search strategy led to the isolation of SS mutants (SUP-1, TG-15) that had pyrophosphorylase activity in the absence of the LS. TG-15 has a leucine to phenylalanine change at position 48 (L(48)F) that corresponds to a phenylalanine residue at the analogous position in the Escherichia coli homotetrameric AGPase as well as a valine to isoleucine change at position 59 (V(59)I). TG-15 was partially purified and kinetic analysis revealed substrate and effector affinities equal to wild type heterotetrameric enzyme with the exception of ATP binding.
Assuntos
Nucleotidiltransferases/química , Nucleotidiltransferases/metabolismo , Solanum tuberosum/enzimologia , Sequência de Aminoácidos , Escherichia coli/enzimologia , Glucose-1-Fosfato Adenililtransferase , Cinética , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Nucleotidiltransferases/genética , Subunidades Proteicas , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Solanum tuberosum/genética , Especificidade por SubstratoRESUMO
Rice seeds, a rich reserve of starch and protein, are a major food source in many countries. Unlike the seeds of other plants, which typically accumulate one major type of storage protein, rice seeds use two major classes, prolamines and globulin-like glutelins. Both storage proteins are synthesized on the endoplasmic reticulum (ER) and translocated to the ER lumen, but are then sorted into separate intracellular compartments. Prolamines are retained in the ER lumen as protein bodies whereas glutelins are transported and stored in protein storage vacuoles. Mechanisms responsible for the retention of prolamines within the ER lumen and their assembly into intracisternal inclusion granules are unknown, but the involvement of RNA localization has been suggested. Here we show that the storage protein RNAs are localized to distinct ER membranes and that prolamine RNAs are targeted to the prolamine protein bodies by a mechanism based on RNA signal(s), a process that also requires a translation initiation codon. Our results indicate that the ER may be composed of subdomains that specialize in the synthesis of proteins directed to different compartments of the plant endomembrane system.
Assuntos
Retículo Endoplasmático/metabolismo , Glutens/genética , Oryza/metabolismo , Proteínas de Plantas/genética , RNA Mensageiro/metabolismo , RNA de Plantas/metabolismo , Regiões 3' não Traduzidas , Glutens/metabolismo , Proteínas de Plantas/metabolismo , Plantas Geneticamente Modificadas , Prolaminas , Estrutura Terciária de Proteína , Transporte Proteico , Sementes , Simplexvirus/genéticaRESUMO
The mRNAs that encode the prolamine storage proteins in rice (Oryza sativa L.) endosperm cells are enriched on the surface of the prolamine protein bodies (PBs), a subcellular structure consisting of a prolamine intracisternal granule surrounded by rough endoplasmic reticulum membrane. Previous biochemical studies (D.G. Muench et al., 1998, Plant Physiol. 116: 559-569) have shown that prolamine mRNAs may be anchored to the PB surface via the cytoskeleton. To better understand the mechanism and role of mRNA localization in rice endosperm cells, we studied the subcellular development of prolamine PBs and their relationship with the cytoskeleton in rice endosperm cells. Confocal microscopy of endosperm cells showed that, unlike the glutelin PBs, the developing prolamine PBs are not randomly distributed within the cell, but instead are often enriched in the cortical region of the cell only a few micrometers beneath the plasma membrane. In addition, the peripheral prolamine PBs are closely associated with the cortical microtubule and actin filament networks. The cortical enrichment of rice prolamine protein bodies represents a unique example of endoplasmic reticulum subdomain localization in plant cells. The interaction of this endoplasmic reticulum subdomain with the cytoskeleton provides new insights on the possible mechanism and role of mRNA localization in plants.
Assuntos
Citoesqueleto/fisiologia , Organelas/fisiologia , Oryza/fisiologia , Proteínas de Plantas/genética , Citoesqueleto/ultraestrutura , Técnica Indireta de Fluorescência para Anticorpo , Regulação da Expressão Gênica de Plantas , Microscopia Confocal , Organelas/ultraestrutura , Oryza/citologia , Oryza/crescimento & desenvolvimento , Proteínas de Plantas/biossíntese , Prolaminas , RNA Mensageiro/metabolismoRESUMO
The characterization and production of starch variants from mutation studies and transgene technology has been invaluable for our understanding of the synthesis of the starch granule. The knowledge gained has allowed for genetic manipulation of the starch biosynthetic pathway in plants. This in vivo approach can be used to generate novel starches and diminishes the need for post-harvest chemically and enzymatically treated starches. Thus, the modification of the starch biosynthetic pathway is a plausible means by which starches with novel properties and applications can be created.
