RESUMO
Metabolites and lipids are the final products of enzymatic processes, distinguishing the different cellular functions and activities of single cells or whole tissues. Understanding these cellular functions within a well-established model system requires a systemic collection of molecular and physiological information. In the current report, the green alga Chlamydomonas reinhardtii was selected to establish a comprehensive workflow for the detailed multi-omics analysis of a synchronously growing cell culture system. After implementation and benchmarking of the synchronous cell culture, a two-phase extraction method was adopted for the analysis of proteins, lipids, metabolites and starch from a single sample aliquot of as little as 10-15 million Chlamydomonas cells. In a proof of concept study, primary metabolites and lipids were sampled throughout the diurnal cell cycle. The results of these time-resolved measurements showed that single compounds were not only coordinated with each other in different pathways, but that these complex metabolic signatures have the potential to be used as biomarkers of various cellular processes. Taken together, the developed workflow, including the synchronized growth of the photoautotrophic cell culture, in combination with comprehensive extraction methods and detailed metabolic phenotyping has the potential for use in in-depth analysis of complex cellular processes, providing essential information for the understanding of complex biological systems.
Assuntos
Ciclo Celular , Chlamydomonas reinhardtii/metabolismo , Metabolismo dos Lipídeos , Aminoácidos/metabolismo , Biomarcadores/metabolismo , Ciclo Celular/fisiologia , Células Cultivadas , Chlamydomonas reinhardtii/fisiologia , Ritmo Circadiano/fisiologia , Metabolismo dos Lipídeos/fisiologia , Lipídeos/isolamento & purificação , Lipídeos/fisiologia , Redes e Vias Metabólicas/fisiologia , Nitrogênio/metabolismo , Proteínas de Plantas/isolamento & purificação , Proteínas de Plantas/metabolismo , Amido/isolamento & purificação , Amido/metabolismo , TemperaturaRESUMO
We applied a top-down systems biology approach to understand how Chlamydomonas reinhardtii acclimates to long-term heat stress (HS) and recovers from it. For this, we shifted cells from 25 to 42°C for 24 h and back to 25°C for ≥8 h and monitored abundances of 1856 proteins/protein groups, 99 polar and 185 lipophilic metabolites, and cytological and photosynthesis parameters. Our data indicate that acclimation of Chlamydomonas to long-term HS consists of a temporally ordered, orchestrated implementation of response elements at various system levels. These comprise (1) cell cycle arrest; (2) catabolism of larger molecules to generate compounds with roles in stress protection; (3) accumulation of molecular chaperones to restore protein homeostasis together with compatible solutes; (4) redirection of photosynthetic energy and reducing power from the Calvin cycle to the de novo synthesis of saturated fatty acids to replace polyunsaturated ones in membrane lipids, which are deposited in lipid bodies; and (5) when sinks for photosynthetic energy and reducing power are depleted, resumption of Calvin cycle activity associated with increased photorespiration, accumulation of reactive oxygen species scavengers, and throttling of linear electron flow by antenna uncoupling. During recovery from HS, cells appear to focus on processes allowing rapid resumption of growth rather than restoring pre-HS conditions.
Assuntos
Aclimatação , Chlamydomonas reinhardtii/fisiologia , Metaboloma , Chaperonas Moleculares/metabolismo , Proteoma , Chlamydomonas reinhardtii/genética , Chlamydomonas reinhardtii/ultraestrutura , Temperatura Alta , Lipídeos/análise , Chaperonas Moleculares/genética , Fotossíntese , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismoRESUMO
Maltose frequently occurs as intermediate of the central carbon metabolism of prokaryotic and eukaryotic cells. Various mutants possess elevated maltose levels. Maltose exists as two anomers, (α- and ß-form) which are rapidly interconverted without requiring enzyme-mediated catalysis. As maltose is often abundant together with other oligoglucans, selective quantification is essential. In this communication, we present a photometric maltose assay using 4-alpha-glucanotransferase (AtDPE2) from Arabidopsis thaliana. Under in vitro conditions, AtDPE2 utilizes maltose as glucosyl donor and glycogen as acceptor releasing the other hexosyl unit as free glucose which is photometrically quantified following enzymatic phosphorylation and oxidation. Under the conditions used, DPE2 does not noticeably react with other di- or oligosaccharides. Selectivity compares favorably with that of maltase frequently used in maltose assays. Reducing end interconversion of the two maltose anomers is in rapid equilibrium and, therefore, the novel assay measures total maltose contents. Furthermore, an AtDPE2-based continuous photometric assay is presented which allows to quantify ß-amylase activity and was found to be superior to a conventional test. Finally, the AtDPE2-based maltose assay was used to quantify leaf maltose contents of both Arabidopsis wild type and AtDPE2-deficient plants throughout the light-dark cycle. These data are presented together with assimilatory starch levels.
