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1.
New Phytol ; 243(1): 162-179, 2024 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-38706429

RESUMEN

Some cyanobacteria can grow photoautotrophically or photomixotrophically by using simultaneously CO2 and glucose. The switch between these trophic modes and the role of glycogen, their main carbon storage macromolecule, was investigated. We analysed the effect of glucose addition on the physiology, metabolic and photosynthetic state of Synechocystis sp. PCC 6803 and mutants lacking phosphoglucomutase and ADP-glucose pyrophosphorylase, with limitations in glycogen synthesis. Glycogen acted as a metabolic buffer: glucose addition increased growth and glycogen reserves in the wild-type (WT), but arrested growth in the glycogen synthesis mutants. Already 30 min after glucose addition, metabolites from the Calvin-Benson-Bassham cycle and the oxidative pentose phosphate shunt increased threefold more in the glycogen synthesis mutants than the WT. These alterations substantially affected the photosynthetic performance of the glycogen synthesis mutants, as O2 evolution and CO2 uptake were both impaired. We conclude that glycogen synthesis is essential during transitions to photomixotrophy to avoid metabolic imbalance that induces inhibition of electron transfer from PSII and subsequently accumulation of reactive oxygen species, loss of PSII core proteins, and cell death. Our study lays foundations for optimising photomixotrophy-based biotechnologies through understanding the coordination of the crosstalk between photosynthetic electron transport and metabolism.


Asunto(s)
Glucógeno , Fotosíntesis , Complejo de Proteína del Fotosistema II , Synechocystis , Synechocystis/metabolismo , Synechocystis/efectos de los fármacos , Synechocystis/crecimiento & desarrollo , Synechocystis/genética , Glucógeno/metabolismo , Transporte de Electrón , Complejo de Proteína del Fotosistema II/metabolismo , Mutación/genética , Glucosa/metabolismo , Dióxido de Carbono/metabolismo , Oxígeno/metabolismo , Glucosa-1-Fosfato Adenililtransferasa/metabolismo , Glucosa-1-Fosfato Adenililtransferasa/genética , Fosfoglucomutasa/metabolismo , Fosfoglucomutasa/genética
2.
J Exp Bot ; 74(5): 1532-1550, 2023 03 13.
Artículo en Inglés | MEDLINE | ID: mdl-36454663

RESUMEN

Glycogen and starch are the main storage polysaccharides, acting as a source of carbon and energy when necessary. Interconversion of glucose-1-phosphate and glucose-6-phosphate by phosphoglucomutases connects the metabolism of these polysaccharides with central carbon metabolism. However, knowledge about how this connection affects the ability of cells to cope with environmental stresses is still scarce. The cyanobacterium Synechocystis sp. PCC 6803 has two enzymes with phosphoglucomutase activity, PGM (phosphoglucomutase) and PMM/PGM (phosphomannomutase/phosphoglucomutase). In this work, we generated a null mutant of PGM (∆PGM) that exhibits very reduced phosphoglucomutase activity (1% of wild type activity). Although this mutant accumulates moderate amounts of glycogen, its phenotype resembles that of glycogen-less mutants, including high light sensitivity and altered response to nitrogen deprivation. Using an on/off arsenite promoter, we demonstrate that PMM/PGM is essential for growth and responsible for the remaining phosphoglucomutase activity in the ∆PGM strain. Furthermore, overexpression of PMM/PGM in the ∆PGM strain is enough to revoke the phenotype of this mutant. These results emphasize the importance of an adequate flux between glycogen and central carbon metabolism to maintain cellular fitness and indicate that although PGM is the main phosphoglucomutase activity, the phosphoglucomutase activity of PMM/PGM can substitute it when expressed in sufficient amounts.


Asunto(s)
Cianobacterias , Fosfoglucomutasa , Fosfoglucomutasa/genética , Fosfoglucomutasa/metabolismo , Glucógeno/metabolismo , Carbono , Almidón , Cianobacterias/metabolismo
3.
J Exp Bot ; 71(6): 2005-2017, 2020 03 25.
Artículo en Inglés | MEDLINE | ID: mdl-31858138

