RESUMO
KEY MESSAGE: This review outlines research performed in the last two decades on the structural, kinetic, regulatory and evolutionary aspects of ADP-glucose pyrophosphorylase, the regulatory enzyme for starch biosynthesis. ADP-glucose pyrophosphorylase (ADP-Glc PPase) catalyzes the first committed step in the pathway of glycogen and starch synthesis in bacteria and plants, respectively. Plant ADP-Glc PPase is a heterotetramer allosterically regulated by metabolites and post-translational modifications. In this review, we focus on the three-dimensional structure of the plant enzyme, the amino acids that bind the regulatory molecules, and the regions involved in transmitting the allosteric signal to the catalytic site. We provide a model for the evolution of the small and large subunits, which produce heterotetramers with distinct catalytic and regulatory properties. Additionally, we review the various post-translational modifications observed in ADP-Glc PPases from different species and tissues. Finally, we discuss the subcellular localization of the enzyme found in grain endosperm from grasses, such as maize and rice. Overall, this work brings together research performed in the last two decades to better understand the multiple mechanisms involved in the regulation of ADP-Glc PPase. The rational modification of this enzyme could improve the yield and resilience of economically important crops, which is particularly important in the current scenario of climate change and food shortage.
Assuntos
Evolução Molecular , Glucose-1-Fosfato Adenililtransferase/química , Glucose-1-Fosfato Adenililtransferase/fisiologia , Plantas/enzimologia , Regulação Alostérica , Glucose-1-Fosfato Adenililtransferase/genética , Modelos Moleculares , Conformação Proteica , Amido/biossíntese , Amido/químicaRESUMO
ADP-glucose pyrophosphorylase (AGPase) is a key allosteric enzyme in plant starch biosynthesis. Plant AGPase is a heterotetrameric enzyme that consists of large (LS) and small subunits (SS), which are encoded by two different genes. In this study, we showed that the conversion of Glu to Gly at position 370 in the LS of AGPase alters the heterotetrameric stability along with the binding properties of substrate and effectors of the enzyme. Kinetic analyses revealed that the affinity of the LSE370GSSWT AGPase for glucose-1-phosphate is 3-fold less than for wild type (WT) AGPase. Additionally, the LSE370GSSWT AGPase requires 3-fold more 3-phosphogyceric acid to be activated. Finally, the LSE370GSSWTAGPase is less heat stable compared with the WT AGPase. Computational analysis of the mutant Gly-370 in the 3D modeled LS AGPase showed that this residue changes charge distribution of the surface and thus affect stability of the LS AGPase and overall heat stability of the heterotetrameric AGPase. In summary, our results show that LSE370 intricately modulate the heat stability and enzymatic activity of potato the AGPase.
Assuntos
Glucose-1-Fosfato Adenililtransferase/fisiologia , Proteínas de Plantas/fisiologia , Solanum tuberosum/enzimologia , Amido/biossíntese , Sítios de Ligação , Estabilidade Enzimática , Glucose-1-Fosfato Adenililtransferase/química , Glicogênio/biossíntese , Temperatura Alta , Cinética , Modelos Moleculares , Mutagênese Sítio-Dirigida , Proteínas de Plantas/química , Estrutura Terciária de Proteína , Solanum tuberosum/genética , Especificidade por SubstratoRESUMO
To elucidate the starch synthesis pathway and the role of this reserve in rice pollen, we characterized mutations in the plastidic phosphoglucomutase, OspPGM, and the plastidic large subunit of ADP-glucose (ADP-Glc) pyrophosphorylase, OsAGPL4 Both genes were up-regulated in maturing pollen, a stage when starch begins to accumulate. Progeny analysis of self-pollinated heterozygous lines carrying the OspPGM mutant alleles, osppgm-1 and osppgm-2, or the OsAGPL4 mutant allele, osagpl4-1, as well as reciprocal crosses between the wild type (WT) and heterozygotes revealed that loss of OspPGM or OsAGPL4 caused male sterility, with the former condition rescued by the introduction of the WT OspPGM gene. While iodine staining and transmission electron microscopy analyses of pollen grains from homozygous osppgm-1 lines produced by anther culture confirmed the starch null phenotype, pollen from homozygous osagpl4 mutant lines, osagpl4-2 and osagpl4-3, generated by the CRISPR/Cas system, accumulated small amounts of starch which were sufficient to produce viable seed. Such osagpl4 mutant pollen, however, was unable to compete against WT pollen successfully, validating the important role of this reserve in fertilization. Our results demonstrate that starch is mainly polymerized from ADP-Glc synthesized from plastidic hexose phosphates in rice pollen and that starch is an essential requirement for successful fertilization in rice.