Assuntos
Arabidopsis/metabolismo , Sistema da Enzima Desramificadora do Glicogênio/metabolismo , Maltose/metabolismo , Fotometria/métodos , Plantas Geneticamente Modificadas/metabolismo , Amido/metabolismo , Sacarose/metabolismo , Citosol/metabolismo , Ensaios Enzimáticos/métodos , Folhas de Planta/metabolismo , Especificidade por SubstratoRESUMO
Lafora disease (LD, OMIM #254780) is a rare, recessively inherited neurodegenerative disease with adolescent onset, resulting in progressive myoclonus epilepsy which is fatal usually within ten years of symptom onset. The disease is caused by loss-of-function mutations in either of the two genes EPM2A (laforin) or EPM2B (malin). It characteristically involves the accumulation of insoluble glycogen-derived particles, named Lafora bodies (LBs), which are considered neurotoxic and causative of the disease. The pathogenesis of LD is therefore centred on the question of how insoluble LBs emerge from soluble glycogen. Recent data clearly show that an abnormal glycogen chain length distribution, but neither hyperphosphorylation nor impairment of general autophagy, strictly correlates with glycogen accumulation and the presence of LBs. This review summarizes results obtained with patients, mouse models, and cell lines and consolidates apparent paradoxes in the LD literature. Based on the growing body of evidence, it proposes that LD is predominantly caused by an impairment in chain-length regulation affecting only a small proportion of the cellular glycogen. A better grasp of LD pathogenesis will further develop our understanding of glycogen metabolism and structure. It will also facilitate the development of clinical interventions that appropriately target the underlying cause of LD.
Assuntos
Proteínas de Transporte/genética , Glucanos/metabolismo , Glicogênio/metabolismo , Doença de Lafora/etiologia , Proteínas Tirosina Fosfatases não Receptoras/genética , Animais , Proteínas de Transporte/metabolismo , Humanos , Doença de Lafora/genética , Doença de Lafora/metabolismo , Proteínas Tirosina Fosfatases não Receptoras/metabolismo , Ubiquitina-Proteína LigasesRESUMO
We have identified a novel means to achieve substantially increased vegetative biomass and oilseed production in the model plant Arabidopsis thaliana. Endogenous isoforms of starch branching enzyme (SBE) were substituted by either one of the endosperm-expressed maize (Zea mays L.) branching isozymes, ZmSBEI or ZmSBEIIb. Transformants were compared with the starch-free background and with the wild-type plants. Each of the maize-derived SBEs restored starch biosynthesis but both morphology and structure of starch particles were altered. Altered starch metabolism in the transformants is associated with enhanced biomass formation and more-than-trebled oilseed production while maintaining seed oil quality. Enhanced oilseed production is primarily due to an increased number of siliques per plant whereas oil content and seed number per silique are essentially unchanged or even modestly decreased. Introduction of cereal starch branching isozymes into oilseed plants represents a potentially useful strategy to increase biomass and oilseed production in related crops and manipulate the structure and properties of leaf starch.