RESUMEN

Cyanobacteria are widely distributed photosynthetic organisms. During the day they store carbon, mainly as glycogen, to provide the energy and carbon source they require for maintenance during the night. Here, we generate a mutant strain of the freshwater cyanobacterium Synechocystis sp. PCC 6803 lacking both glycogen synthases. This mutant has a lethal phenotype due to massive accumulation of ADP-glucose, the substrate of glycogen synthases. This accumulation leads to alterations in its photosynthetic capacity and a dramatic decrease in the adenylate energy charge of the cell to values as low as 0.1. Lack of ADP-glucose pyrophosphorylase, the enzyme responsible for ADP-glucose synthesis, or reintroduction of any of the glycogen synthases abolishes the lethal phenotype. Viability of the glycogen synthase mutant is also fully recovered in NaCl-supplemented medium, which redirects the surplus of ADP-glucose to synthesize the osmolite glucosylglycerol. This alternative metabolic sink also suppresses phenotypes associated with the defective response to nitrogen deprivation characteristic of glycogen-less mutants, restoring the capacity to degrade phycobiliproteins. Thus, our system is an excellent example of how inadequate management of the adenine nucleotide pools results in a lethal phenotype, and the influence of metabolic carbon flux in cell viability and fitness.


Asunto(s)
Adenosina Difosfato Glucosa , Synechocystis , Carbono , Ciclo del Carbono , Glucosa , Cloruro de Sodio , Synechocystis/genética
4.
Plant Physiol ; 171(3): 1879-92, 2016 07.
Artículo en Inglés | MEDLINE | ID: mdl-27208262

RESUMEN

At variance with the starch-accumulating plants and most of the glycogen-accumulating cyanobacteria, Cyanobacterium sp. CLg1 synthesizes both glycogen and starch. We now report the selection of a starchless mutant of this cyanobacterium that retains wild-type amounts of glycogen. Unlike other mutants of this type found in plants and cyanobacteria, this mutant proved to be selectively defective for one of the two types of glycogen/starch synthase: GlgA2. This enzyme is phylogenetically related to the previously reported SSIII/SSIV starch synthase that is thought to be involved in starch granule seeding in plants. This suggests that, in addition to the selective polysaccharide debranching demonstrated to be responsible for starch rather than glycogen synthesis, the nature and properties of the elongation enzyme define a novel determinant of starch versus glycogen accumulation. We show that the phylogenies of GlgA2 and of 16S ribosomal RNA display significant congruence. This suggests that this enzyme evolved together with cyanobacteria when they diversified over 2 billion years ago. However, cyanobacteria can be ruled out as direct progenitors of the SSIII/SSIV ancestral gene found in Archaeplastida. Hence, both cyanobacteria and plants recruited similar enzymes independently to perform analogous tasks, further emphasizing the importance of convergent evolution in the appearance of starch from a preexisting glycogen metabolism network.


Asunto(s)
Proteínas Bacterianas/metabolismo , Evolución Biológica , Cianobacterias/metabolismo , Glucógeno/metabolismo , Almidón Sintasa/metabolismo , Proteínas Bacterianas/genética , Cianobacterias/fisiología , Escherichia coli/genética , Escherichia coli/metabolismo , Genoma Bacteriano , Glucógeno/química , Glucógeno Sintasa/genética , Glucógeno Sintasa/metabolismo , Mutación , Filogenia , Polisacáridos Bacterianos/genética , Polisacáridos Bacterianos/metabolismo , Almidón/metabolismo , Almidón Sintasa/genética , Synechocystis/genética , Synechocystis/metabolismo
5.
Microb Biotechnol ; 17(8): e14546, 2024 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-39126420

RESUMEN

The latest assessment of progress towards the Sustainable Development Goals (SDGs) has identified major obstacles, such as climate change, global instability and pandemics, which threaten efforts to achieve the SDGs even by 2050. Urgent action is needed, particularly to reduce poverty, hunger and climate change. In this context, microalgae are emerging as a promising solution, particularly in the context of food security and environmental sustainability. As versatile organisms, microalgae offer nutritional benefits such as high-quality proteins and essential fatty acids, and can be cultivated in non-arable areas, reducing competition for resources and improving the sustainability of food systems. The role of microalgae also includes other applications in aquaculture, where they serve as sustainable alternatives to animal feed, and in agriculture, where they act as biofertilizers and biostimulants. These microorganisms also play a key role in interventions on degraded land, stabilizing soils, improving hydrological function and increasing nutrient and carbon availability. Microalgae therefore support several SDGs by promoting sustainable agricultural practices and contributing to land restoration and carbon sequestration efforts. The integration of microalgae in these areas is essential to mitigate environmental impacts and improve global food security, highlighting the need for increased research and development, as well as public and political support, to exploit their full potential to advance the SDGs.