Assuntos
Engenharia Genética , Amido/genética , Sequência de Carboidratos , Dados de Sequência Molecular , Mutagênese , Amido/biossíntese , Amido/químicaRESUMO
Wild-type Arabidopsis plants, the starch-deficient mutant TL46, and the near-starchless mutant TL25 were evaluated by noninvasive in situ methods for their capacity for net CO2 assimilation, true rates of photosynthetic O2 evolution (determined from chlorophyll fluorescence measurements of photosystem II), partitioning of photosynthate into sucrose and starch, and plant growth. Compared with wild-type plants, the starch mutants showed reduced photosynthetic capacity, with the largest reduction occurring in mutant TL25 subjected to high light and increased CO2 partial pressure. The extent of stimulation of CO2 assimilation by increasing CO2 or by reducing O2 partial pressure was significantly less for the starch mutants than for wild-type plants. Under high light and moderate to high levels of CO2, the rates of CO2 assimilation and O2 evolution and the percentage inhibition of photosynthesis by low O2 were higher for the wild type than for the mutants. The relative rates of 14CO2 incorporation into starch under high light and high CO2 followed the patterns of photosynthetic capacity, with TL46 showing 31% to 40% of the starch-labeling rates of the wild type and TL25 showing less than 14% incorporation. Overall, there were significant correlations between the rates of starch synthesis and CO2 assimilation and between the rates of starch synthesis and cumulative leaf area. These results indicate that leaf starch plays an important role as a transient reserve, the synthesis of which can ameliorate any potential reduction in photosynthesis caused by feedback regulation.
Assuntos
Arabidopsis/metabolismo , Carbono/metabolismo , Nucleotidiltransferases/metabolismo , Arabidopsis/genética , Arabidopsis/crescimento & desenvolvimento , Proteínas de Arabidopsis , Dióxido de Carbono/metabolismo , Transporte de Elétrons , Retroalimentação , Glucose-1-Fosfato Adenililtransferase , Mutação , Nucleotidiltransferases/genética , Oxigênio/metabolismo , Fotoperíodo , Fotossíntese , Folhas de Planta/crescimento & desenvolvimento , Amido/metabolismo , Sacarose/metabolismoRESUMO
Mutagenesis of the large subunit (LS) of the potato ADP-glucose pyrophosphorylase generated an enzyme, P52L, that was insensitive to 3-phosphoglycerate (3-PGA). To identify additional residues involved in 3-PGA interaction, we subjected P52L LS DNA to a second round of mutagenesis and identified second-site revertants by their ability to restore glycogen accumulation as assessed by iodine (I2) staining. Enzymes from class I revertants with normal I2-staining had an 11- to 49-fold greater affinity for the activator 3-PGA compared with the P52L mutant and a decreased sensitivity to the inhibitor orthophosphate. Sequence analysis of these class I revertants identified a P66L mutation in R4, an E38K mutation in R20, and a G101N mutation in R10 and R32. These mutations appear to restore 3-PGA binding by counteracting the effect of the P52L mutation because introducing E38K or G101N into the wild-type LS led to enzyme variants with higher affinity for the activator 3-PGA and increased resistance to the inhibitor orthophosphate. The generation of these revertant enzymes provides additional structure-function information on the allosteric regulation of higher plant ADP-glucose pyrophosphorylases and validates a strategy for developing novel variants of the enzyme that may be useful in manipulating starch biosynthesis in higher plants.