Assuntos
Arabidopsis/genética , Arabidopsis/metabolismo , Biomassa , Óleos de Plantas/metabolismo , Sementes/crescimento & desenvolvimento , Amido/metabolismo , Enzima Ramificadora de 1,4-alfa-Glucana/metabolismo , Cloroplastos/enzimologia , Endosperma/metabolismo , Regulação da Expressão Gênica de Plantas , Teste de Complementação Genética , Fenótipo , Folhas de Planta/metabolismo , Plantas Geneticamente Modificadas , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Sementes/metabolismo , Transformação Genética , Transgenes , Zea mays/metabolismoRESUMO
Transitory starch metabolism is a nonlinear and highly regulated process. It originated very early in the evolution of chloroplast-containing cells and is largely based on a mosaic of genes derived from either the eukaryotic host cell or the prokaryotic endosymbiont. Initially located in the cytoplasm, starch metabolism was rewired into plastids in Chloroplastida. Relocation was accompanied by gene duplications that occurred in most starch-related gene families and resulted in subfunctionalization of the respective gene products. Starch-related isozymes were then evolutionary conserved by constraints such as internal starch structure, posttranslational protein import into plastids and interactions with other starch-related proteins. 25 starch-related genes in 26 accessions of Arabidopsis thaliana were sequenced to assess intraspecific diversity, phylogenetic relationships, and modes of selection. Furthermore, sequences derived from additional 80 accessions that are publicly available were analyzed. Diversity varies significantly among the starch-related genes. Starch synthases and phosphorylases exhibit highest nucleotide diversities, while pyrophosphatases and debranching enzymes are most conserved. The gene trees are most compatible with a scenario of extensive recombination, perhaps in a Pleistocene refugium. Most genes are under purifying selection, but disruptive selection was inferred for a few genes/substitutiones. To study transcript levels, leaves were harvested throughout the light period. By quantifying the transcript levels and by analyzing the sequence of the respective accessions, we were able to estimate whether transcript levels are mainly determined by genetic (i.e., accession dependent) or physiological (i.e., time dependent) parameters. We also identified polymorphic sites that putatively affect pattern or the level of transcripts.
Assuntos
Proteínas de Arabidopsis/genética , Arabidopsis/genética , Evolução Molecular , Regulação da Expressão Gênica de Plantas , Variação Genética , Amido/metabolismo , Arabidopsis/classificação , Arabidopsis/metabolismo , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/metabolismoRESUMO
In leaves of two starch-related single-knockout lines lacking either the cytosolic transglucosidase (also designated as disproportionating enzyme 2, DPE2) or the maltose transporter (MEX1), the activity of the plastidial phosphorylase isozyme (PHS1) is increased. In both mutants, metabolism of starch-derived maltose is impaired but inhibition is effective at different subcellular sites. Two constitutive double knockout mutants were generated (designated as dpe2-1×phs1a and mex1×phs1b) both lacking functional PHS1. They reveal that in normally grown plants, the plastidial phosphorylase isozyme participates in transitory starch degradation and that the central carbon metabolism is closely integrated into the entire cell biology. All plants were grown either under continuous illumination or in a light-dark regime. Both double mutants were compromised in growth and, compared with the single knockout plants, possess less average leaf starch when grown in a light-dark regime. Starch and chlorophyll contents decline with leaf age. As revealed by transmission electron microscopy, mesophyll cells degrade chloroplasts, but degradation is not observed in plants grown under continuous illumination. The two double mutants possess similar but not identical phenotypes. When grown in a light-dark regime, mesophyll chloroplasts of dpe2-1×phs1a contain a single starch granule but under continuous illumination more granules per chloroplast are formed. The other double mutant synthesizes more granules under either growth condition. In continuous light, growth of both double mutants is similar to that of the parental single knockout lines. Metabolite profiles and oligoglucan patterns differ largely in the two double mutants.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/crescimento & desenvolvimento , Técnicas de Inativação de Genes , Sistema da Enzima Desramificadora do Glicogênio/metabolismo , Mutação/genética , Plastídeos/enzimologia , Proteínas Tirosina Fosfatases/metabolismo , Amido/metabolismo , Arabidopsis/enzimologia , Arabidopsis/genética , Arabidopsis/ultraestrutura , Biomassa , Metabolismo dos Carboidratos , Carbono/metabolismo , Clorofila/metabolismo , Cromatografia de Afinidade , Cruzamentos Genéticos , Isoenzimas/metabolismo , Maltose/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Células do Mesofilo/metabolismo , Células do Mesofilo/ultraestrutura , Metabolômica , Fenótipo , Fotoperíodo , Plastídeos/ultraestrutura , Sacarose/metabolismoRESUMO
Controlled conversion of leaf starch to sucrose at night is essential for the normal growth of Arabidopsis. The conversion involves the cytosolic metabolism of maltose to hexose phosphates via an unusual, multidomain protein with 4-glucanotransferase activity, DPE2, believed to transfer glucosyl moieties to a complex heteroglycan prior to their conversion to hexose phosphate via a cytosolic phosphorylase. The significance of this complex pathway is unclear; conversion of maltose to hexose phosphate in bacteria proceeds via a more typical 4-glucanotransferase that does not require a heteroglycan acceptor. It has recently been suggested that DPE2 generates a heterogeneous series of terminal glucan chains on the heteroglycan that acts as a "glucosyl buffer" to ensure a constant rate of sucrose synthesis in the leaf at night. Alternatively, DPE2 and/or the heteroglycan may have specific properties important for their function in the plant. To distinguish between these ideas, we compared the properties of DPE2 with those of the Escherichia coli glucanotransferase MalQ. We found that MalQ cannot use the plant heteroglycan as an acceptor for glucosyl transfer. However, experimental and modeling approaches suggested that it can potentially generate a glucosyl buffer between maltose and hexose phosphate because, unlike DPE2, it can generate polydisperse malto-oligosaccharides from maltose. Consistent with this suggestion, MalQ is capable of restoring an essentially wild-type phenotype when expressed in mutant Arabidopsis plants lacking DPE2. In light of these findings, we discuss the possible evolutionary origins of the complex DPE2-heteroglycan pathway.