Asunto(s)
Microalgas , Desarrollo Sostenible , Microalgas/metabolismo , Microalgas/crecimiento & desarrollo , Agricultura/métodos , Acuicultura , Cambio Climático , Conservación de los Recursos Naturales , Seguridad Alimentaria
6.
Plant Physiol ; 157(2): 730-41, 2011 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-21825107

RESUMEN

The target of rapamycin (TOR) kinase integrates nutritional and stress signals to coordinately control cell growth in all eukaryotes. TOR associates with highly conserved proteins to constitute two distinct signaling complexes termed TORC1 and TORC2. Inactivation of TORC1 by rapamycin negatively regulates protein synthesis in most eukaryotes. Here, we report that down-regulation of TOR signaling by rapamycin in the model green alga Chlamydomonas reinhardtii resulted in pronounced phosphorylation of the endoplasmic reticulum chaperone BiP. Our results indicated that Chlamydomonas TOR regulates BiP phosphorylation through the control of protein synthesis, since rapamycin and cycloheximide have similar effects on BiP modification and protein synthesis inhibition. Modification of BiP by phosphorylation was suppressed under conditions that require the chaperone activity of BiP, such as heat shock stress or tunicamycin treatment, which inhibits N-linked glycosylation of nascent proteins in the endoplasmic reticulum. A phosphopeptide localized in the substrate-binding domain of BiP was identified in Chlamydomonas cells treated with rapamycin. This peptide contains a highly conserved threonine residue that might regulate BiP function, as demonstrated by yeast functional assays. Thus, our study has revealed a regulatory mechanism of BiP in Chlamydomonas by phosphorylation/dephosphorylation events and assigns a role to the TOR pathway in the control of BiP modification.


Asunto(s)
Chlamydomonas reinhardtii/metabolismo , Proteínas de Choque Térmico/metabolismo , Serina-Treonina Quinasas TOR/metabolismo , Sitios de Unión , Chlamydomonas reinhardtii/efectos de los fármacos , Cicloheximida/farmacología , Chaperón BiP del Retículo Endoplásmico , Glicosilación/efectos de los fármacos , Respuesta al Choque Térmico , Fosforilación , Biosíntesis de Proteínas/efectos de los fármacos , Inhibidores de la Síntesis de la Proteína/farmacología , Sirolimus/farmacología , Serina-Treonina Quinasas TOR/antagonistas & inhibidores , Treonina , Tunicamicina/farmacología
7.
Eukaryot Cell ; 7(2): 212-22, 2008 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-18039939

RESUMEN

The highly conserved target of rapamycin (TOR) kinase is a central controller of cell growth in all eukaryotes. TOR exists in two functionally and structurally distinct complexes, termed TOR complex 1 (TORC1) and TORC2. LST8 is a TOR-interacting protein that is present in both TORC1 and TORC2. Here we report the identification and characterization of TOR and LST8 in large protein complexes in the model photosynthetic green alga Chlamydomonas reinhardtii. We demonstrate that Chlamydomonas LST8 is part of a rapamycin-sensitive TOR complex in this green alga. Biochemical fractionation and indirect immunofluorescence microscopy studies indicate that TOR and LST8 exist in high-molecular-mass complexes that associate with microsomal membranes and are particularly abundant in the peri-basal body region in Chlamydomonas cells. A Saccharomyces cerevisiae complementation assay demonstrates that Chlamydomonas LST8 is able to functionally and structurally replace endogenous yeast LST8 and allows us to propose that binding of LST8 to TOR is essential for cell growth.


Asunto(s)
Proteínas Algáceas/metabolismo , Membrana Celular/metabolismo , Chlamydomonas reinhardtii/metabolismo , Retículo Endoplásmico/metabolismo , Proteínas Algáceas/genética , Secuencia de Aminoácidos , Animales , Northern Blotting , Chlamydomonas reinhardtii/genética , Chlamydomonas reinhardtii/crecimiento & desarrollo , Técnica del Anticuerpo Fluorescente , Biblioteca de Genes , Prueba de Complementación Genética , Inmunoprecipitación , Datos de Secuencia Molecular , Saccharomyces cerevisiae/crecimiento & desarrollo , Saccharomyces cerevisiae/metabolismo
8.
Regul Pept ; 125(1-3): 41-6, 2005 Feb 15.
Artículo en Inglés | MEDLINE | ID: mdl-15582712