Assuntos
Nucleotidiltransferases/genética , Solanum tuberosum/enzimologia , Solanum tuberosum/genética , Sítio Alostérico/genética , Sequência de Aminoácidos , Sequência de Bases , Primers do DNA/genética , Ativação Enzimática/efeitos dos fármacos , Ativação Enzimática/genética , Estabilidade Enzimática/genética , Genes de Plantas , Variação Genética , Glucose-1-Fosfato Adenililtransferase , Ácidos Glicéricos/metabolismo , Ácidos Glicéricos/farmacologia , Temperatura Alta , Cinética , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Nucleotidiltransferases/química , Nucleotidiltransferases/metabolismo , Homologia de Sequência de Aminoácidos , Amido/biossíntese , Regulação para CimaRESUMO
ADP-glucose pyrophosphorylase is a key regulatory enzyme in starch synthesis in most plant tissues. Unlike the allosteric regulatory dependent properties of the leaf enzyme, the enzymes from non-photosynthetic tissues exhibit varying levels of sensitivity to allosteric regulation, a behavior which may be an inherent property of the enzyme or a product of post-translational modification. As partial proteolysis of the holoenzyme may account for the wide variation of allosteric regulatory behavior exhibited by enzymes from non-photosynthetic tissues, small N- and C-terminal peptide deletions were made on either the potato large and small subunit and co-expressed with the counterpart wild-type subunit in Escherichia coli. Removal of the putative carboxy-terminal allosteric binding region from either subunit type results in an abolishment of enzyme formation indicating that the carboxy terminus of each subunit type is essential for proper subunit folding and/or enzyme assembly as well as its suggested role in allosteric regulation. Removal of a small 10 amino acid peptide from the N-terminus of the small subunit increased its resistance to the allosteric inhibitor Pi as well as its sensitivity to heat treatment. Likewise, removal of the corresponding peptide (17 residues) at the N-terminus of the large subunit also increased its resistance towards Pi inhibition but, in addition, increased its sensitivity to 3-PGA activation. Deletion of an additional 11 residues reversed these changes in allosteric properties but at the expense of a reduced catalytic turnover rate. Combined, these results indicate that the N- and C-terminal regions are essential for the proper catalytic and allosteric regulatory properties of the potato ADP-glucose pyrophosphorylase. The possible significance of these results on the observed insensitivity to effector molecules by ADP-glucose pyrophosphorylases from other non-photosynthetic tissues is discussed.
Assuntos
Nucleotidiltransferases/metabolismo , Fragmentos de Peptídeos/fisiologia , Regulação Alostérica/efeitos dos fármacos , Regulação Alostérica/genética , Sequência de Aminoácidos , Aminoácidos/genética , Aminoácidos/fisiologia , Catálise , Glucose-1-Fosfato Adenililtransferase , Temperatura Alta , Cinética , Dados de Sequência Molecular , Nucleotidiltransferases/genética , Fragmentos de Peptídeos/genética , Deleção de Sequência , Solanum tuberosum/enzimologia , Spinacia oleracea/enzimologiaRESUMO
To explore the structure-function relationships of the heterotetrameric higher plant ADP-glucose pyrophosphorylase, composed of a pair of large and small subunits, the small subunit cDNA was subjected to chemical mutagenesis and then co-expressed with the wild-type large subunit cDNA. Mutants were selected for their inability to complement a defective bacterial ADP-glucose pyrophosphorylase gene and, in turn, to accumulate glycogen as viewed by iodine staining of the cells. Based on these initial analyses, we subsequently identified four distinct classes of mutations which were glycogen-deficient but exhibited enzyme activity levels comparable to the normal recombinant enzyme under saturating reaction conditions. Three classes, each a product of single amino acid substitution, showed altered kinetic constants for substrates. Substitution of Asp252 to Asn conferred the enzyme lower affinity for glucose-1-phosphate, replacement of Asp121 to Asn resulted in an enzyme less responsive to both glucose-1-phosphate and ATP, while the Ala106 to Thr substituted enzyme contains altered sensitivity primarily to ATP. The fourth class, a Pro43 to Ser substitution, resulted in an enzyme with decreased sensitivity (8-fold) to the activator 3-PGA. Overall, the results of this study suggests that the two subunit types do not have identical roles in enzyme function and that the small subunit plays a more dominant role in catalysis than the large subunit.