Assuntos
Escuridão , Escherichia coli/enzimologia , Glucosiltransferases/metabolismo , Maltose/metabolismo , Folhas de Planta/metabolismo , Amido/metabolismo , Sacarose/metabolismo , Arabidopsis/genética , Arabidopsis/crescimento & desenvolvimento , Soluções Tampão , Citosol/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Evolução Molecular , Glucosiltransferases/química , Metabolômica , Mutação/genética , Oligossacarídeos/metabolismo , Fenótipo , Plantas Geneticamente Modificadas , Estrutura Terciária de Proteína , Proteínas Recombinantes/isolamento & purificação , Relação Estrutura-Atividade , Especificidade por SubstratoRESUMO
BACKGROUND: The versatile Vacuole Membrane Protein 1 (VMP1) has been previously investigated in six species. It has been shown to be essential in macroautophagy, where it takes part in autophagy initiation. In addition, VMP1 has been implicated in organellar biogenesis; endo-, exo- and phagocytosis, and protein secretion; apoptosis; and cell adhesion. These roles underly its proven involvement in pancreatitis, diabetes and cancer in humans. RESULTS: In this study we analyzed a VMP1 homologue from the green alga Chlamydomonas reinhardtii. CrVMP1 knockdown lines showed severe phenotypes, mainly affecting cell division as well as the morphology of cells and organelles. We also provide several pieces of evidence for its involvement in macroautophagy. CONCLUSION: Our study adds a novel role to VMP1's repertoire, namely the regulation of cytokinesis. Though the directness of the observed effects and the mechanisms underlying them remain to be defined, the protein's involvement in macroautophagy in Chlamydomonas, as found by us, suggests that CrVMP1 shares molecular characteristics with its animal and protist counterparts.
Assuntos
Forma Celular , Chlamydomonas/citologia , Chlamydomonas/metabolismo , Citocinese , Proteínas de Plantas/metabolismo , Sequência de Aminoácidos , Autofagia/genética , Ciclo Celular/genética , Chlamydomonas/genética , Chlamydomonas/ultraestrutura , Cromatografia Líquida de Alta Pressão , Regulação da Expressão Gênica de Plantas , Técnicas de Silenciamento de Genes , Genes de Plantas , Espectrometria de Massas , Metabolômica , Dados de Sequência Molecular , Mutação/genética , Fenótipo , Proteínas de Plantas/química , Análise de Componente Principal , Proteólise , Alinhamento de SequênciaRESUMO
Many plants accumulate substantial starch reserves in their leaves during the day and remobilize them at night to provide carbon and energy for maintenance and growth. In this paper, we explore the role of a sugar-signaling metabolite, trehalose-6-phosphate (Tre6P), in regulating the accumulation and turnover of transitory starch in Arabidopsis (Arabidopsis thaliana) leaves. Ethanol-induced overexpression of trehalose-phosphate synthase during the day increased Tre6P levels up to 11-fold. There was a transient increase in the rate of starch accumulation in the middle of the day, but this was not linked to reductive activation of ADP-glucose pyrophosphorylase. A 2- to 3-fold increase in Tre6P during the night led to significant inhibition of starch degradation. Maltose and maltotriose did not accumulate, suggesting that Tre6P affects an early step in the pathway of starch degradation in the chloroplasts. Starch granules isolated from induced plants had a higher orthophosphate content than granules from noninduced control plants, consistent either with disruption of the phosphorylation-dephosphorylation cycle that is essential for efficient starch breakdown or with inhibition of starch hydrolysis by ß-amylase. Nonaqueous fractionation of leaves showed that Tre6P is predominantly located in the cytosol, with estimated in vivo Tre6P concentrations of 4 to 7 µm in the cytosol, 0.2 to 0.5 µm in the chloroplasts, and 0.05 µm in the vacuole. It is proposed that Tre6P is a component in a signaling pathway that mediates the feedback regulation of starch breakdown by sucrose, potentially linking starch turnover to demand for sucrose by growing sink organs at night.