RESUMEN

Pancreastatin (PST), a chromogranin A-derived peptide, has an anti-insulin metabolic effect and inhibits growth and proliferation by producing nitric oxide (NO) in HTC rat hepatoma cells. When NO production is blocked, a proliferative effect prevails due to the activation a Galphaq/11-phospholipase C-beta (PLC-beta) pathway, which leads to an increase in [Ca2+]i, protein kinase C (PKC) and mitogen-activated protein kinase (MAPK) activation. The aim of the present study was to investigate the NO synthase (NOS) isoform that mediates these effects of PST on HTC hepatoma cells and the possible roles of cyclic GMP (cGMP) and cGMP-dependent protein kinase. DNA and protein synthesis in response to PST were measured as [3H]-thymidine and [3H]-leucine incorporation in the presence of various pharmacological inhibitors: N-monomethyl-L-arginine (NMLA, nonspecific NOS inhibitor), L-NIO (endothelial nitric oxide synthase (eNOS) inhibitor), espermidine (neuronal nitric oxide synthase (nNOS) inhibitor), LY83583 (guanylyl cyclase inhibitor), and KT5823 (protein kinase G inhibitor, (PKG)). L-NIO, similarly to NMLA, reverted the inhibitory effect of PST on hepatoma cell into a stimulatory effect on growth and proliferation. Nevertheless, espermidine also prevented the inhibitory effect of PST, but there was no stimulation of growth and proliferation. When guanylyl cyclase activity was blocked, there was again a reversion of the inhibitory effect into a stimulatory action, suggesting that the effect of NO was mediated by the production of cGMP. PKG inhibition prevented the inhibitory effect of PST, but there was no stimulatory effect. Therefore, the inhibitory effect of PST on growth and proliferation of hepatoma cells may be mainly mediated by eNOS activation. In turn, the effect of NO may be mediated by cGMP, whereas other pathways in addition to PKG activation seem to mediate the inhibition of DNA and protein synthesis by PST in HTC hepatoma cells.


Asunto(s)
Carcinoma Hepatocelular/metabolismo , Cromograninas/fisiología , Proteínas Quinasas Dependientes de GMP Cíclico/fisiología , GMP Cíclico/fisiología , Hígado/citología , Proteínas del Tejido Nervioso/fisiología , Óxido Nítrico Sintasa/fisiología , Ornitina/análogos & derivados , Hormonas Pancreáticas/metabolismo , Hormonas Pancreáticas/fisiología , Aminoquinolinas/farmacología , Animales , Arginina/química , Calcio/metabolismo , Carbazoles/farmacología , Aumento de la Célula , Proliferación Celular , Cromogranina A , Proteínas Quinasas Dependientes de GMP Cíclico/farmacología , ADN/química , ADN/metabolismo , Inhibidores Enzimáticos/farmacología , Guanilato Ciclasa/metabolismo , Indoles/farmacología , Isoenzimas/metabolismo , Leucina/química , Sistema de Señalización de MAP Quinasas , Óxido Nítrico/química , Óxido Nítrico/metabolismo , Óxido Nítrico Sintasa de Tipo I , Óxido Nítrico Sintasa de Tipo III , Ornitina/farmacología , Péptidos/química , Fosfolipasa C beta , Isoformas de Proteínas , Ratas , Receptores del Factor Natriurético Atrial/metabolismo , Espermidina/farmacología , Timidina/química , Factores de Tiempo , Fosfolipasas de Tipo C/metabolismo , omega-N-Metilarginina/farmacología
9.
Mol Plant ; 7(1): 87-100, 2014 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-24121290

RESUMEN

Glycogen constitutes the major carbon storage source in cyanobacteria, as starch in algae and higher plants. Glycogen and starch synthesis is linked to active photosynthesis and both of them are degraded to glucose in the dark to maintain cell metabolism. Control of glycogen biosynthesis in cyanobacteria could be mediated by the regulation of the enzymes involved in this process, ADP-glucose pyrophosphorylase (AGP) and glycogen synthase, which were identified as putative thioredoxin targets. We have analyzed whether both enzymes were subjected to redox modification using purified recombinant enzymes or cell extracts in the model cyanobacterium Synechocystis sp. PCC 6803. Our results indicate that both AGP and glycogen synthases are sensitive to copper oxidation. However, only AGP exhibits a decrease in its enzymatic activity, which is recovered after reduction by DTT or reduced thioredoxin (TrxA), suggesting a redox control of AGP. In order to elucidate the role in redox control of the cysteine residues present on the AGP sequence (C45, C185, C320, and C337), they were replaced with serine. All AGP mutant proteins remained active when expressed in Synechocystis, although they showed different electrophoretic mobility profiles after copper oxidation, reflecting a complex pattern of cysteines interaction.