Assuntos
Nucleotidiltransferases/química , Nucleotidiltransferases/metabolismo , Solanum tuberosum/enzimologia , Sequência de Aminoácidos , Substituição de Aminoácidos , Sítios de Ligação , Clonagem Molecular , DNA Complementar , Glucose-1-Fosfato Adenililtransferase , Cinética , Substâncias Macromoleculares , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Especificidade por SubstratoRESUMO
Recent evidence has shown that the prolamine polysomes are attached not only to the endoplasmic reticulum membranes that bound the prolamine protein bodies (PBs) but also to cytoskeleton elements associated with this subcellular fraction. To learn more about the nature of the proteins that are associated with this supra-macromolecular complex, proteins extracted from an enriched cytoskeleton-PB fraction were resolved by two-dimensional polyacrylamide gel electrophoresis under non-equilibrium conditions and analyzed for their composition by immunological and biochemical methods. Immunoblot analysis indicated the presence of the cytoskeletal proteins, actin and tubulin, and the cytoskeletal-associated protein EF1 alpha in this fraction. Microsequencing of selected polypeptides revealed a diversity of protein sequences. In addition to contaminating storage proteins which are selectively solubilized by the isolation procedure, several ribosomal proteins and histone H3 were also identified. Some of the remaining polypeptides showed partial homology to protein sequences deposited in the database, several of which are cytoskeleton-associated proteins.
Assuntos
Citoesqueleto/química , Organelas/química , Oryza/química , Proteínas de Plantas/química , Sementes/química , Sequência de Aminoácidos , Eletroforese em Gel Bidimensional , Immunoblotting , Dados de Sequência Molecular , Peptídeos/isolamento & purificação , Análise de Sequência , Homologia de Sequência de Aminoácidos , Frações Subcelulares/químicaRESUMO
The ER luminal binding protein, BiP, has been linked to prolamine protein body formation in rice. To obtain further information on the possible role of this chaperone in protein body formation we have cloned and sequenced a BiP cDNA homolog from rice endosperm. The rice sequence is very similar to the maize BiP exhibiting 92% nucleotide identity and 96% deduced amino acid sequence identity in the coding region. Substantial amino acid sequence homology exists between rice BiP and BiP homologs from several other plant and animal species including long stretches of conservation through the amino-terminal ATPase domain. Considerable variation, however, is observed within the putative carboxy-terminal peptide-binding domain between the plant and nonplant BiP sequences. A single hand of approximately 2.4 kb was visible when RNA gel blots of total RNA purified from seed tissue were probed with radiolabeled rice BiP cDNA. This band increased in intensity during seed development up to 10 days after flowering, and then decreased gradually until seed maturity. Protein gel blots indicated that BiP polypeptide accumulation parallels that of the prolamine polypeptides throughout seed development. Immunocytochemical analysis demonstrated that BiP is localized in a non-stochastic fashion in the endoplasmic reticulum membrane complex of developing endosperm cells. It is abundant on the periphery of the protein inclusion body but not in the central portion of the protein body or in the cisternal ER membranes connecting the protein bodies. These data support a model which proposes that BiP associates with the newly synthesized prolamine polypeptide to facilitate its folding and assembly into a protein inclusion body, and is then recycled.
Assuntos
Proteínas de Transporte/genética , Proteínas de Choque Térmico/genética , Oryza/genética , Proteínas de Plantas/genética , Sequência de Aminoácidos , Proteínas de Arabidopsis , Sequência de Bases , Clonagem Molecular , DNA de Plantas , Expressão Gênica , Dados de Sequência Molecular , Oryza/ultraestrutura , RNA Mensageiro/análise , Homologia de Sequência de AminoácidosRESUMO
As part of a structure-function analysis of the higher-plant ADP-glucose pyrophosphorylase (AGP), we used a random mutagenesis approach in combination with a novel bacterial complementation system to isolate over 100 mutants that were defective in glycogen production (T.W. Greene, S.E. Chantler, M.L. Khan, G.F. Barry, J. Preiss, T.W. Okita [1996] Proc Natl Acad Sci USA 93: 1509-1513). One mutant of the large subunit M27 was identified by its capacity to only partially complement a mutation in the structural gene for the bacterial AGP (glg C), as determined by its light-staining phenotype when cells were exposed to l3 vapors. Enzyme-linked immunosorbent assay and enzymatic pyrophosphorylysis assays of M27 cell extracts showed that the level of expression and AGP activity was comparable to those of cells that expressed the wild-type recombinant enzyme. Kinetic analysis indicated that the M27 AGP displays normal Michaelis constant values for the substrates glucose-1-phosphate and ATP but requires 6- to 10-fold greater levels of 3-phosphoglycerate (3-PGA) than the wild-type recombinant enzyme for maximum activation. DNA sequence analysis showed that M27 contains a single point mutation that resulted in the replacement of aspartic acid 413 to alanine. Substitution of a lysine residue at this site almost completely abolished activation by 3-PGA. Aspartic acid 413 is adjacent to a lysine residue that was previously identified by chemical modification studies to be important in the binding of 3-PGA (K. Ball, J. Preiss [1994] J Biol Chem 269: 24706-24711). The kinetic properties of M27 corroborate the importance of this region in the allosteric regulation of a higher-plant AGP.