Assuntos
Arabidopsis/metabolismo , Retroalimentação Fisiológica/fisiologia , Folhas de Planta/metabolismo , Amido/metabolismo , Fosfatos Açúcares/metabolismo , Trealose/análogos & derivados , Arabidopsis/efeitos dos fármacos , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Cloroplastos/metabolismo , Grânulos Citoplasmáticos/metabolismo , Grânulos Citoplasmáticos/ultraestrutura , Citosol/metabolismo , Etanol/farmacologia , Glucosiltransferases/metabolismo , Hidrólise/efeitos dos fármacos , Immunoblotting , Maltose/metabolismo , Microscopia Eletrônica de Varredura , Fosfatos/metabolismo , Folhas de Planta/efeitos dos fármacos , Plantas Geneticamente Modificadas , Amido/ultraestrutura , Fatores de Tempo , Trealose/metabolismo , Trissacarídeos/metabolismoRESUMO
The crucial role of carbohydrate in plant growth and morphogenesis is widely recognized. In this study, we describe the characterization of nana, a dwarf Arabidopsis (Arabidopsis thaliana) mutant impaired in carbohydrate metabolism. We show that the nana dwarf phenotype was accompanied by altered leaf morphology and a delayed flowering time. Our genetic and molecular data indicate that the mutation in nana is due to a transfer DNA insertion in the promoter region of a gene encoding a chloroplast-located aspartyl protease that alters its pattern of expression. Overexpression of the gene (oxNANA) phenocopies the mutation. Both nana and oxNANA display alterations in carbohydrate content, and the extent of these changes varies depending on growth light intensity. In particular, in low light, soluble sugar levels are lower and do not show the daily fluctuations observed in wild-type plants. Moreover, nana and oxNANA are defective in the expression of some genes implicated in sugar metabolism and photosynthetic light harvesting. Interestingly, some chloroplast-encoded genes as well as genes whose products seem to be involved in retrograde signaling appear to be down-regulated. These findings suggest that the NANA aspartic protease has an important regulatory function in chloroplasts that not only influences photosynthetic carbon metabolism but also plastid and nuclear gene expression.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/enzimologia , Arabidopsis/crescimento & desenvolvimento , Ácido Aspártico Proteases/metabolismo , Metabolismo dos Carboidratos , Cloroplastos/enzimologia , Arabidopsis/efeitos dos fármacos , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Ácido Aspártico Proteases/genética , Metabolismo dos Carboidratos/efeitos dos fármacos , Cloroplastos/efeitos dos fármacos , Ritmo Circadiano/efeitos dos fármacos , Ritmo Circadiano/genética , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Mutação/genética , Fenótipo , Fotossíntese/efeitos dos fármacos , Fotossíntese/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Amido/metabolismo , Sacarose/farmacologiaRESUMO
Plants metabolize transitory starch by precisely coordinated plastidial and cytosolic processes. The latter appear to include the action of water-soluble heteroglycans (SHGin ) whose monosaccharide pattern is similar to that of apoplastic glycans (SHGex ) but, unlike SHGex , SHGin strongly interacts with glucosyl transferases. In this study, we analyzed starch metabolism using mesophyll protoplasts from wild-type plants and two knock-out mutants [deficient in the cytosolic transglucosidase, disproportionating isoenzyme 2 (DPE2) or the plastidial phosphoglucomutase (PGM1)] from Arabidopsis thaliana. Protoplasts prelabeled by photosynthetic (14) CO2 fixation were transferred to an unlabeled medium and were darkened or illuminated. Carbon transitions from the Calvin cycle or from starch to both SHGin and SHGex were analyzed. In illuminated protoplasts, starch turn-over was undetectable but darkened protoplasts continuously degraded starch. During illumination, neither the total (14) C content nor the labeling patterns of the sugar residues of SHGin were significantly altered but both the total amount and the labeling of the constituents of SHGex increased with time. In darkened protoplasts, the (14) C-content of most of the sugar residues of SHGin transiently and strongly increased and then declined. This effect was not observed in any SHGex constituent. In darkened DPE2-deficient protoplasts, none of the SHGin constituents exhibited an essential transient increase in labeling. In contrast, some residues of SHGin from the PGM1 mutant exhibited a transient increase in label but this effect significantly differed from that of the wild type. Two conclusions are reached: first, SHGin and SHGex exert different metabolic functions and second, SHGin is directly involved in starch degradation.