Assuntos
Glucose-1-Fosfato Adenililtransferase/metabolismo , Oryza/fisiologia , Fosfoglucomutase/metabolismo , Pólen/metabolismo , Amido/biossíntese , Fertilidade/fisiologia , Glucose-1-Fosfato Adenililtransferase/fisiologia , Microscopia , Mutação , Oryza/enzimologia , Oryza/metabolismo , Fosfoglucomutase/fisiologia , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
ADP-glucose pyrophosphorylase (AGPase) controls a rate-limiting step in the starch biosynthetic pathway in higher plants. Here we isolated a shrunken rice mutant w24. Map-based cloning identified OsAGPL2, a large subunit of the cytosolic AGPase in rice endosperm, as the gene responsible for the w24 mutation. In addition to severe inhibition of starch synthesis and significant accumulation of sugar, the w24 endosperm showed obvious defects in compound granule formation and storage protein synthesis. The defect in OsAGPL2 enhanced the expression levels of the AGPase family. Meanwhile, the elevated activities of starch phosphorylase 1 and sucrose synthase in the w24 endosperm might possibly partly account for the residual starch content in the mutant seeds. Moreover, the expression of OsAGPL2 and its counterpart, OsAGPS2b, was highly coordinated in rice endosperm. Yeast two-hybrid and BiFC assays verified direct interactions between OsAGPL2 and OsAGPS2b as well as OsAGPL1 and OsAGPS1, supporting the model for spatiotemporal complex formation of AGPase isoforms in rice endosperm. Besides, our data provided no evidence for the self-binding of OsAGPS2b, implying that OsAGPS2b might not interact to form higher molecular mass aggregates in the absence of OsAGPL2. Therefore, the molecular mechanism of rice AGPase assembly might differ from that of Arabidopsis.
Assuntos
Glucose-1-Fosfato Adenililtransferase/fisiologia , Oryza/metabolismo , Proteínas de Plantas/fisiologia , Mapeamento Cromossômico , Clonagem Molecular , Endosperma/metabolismo , Glucose-1-Fosfato Adenililtransferase/genética , Glucose-1-Fosfato Adenililtransferase/metabolismo , Oryza/enzimologia , Oryza/genética , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Amido/metabolismoRESUMO
KEY MESSAGE: AGPase, a key enzyme of starch biosynthetic pathway, has a significant role in crop productivity. Thermotolerant variants of AGPase in cereals may be used for developing cultivars, which may enhance productivity under heat stress. Improvement of crop productivity has always been the major goal of plant breeders to meet the global demand for food. However, crop productivity itself is influenced in a large measure by a number of abiotic stresses including heat, which causes major losses in crop productivity. In cereals, crop productivity in terms of grain yield mainly depends upon the seed starch content so that starch biosynthesis and the enzymes involved in this process have been a major area of investigation for plant physiologists and plant breeders alike. Considerable work has been done on AGPase and its role in crop productivity, particularly under heat stress, because this enzyme is one of the major enzymes, which catalyses the rate-limiting first committed key enzymatic step of starch biosynthesis. Keeping the above in view, this review focuses on the basic features of AGPase including its structure, regulatory mechanisms involving allosteric regulators, its sub-cellular localization and its genetics. Major emphasis, however, has been laid on the genetics of AGPases and its manipulation for developing high yielding cultivars that will have comparable productivity under heat stress. Some important thermotolerant variants of AGPase, which mainly involve specific amino acid substitutions, have been highlighted, and the prospects of using these thermotolerant variants of AGPase in developing cultivars for heat prone areas have been discussed. The review also includes a brief account on transgenics for AGPase, which have been developed for basic studies and crop improvement.