Assuntos
Ácido Aspártico , Nucleotidiltransferases/química , Nucleotidiltransferases/metabolismo , Solanum tuberosum/enzimologia , Regulação Alostérica , Sítio Alostérico , Sequência de Aminoácidos , Primers do DNA , Glucose-1-Fosfato Adenililtransferase , Cinética , Substâncias Macromoleculares , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Mutação Puntual , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Homologia de Sequência de Aminoácidos , Spinacia oleracea/enzimologiaRESUMO
ADP-glucose pyrophosphorylase (AGP) is a key regulatory enzyme in the biosynthesis of starch in higher plants. Previous studies have suggested that, unlike other plants that display tissue-specific AGP genes, potato expresses the same AGP small-subunit gene (sAGP) in multiple tissues. This view was confirmed by the spatial patterns of expression of the sAGP gene in transgenic potato plants observed when a promoter-dependent-beta-glucuronidase (beta-GUS) system was used. sAGP-beta-GUS chimeric gene fusions were expressed at high levels in tubers and in many other starch-containing cells throughout the plant. Deletional analysis of the 5'-upstream region of sAGP revealed that the observed spatial patterns of expression were due to different regions of the promoter of sAGP functioning in combination to confer cell- and organ-specific patterns of expression. Depending on the tissue examined, the patterns of reporter-gene expression were enhanced, suppressed, or altered when the 3'-nopaline-synthase terminator was replaced by the 3'-flanking sequence of sAGP. The observed cellular expression patterns of sAGP only partially overlap with the reported expression patterns of the major large-subunit gene (lAGP) in leaves. Since AGP is a heterotetrameric enzyme, composed of two sAGP and two lAGP subunits, this difference in the cellular expression patterns as well as quantitative differences in expression of the two AGP genes may account for the observed post-transcriptional regulation, i.e., relatively high levels of transcript but low levels of sAGP subunit in leaves.
Assuntos
Regulação Enzimológica da Expressão Gênica , Genes de Plantas , Nucleotidiltransferases/biossíntese , Regiões Promotoras Genéticas , Sequências Reguladoras de Ácido Nucleico , Solanum tuberosum/enzimologia , Agrobacterium tumefaciens , Sequência de Bases , Clonagem Molecular , Sequência Consenso , Primers do DNA , Regulação da Expressão Gênica de Plantas , Glucose-1-Fosfato Adenililtransferase , Glucuronidase/biossíntese , Substâncias Macromoleculares , Dados de Sequência Molecular , Nucleotidiltransferases/genética , Folhas de Planta , Raízes de Plantas , Caules de Planta , Plantas Geneticamente Modificadas , Reação em Cadeia da Polimerase , Proteínas Recombinantes de Fusão/biossínteseRESUMO
ADPglucose pyrophosphorylase (glucose-1-phosphate adenylyltransferase; ADP:alpha-D-glucose-1-phosphate adenylyltransferase, EC 2.7.7.27) catalyzes a key regulatory step in alpha-glucan synthesis in bacteria and higher plants. We have previously shown that the expression of the cDNA sequences of the potato tuber large (LS) and small (SS) subunits yielded a functional heterotetrameric enzyme capable of complementing a mutation in the single AGP (glgC) structural gene of Escherichia coli. This heterologous complementation provides a powerful genetic approach to obtain biochemical information on the specific roles of LS and SS in enzyme function. By mutagenizing the LS cDNA with hydroxylamine and then coexpressing with wild-type SS in an E. coli glgC- strain, >350 mutant colonies were identified that were impaired in glycogen production. One mutant exhibited enzymatic and antigen levels comparable to the wild-type recombinant enzyme but required 45-fold greater levels of the activator 3-phosphoglycerate for maximum activity. Sequence analysis identified a single nucleotide change that resulted in the change of Pro-52 to Leu. This heterologous genetic system provides an efficient means to identify residues important for catalysis and allosteric functioning and should lead to novel approaches to increase plant productivity.