Assuntos
Arabidopsis/metabolismo , Carbono/metabolismo , Fotossíntese/fisiologia , Polissacarídeos/metabolismo , Amido/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Metabolismo dos Carboidratos , Radioisótopos de Carbono , Escuridão , Técnicas de Inativação de Genes , Marcação por Isótopo , Luz , Células do Mesofilo/metabolismo , Mutação , Fosfoglucomutase/genética , Fosfoglucomutase/metabolismo , Plantas Geneticamente Modificadas , Polissacarídeos/química , SolubilidadeRESUMO
Phosphorylation and dephosphorylation of starch and glycogen are important for their physicochemical properties and also their physiological functions. It is therefore desirable to reliably determine the phosphorylation sites. Heteronuclear multidimensional NMR-spectroscopy is in principle a straightforward analytical approach even for complex carbohydrate molecules. With heterogeneous samples from natural sources, however, the task becomes more difficult because a full assignment of the resonances of the carbohydrates is impossible to obtain. Here, we show that the combination of heteronuclear (1) H,(13) C and (1) H,(13) C,(31) P techniques and information derived from spectra of a set of reference compounds can lead to an unambiguous determination of the phosphorylation sites even in heterogeneous samples.
Assuntos
Glucanos/síntese química , Isótopos de Carbono , Glucanos/química , Espectroscopia de Ressonância Magnética/normas , Isótopos de Fósforo , Fosforilação , Prótons , Padrões de ReferênciaRESUMO
In a synchronized photoautotrophic culture of Chlamydomonas reinhardtii, cell size, cell number, and the averaged starch content were determined throughout the light-dark cycle. For single-cell analyses, the relative cellular starch was quantified by measuring the second harmonic generation (SHG). In destained cells, amylopectin essentially represents the only biophotonic structure. As revealed by various validation procedures, SHG signal intensities are a reliable relative measure of the cellular starch content. During photosynthesis-driven starch biosynthesis, synchronized Chlamydomonas cells possess an unexpected cell-to-cell diversity both in size and starch content, but the starch-related heterogeneity largely exceeds that of size. The cellular volume, starch content, and amount of starch/cell volume obey lognormal distributions. Starch degradation was initiated by inhibiting the photosynthetic electron transport in illuminated cells or by darkening. Under both conditions, the averaged rate of starch degradation is almost constant, but it is higher in illuminated than in darkened cells. At the single-cell level, rates of starch degradation largely differ but are unrelated to the initial cellular starch content. A rate equation describing the cellular starch degradation is presented. SHG-based three-dimensional reconstructions of Chlamydomonas cells containing starch granules are shown.
Assuntos
Técnicas de Cultura de Células/métodos , Chlamydomonas reinhardtii/citologia , Chlamydomonas reinhardtii/metabolismo , Análise de Célula Única/métodos , Amilopectina/metabolismo , Contagem de Células , Tamanho Celular , Chlamydomonas reinhardtii/enzimologia , Cinética , Microscopia Confocal , Reprodutibilidade dos Testes , Fatores de TempoRESUMO
The present study established the way in which plastidial α-glucan phosphorylase (Pho1) synthesizes maltodextrin (MD) which can be the primer for starch biosynthesis in rice endosperm. The synthesis of MD by Pho1 was markedly accelerated by branching enzyme (BE) isozymes, although the greatest effect was exhibited by the presence of branching isozyme I (BEI) rather than by isozyme IIa (BEIIa) or isozyme IIb (BEIIb). The enhancement of the activity of Pho1 by BE was not merely due to the supply of a non-reducing ends. At the same time, Pho1 greatly enhanced the BE activity, possibly by generating a branched carbohydrate substrate which is used by BE with a higher affinity. The addition of isoamylase to the reaction mixture did not prevent the concerted action of Pho1 and BEI. Furthermore, in the product, the branched structure was, at least to some extent, maintained. Based on these results we propose that the interaction between Pho1 and BE is not merely due to chain-elongating and chain-branching reactions, but occurs in a physically and catalytically synergistic manner by each activating the mutual capacity of the other, presumably forming a physical association of Pho1, BEI and branched MDs. This close interaction might play a crucial role in the synthesis of branched MDs and the branched MDs can act as a primer for the biosynthesis of amylopectin molecules.