Assuntos
Grão Comestível/fisiologia , Glucose-1-Fosfato Adenililtransferase/fisiologia , Temperatura Alta , Proteínas de Plantas/fisiologia , Citosol/enzimologia , Grão Comestível/enzimologia , Glucose-1-Fosfato Adenililtransferase/genética , Resposta ao Choque Térmico , Melhoramento Vegetal , Proteínas de Plantas/genética , Plantas Geneticamente Modificadas/enzimologia , Estrutura Terciária de ProteínaRESUMO
Molecular farming refers to the process of creating bioengineered plants with the capability of producing potentially valuable products, such as drugs, vaccines, and chemicals. We have investigated the potential of the sweet potato ADP-glucose pyrophosphorylase gene (ibAGP1) promoter and its transit peptide (TP) as an expression system for the mass production of foreign proteins in potato. The ibAGP1 promoter and its TP sequence were transformed into potato along with beta-glucuronidase (GUS) as a reporter gene, and GUS activity was subsequently analyzed in the transgenic potato plants. In tuber tissues, GUS activity in transgenic plants carrying only the ibAGP1 promoter (ibAGP1::GUS) increased up to 15.6-fold compared with that of transgenic plants carrying only the CaMV35S promoter (CaMV35S::GUS). GUS activity in transgenic plants was further enhanced by the addition of the sweetpotato TP to the recombinant vector (ibAGP1::TP::GUS), with tuber tissues showing a 26-fold increase in activity compared with that in the CaMV35S::GUS-transgenic lines. In leaf tissues, the levels of GUS activity found in ibAGP1::GUS-transgenic lines were similar to those in CaMV35S::GUS-lines, but they were significantly enhanced in ibAGP1::TP::GUS-lines. GUS activity gradually increased with increasing tuber diameter in ibAGP1::GUS-transgenic plants, reaching a maximum level when the tuber was 35 mm in diameter. In contrast, extremely elevated levels of GUS activity - up to about 10-fold higher than that found in CaMV35S::GUS-lines - were found in ibAGP1::TP::GUS-transgenic lines at a much earlier stage of tuber development (diameter 4 mm), and these higher levels were maintained throughout the entire tuber developmental stage. These results suggest that the sweetpotato ibAGP1 promoter and its TP are a potentially strong foreign gene expression system that can be used for molecular farming in potato plants.
Assuntos
Regulação da Expressão Gênica de Plantas/genética , Glucose-1-Fosfato Adenililtransferase/genética , Glucuronidase/genética , Ipomoea batatas/genética , Proteínas de Plantas/genética , Tubérculos/enzimologia , Regiões Promotoras Genéticas/genética , Solanum tuberosum/enzimologia , Agrobacterium tumefaciens/genética , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Regulação da Expressão Gênica no Desenvolvimento/genética , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Genes Reporter , Vetores Genéticos/genética , Glucose-1-Fosfato Adenililtransferase/fisiologia , Glucuronidase/biossíntese , Ipomoea batatas/enzimologia , Folhas de Planta/enzimologia , Proteínas de Plantas/fisiologia , Tubérculos/crescimento & desenvolvimento , Plantas Geneticamente Modificadas/enzimologia , Plantas Geneticamente Modificadas/genética , Regiões Promotoras Genéticas/efeitos dos fármacos , RNA Mensageiro/biossíntese , RNA de Plantas/biossíntese , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/genética , Solanum tuberosum/crescimento & desenvolvimento , Especificidade da Espécie , Sacarose/farmacologia , Transformação GenéticaRESUMO
The higher plant ADP-glucose pyrophosphorylase is a heterotetramer consisting of two subunit types, which have evolved at different rates from a common ancestral gene. The potato tuber small subunit (SS) displays both catalytic and regulatory properties, whereas the exact role of the large subunit (LS), which contains substrate and effector binding sites, remains unresolved. We identified a mutation, S302N, which increased the solubility of the recombinant potato tuber LS and, in turn, enabling it to form a homotetrameric structure. The LS302N homotetramer possesses very little enzyme activity at a level 100-fold less than that seen for the unactivated SS homotetramer. Unlike the SS enzyme, however, the LS302N homotetramer enzyme is neither activated by the effector 3-phosphoglycerate nor inhibited by P(i). When combined with the catalytically silenced SS, S D143N, however, the LS302N-containing enzyme shows significantly enhanced catalytic activity and restored 3-PGA activation. This unmasking of catalytic and regulatory potential of the LS is conspicuously evident when the activities of the resurrected L(K41R.T51K.S302N) homotetramer are compared with its heterotetrameric form assembled with S D143N. Overall, these results indicate that the LS possesses catalytic and regulatory properties only when assembled with SS and that the net properties of the heterotetrameric enzyme is a product of subunit synergy.