Asunto(s)
Glucosa-1-Fosfato Adenililtransferasa/metabolismo , Glucógeno Sintasa/metabolismo , Glucógeno/biosíntesis , Synechocystis/metabolismo , Cisteína/metabolismo , Glucosa-1-Fosfato Adenililtransferasa/química , Oxidación-Reducción , Synechocystis/enzimología , Tiorredoxinas/metabolismo
11.
Autophagy ; 4(7): 851-65, 2008 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-18670193

RESUMEN

The target of rapamycin (TOR) is a conserved Ser/Thr kinase that controls cell growth by activating an array of anabolic processes including protein synthesis, transcription and ribosome biogenesis, and by inhibiting catabolic processes such as mRNA degradation and autophagy. Control of autophagy by TOR occurs primarily at the induction step, and involves activation of the ATG1 kinase, a conserved component of the autophagic machinery. A substantial number of genes participating in autophagy have been originally identified in yeast. Most of these genes have mammalian homologues and many have apparent homologues in plants, indicating that autophagy is conserved among eukaryotes. The recent identification of TOR as a key element in cell growth control in plants and algae opens the way for future studies to investigate whether this signaling pathway may also control autophagy in photosynthetic organisms.


Asunto(s)
Arabidopsis/fisiología , Autofagia , Chlamydomonas reinhardtii/fisiología , Proteínas Quinasas/metabolismo , Saccharomyces cerevisiae/fisiología , Proteínas Algáceas/genética , Proteínas Algáceas/metabolismo , Animales , Arabidopsis/enzimología , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Autofagia/genética , Chlamydomonas reinhardtii/enzimología , Humanos , Proteínas Quinasas/genética , Saccharomyces cerevisiae/enzimología , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Transducción de Señal , Serina-Treonina Quinasas TOR
12.
Plant Physiol ; 139(4): 1736-49, 2005 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-16299168

RESUMEN

The macrolide rapamycin specifically binds the 12-kD FK506-binding protein (FKBP12), and this complex potently inhibits the target of rapamycin (TOR) kinase. The identification of TOR in Arabidopsis (Arabidopsis thaliana) revealed that TOR is conserved in photosynthetic eukaryotes. However, research on TOR signaling in plants has been hampered by the natural resistance of plants to rapamycin. Here, we report TOR inactivation by rapamycin treatment in a photosynthetic organism. We identified and characterized TOR and FKBP12 homologs in the unicellular green alga Chlamydomonas reinhardtii. Whereas growth of wild-type Chlamydomonas cells is sensitive to rapamycin, cells lacking FKBP12 are fully resistant to the drug, indicating that this protein mediates rapamycin action to inhibit cell growth. Unlike its plant homolog, Chlamydomonas FKBP12 exhibits high affinity to rapamycin in vivo, which was increased by mutation of conserved residues in the drug-binding pocket. Furthermore, pull-down assays demonstrated that TOR binds FKBP12 in the presence of rapamycin. Finally, rapamycin treatment resulted in a pronounced increase of vacuole size that resembled autophagic-like processes. Thus, our findings suggest that Chlamydomonas cell growth is positively controlled by a conserved TOR kinase and establish this unicellular alga as a useful model system for studying TOR signaling in photosynthetic eukaryotes.


Asunto(s)
Proteínas Algáceas/antagonistas & inhibidores , Proteínas de Ciclo Celular/antagonistas & inhibidores , Chlamydomonas reinhardtii/efectos de los fármacos , Chlamydomonas reinhardtii/metabolismo , Proteínas Protozoarias/antagonistas & inhibidores , Sirolimus/farmacología , Proteínas Algáceas/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Proteínas de Ciclo Celular/metabolismo , Chlamydomonas reinhardtii/genética , Chlamydomonas reinhardtii/crecimiento & desarrollo , Clonación Molecular , Secuencia Conservada , ADN de Algas/genética , ADN Complementario/genética , ADN Protozoario/genética , Modelos Moleculares , Datos de Secuencia Molecular , Mutación , Filogenia , Proteínas Protozoarias/metabolismo , Homología de Secuencia de Aminoácido , Transducción de Señal/efectos de los fármacos , Proteína 1A de Unión a Tacrolimus/química , Proteína 1A de Unión a Tacrolimus/genética , Proteína 1A de Unión a Tacrolimus/metabolismo
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