Assuntos
Enzima Ramificadora de 1,4-alfa-Glucana/metabolismo , Oryza/enzimologia , Plastídeos/enzimologia , Polissacarídeos/biossíntese , Amido Fosforilase/metabolismo , Glucanos/biossíntese , Isoamilase/metabolismo , Ligação Proteica , Pseudomonas/enzimologiaRESUMO
Almost all glucosyl transfer reactions rely on glucose-1-phosphate (Glc-1-P) that either immediately acts as glucosyl donor or as substrate for the synthesis of the more widely used Glc dinucleotides, ADPglucose or UDPglucose. In this communication, we have analyzed two Glc-1-P-related processes: the carbon flux from externally supplied Glc-1-P to starch by either mesophyll protoplasts or intact chloroplasts from Arabidopsis (Arabidopsis thaliana). When intact protoplasts or chloroplasts are incubated with [U-(14)C]Glc-1-P, starch is rapidly labeled. Incorporation into starch is unaffected by the addition of unlabeled Glc-6-P or Glc, indicating a selective flux from Glc-1-P to starch. However, illuminated protoplasts incorporate less (14)C into starch when unlabeled bicarbonate is supplied in addition to the (14)C-labeled Glc-1-P. Mesophyll protoplasts incubated with [U-(14)C]Glc-1-P incorporate (14)C into the plastidial pool of adenosine diphosphoglucose. Protoplasts prepared from leaves of mutants of Arabidopsis that lack either the plastidial phosphorylase or the phosphoglucomutase isozyme incorporate (14)C derived from external Glc-1-P into starch, but incorporation into starch is insignificant when protoplasts from a mutant possessing a highly reduced ADPglucose pyrophosphorylase activity are studied. Thus, the path of assimilatory starch biosynthesis initiated by extraplastidial Glc-1-P leads to the plastidial pool of adenosine diphosphoglucose, and at this intermediate it is fused with the Calvin cycle-driven route. Mutants lacking the plastidial phosphoglucomutase contain a small yet significant amount of transitory starch.
Assuntos
Arabidopsis/metabolismo , Cloroplastos/metabolismo , Glucofosfatos/metabolismo , Folhas de Planta/metabolismo , Protoplastos/metabolismo , Arabidopsis/enzimologia , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Transporte Biológico , Isótopos de Carbono/análise , Cloroplastos/enzimologia , Glucose-1-Fosfato Adenililtransferase/metabolismo , Glucosiltransferases/genética , Glucosiltransferases/metabolismo , Mutação , Amido/biossínteseRESUMO
Parenchyma cells from tubers of Solanum tuberosum L. convert several externally supplied sugars to starch but the rates vary largely. Conversion of glucose 1-phosphate to starch is exceptionally efficient. In this communication, tuber slices were incubated with either of four solutions containing equimolar [U-¹4C]glucose 1-phosphate, [U-¹4C]sucrose, [U-¹4C]glucose 1-phosphate plus unlabelled equimolar sucrose or [U-¹4C]sucrose plus unlabelled equimolar glucose 1-phosphate. C¹4-incorporation into starch was monitored. In slices from freshly harvested tubers each unlabelled compound strongly enhanced ¹4C incorporation into starch indicating closely interacting paths of starch biosynthesis. However, enhancement disappeared when the tubers were stored. The two paths (and, consequently, the mutual enhancement effect) differ in temperature dependence. At lower temperatures, the glucose 1-phosphate-dependent path is functional, reaching maximal activity at approximately 20 °C but the flux of the sucrose-dependent route strongly increases above 20 °C. Results are confirmed by in vitro experiments using [U-¹4C]glucose 1-phosphate or adenosine-[U-¹4C]glucose and by quantitative zymograms of starch synthase or phosphorylase activity. In mutants almost completely lacking the plastidial phosphorylase isozyme(s), the glucose 1-phosphate-dependent path is largely impeded. Irrespective of the size of the granules, glucose 1-phosphate-dependent incorporation per granule surface area is essentially equal. Furthermore, within the granules no preference of distinct glucosyl acceptor sites was detectable. Thus, the path is integrated into the entire granule biosynthesis. In vitro C¹4C-incorporation into starch granules mediated by the recombinant plastidial phosphorylase isozyme clearly differed from the in situ results. Taken together, the data clearly demonstrate that two closely but flexibly interacting general paths of starch biosynthesis are functional in potato tuber cells.