Assuntos
Glucose-1-Fosfato Adenililtransferase/genética , Glucose-1-Fosfato Adenililtransferase/fisiologia , Mutação , Solanum tuberosum/enzimologia , Sítios de Ligação , Catálise , Dimerização , Relação Dose-Resposta a Droga , Ácidos Glicéricos/química , Glicogênio/química , Cinética , Modelos Moleculares , Mutagênese Sítio-Dirigida , Estrutura Terciária de Proteína , Proteínas Recombinantes/química , Relação Estrutura-AtividadeRESUMO
'Nudix' hydrolases are widely distributed nucleotide pyrophosphatases that possess a conserved GX5EX7REUXEEXGU motif where U is usually isoleucine, leucine or valine. Among them, Escherichia coli ADP-sugar pyrophosphatase (ASPP) has been shown to catalyze the hydrolytic breakdown of ADP-glucose linked to bacterial glycogen biosynthesis. Comparisons of the 31 different Nudix-encoding sequences of the Arabidopsis genome with those coding for known bacterial and mammalian ASPPs identified one sequence possessing important divergences in the Nudix motif that, once expressed in E. coli, produced a protein with ASPP activity. This protein, designated as AtASPP, shares strong homology with hypothetical rice and potato proteins, indicating that ASPPs are widely distributed in both mono- and dicotyledonous plants. As a first step to test the possible involvement of plant ASPPs in regulating the intracellular levels of ADP-glucose linked to starch biosynthesis, we produced and characterized AtASPP-overexpressing Arabidopsis plants. Source leaves from these plants exhibited a large reduction in the levels of both ADP-glucose and starch, indicating that plant ASPPs catalyze the hydrolytic breakdown of a sizable pool of ADP-glucose linked to starch biosynthesis. No pleiotropic changes in maximum catalytic activities of enzymes closely linked to starch metabolism could be detected in AtASPP-overexpressing leaves. The overall information provides the first evidence for the existence of plant Nudix hydrolases that have access to an intracellular pool of ADP-glucose linked to starch biosynthesis.
Assuntos
Adenosina Difosfato Glucose/metabolismo , Arabidopsis/genética , Arabidopsis/metabolismo , Pirofosfatases/genética , Amido/metabolismo , Motivos de Aminoácidos/genética , Sequência de Aminoácidos , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/fisiologia , Clonagem Molecular , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/fisiologia , Perfilação da Expressão Gênica , Regulação Enzimológica da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Genes de Plantas/genética , Glucose-1-Fosfato Adenililtransferase/genética , Glucose-1-Fosfato Adenililtransferase/fisiologia , Dados de Sequência Molecular , Folhas de Planta/metabolismo , Pirofosfatases/fisiologia , Proteínas Recombinantes/genética , Especificidade por Substrato , Sacarose/metabolismo , Nudix HidrolasesRESUMO
Starch, the most common form of stored carbon in plants, is both the major food source for mankind and important raw material for many industries. It is composed of two types of alpha-1,4-linked glucan polymer: essentially unbranched amylose and regularly branched amylopectin, and synthesized in photosynthetic and non-photosynthetic organs. Starch is synthesized via four committed enzyme steps: ADP-Glc pyrophosphorylase, which synthesizes sugar nucleotide precursors; starch synthase, which extends the alpha-1,4-linked glucan chains using ADP-Glc; starch-branching enzymes, which introduce alpha-1,6 branch points to form amylopectin; and starch debranching enzymes, which hydrolyze alpha-1,6 branches in glucans. In this paper, recent advances in biochemical characterizations and gene engineering concerning these enzymes were reviewed, and the achievements in gene engineering involved in manipulation of starch amount and quality were also cited.