Assuntos
Ácidos Glicéricos/metabolismo , Nucleotidiltransferases/genética , Proteínas de Plantas/genética , Solanum tuberosum/enzimologia , Regulação Alostérica , Sequência de Aminoácidos , Proteínas de Bactérias/metabolismo , Sequência Consenso , DNA Complementar/genética , DNA de Plantas/genética , Ativação Enzimática , Escherichia coli/enzimologia , Teste de Complementação Genética , Glucose-1-Fosfato Adenililtransferase , Glicogênio/biossíntese , Dados de Sequência Molecular , Mutagênese , Nucleotidiltransferases/metabolismo , Proteínas de Plantas/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Solanum tuberosum/genéticaRESUMO
Three DNA binding activities, BP-1, BP-2 and BP-3, which interact with the 5' flanking region of the rice glutelin Gt3 gene, were identified by gel retardation assays of rice seed nuclear extracts. The DNA binding activities were seed-specific as identical DNA-protein complexes were not observed when nuclear extracts from leaf tissue or suspension culture cells were analyzed, suggesting that these DNA binding activities are involved in seed-specific expression of the Gt3 gene. The DNA sequences recognized by these DNA binding activities were identified by DNaseI foot-printing and Bal 31 nuclease mapping analyses. BP-1 recognizes DNA sequences located at -272 bp to -259 bp relative to the transcriptional start site. This DNA segment contains a sequence motif that is conserved among several seed protein genes, implicating that the motif may be a common cis-regulatory element determining seed-specific expression of these genes. BP-2 interacts with sequences located between -861 bp to -838 bp while BP-3 interacts with sequences upstream of -788 bp. The temporal levels of BP-2 binding activity parallel the steady state levels of the Gt3 mRNAs during seed development. Overall, these results and those obtained from in vivo promoter analysis in transgenic plants [Zhao et al. (1994) Plant Mol. Biol. 25: 429] indicate that multiple regulatory elements located at two spatially separated regions of the Gt3 promoter are involved in endosperm-specific and temporal regulation.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Glutens/genética , Proteínas Nucleares/metabolismo , Oryza/genética , Regiões Promotoras Genéticas , Sequência de Bases , DNA de Plantas/metabolismo , Regulação da Expressão Gênica de Plantas , Genes de Plantas , Dados de Sequência Molecular , Oryza/metabolismo , Ligação Proteica , Sementes/químicaRESUMO
Using a cDNA library prepared from poly(A)+ RNA from 10-day-old rice endosperm, partial nucleotide sequences of randomly isolated clones were analyzed. A total of 153 (30.6%) out of 500 cDNA clones showed high amino acid identity to previously identified genes. There was significant redundancy in cDNAs encoding prolamine and glutelin. About 21.0% of the cDNA clones were found to code for seed storage protein genes. Consequently, 37 independent genes were identified. Using cDNA clones encoding glutelin, prolamine, seed allergen, alpha-1,4-glucan branching enzyme, glycine-rich RNA binding protein, metallothionein, non-specific lipid-transfer protein and ubiquitin conjugating enzyme the accumulation of mRNA during rice seed development was compared. Genes associated with seed storage protein and starch biosynthesis were expressed according to expected developmental stages. Glycine-rich RNA binding protein genes as well as metallothionein-like protein genes were highly expressed in developing seeds, but low in leaves of whole plants.