Assuntos
Ciclo do Carbono , Solanum tuberosum/citologia , Solanum tuberosum/metabolismo , Amido/metabolismo , Ciclo do Carbono/efeitos dos fármacos , Isótopos de Carbono , Misturas Complexas , Glucanos/metabolismo , Glucofosfatos/farmacologia , Isoenzimas/metabolismo , Tubérculos/citologia , Tubérculos/efeitos dos fármacos , Tubérculos/fisiologia , Tubérculos/ultraestrutura , Plantas Geneticamente Modificadas , Plastídeos/efeitos dos fármacos , Plastídeos/enzimologia , Polissacarídeos/metabolismo , Solanum tuberosum/genética , Solanum tuberosum/fisiologia , Solubilidade/efeitos dos fármacos , Amido/ultraestrutura , Amido Fosforilase/metabolismo , Sintase do Amido/metabolismo , Sacarose/farmacologia , TemperaturaRESUMO
The biochemical function of the Laforin-like dual-specific phosphatase AtSEX4 (EC 3.1.3.48) has been studied. Crystalline maltodextrins representing the A- or the B-type allomorph were prephosphorylated using recombinant glucan, water dikinase (StGWD) or the successive action of both plastidial dikinases (StGWD and AtPWD). AtSEX4 hydrolyzed carbon 6-phosphate esters from both the prephosphorylated A- and B-type allomorphs and the kinetic constants are similar. The phosphatase also acted on prelabeled carbon-3 esters from both crystalline maltodextrins. Similarly, native starch granules prelabeled in either the carbon-6 or carbon-3 position were also dephosphorylated by AtSEX4. The phosphatase did also hydrolyze phosphate esters of both prephosphorylated maltodextrins when the (phospho)glucans had been solubilized by heat treatment. Submillimolar concentrations of nonphosphorylated maltodextrins inhibited AtSEX4 provided they possessed a minimum of length and had been solubilized. As opposed to the soluble phosphomaltodextrins, the AtSEX4-mediated dephosphorylation of the insoluble substrates was incomplete and at least 50% of the phosphate esters were retained in the pelletable (phospho)glucans. The partial dephosphorylation of the insoluble glucans also strongly reduced the release of nonphosphorylated chains into solution. Presumably, this effect reflects fast structural changes that following dephosphorylation occur near the surface of the maltodextrin particles. A model is proposed defining distinct stages within the phosphorylation/dephosphorylation-dependent transition of alpha-glucans from the insoluble to the soluble state.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/enzimologia , Fosfatases de Especificidade Dupla/metabolismo , Glucanos/metabolismo , Amido/metabolismo , FosforilaçãoRESUMO
A putative phosphatase, LSF1 (for LIKE SEX4; previously PTPKIS2), is closely related in sequence and structure to STARCH-EXCESS4 (SEX4), an enzyme necessary for the removal of phosphate groups from starch polymers during starch degradation in Arabidopsis (Arabidopsis thaliana) leaves at night. We show that LSF1 is also required for starch degradation: lsf1 mutants, like sex4 mutants, have substantially more starch in their leaves than wild-type plants throughout the diurnal cycle. LSF1 is chloroplastic and is located on the surface of starch granules. lsf1 and sex4 mutants show similar, extensive changes relative to wild-type plants in the expression of sugar-sensitive genes. However, although LSF1 and SEX4 are probably both involved in the early stages of starch degradation, we show that LSF1 neither catalyzes the same reaction as SEX4 nor mediates a sequential step in the pathway. Evidence includes the contents and metabolism of phosphorylated glucans in the single mutants. The sex4 mutant accumulates soluble phospho-oligosaccharides undetectable in wild-type plants and is deficient in a starch granule-dephosphorylating activity present in wild-type plants. The lsf1 mutant displays neither of these phenotypes. The phenotype of the lsf1/sex4 double mutant also differs from that of both single mutants in several respects. We discuss the possible role of the LSF1 protein in starch degradation.