Plastidic phosphoglucose isomerase is an important determinant of starch accumulation in mesophyll cells, growth, photosynthetic capacity, and biosynthesis of plastidic cytokinins in Arabidopsis

Póster presentado en el XXI Reunión de la Sociedad Española de Fisiología Vegetal y el XIV Congreso Hispano-Luso Fisiología Vegetal, celebrado en Toledo del 14 al 17 de junio de 2015.Peer Reviewe

The role of plastidic phosphoglucose isomerase in the response of Arabidopsis thaliana to volatile compounds emitted by pathogenic microorganisms

El almidón es un homopolímero ramificado de residuos de glucosa unidos covalentemente a través de enlaces de tipo α-1,4 y α-1,6. Sintetizado en el plastidio, este polisacárido de reserva constituye la forma principal de almacenamiento de carbohidratos en plantas superiores y un determinante importante tanto del crecimiento de la planta como de su relación con el entorno. Está ampliamente aceptado que el proceso de biosíntesis del almidón en hojas tiene lugar exclusivamente en el cloroplasto. Según esta interpretación, el almidón es el producto fi nal de una ruta metabólica conectada con el ciclo de Calvin- Benson (CBC) a través de la fosfoglucosa isomerasa plastidial (pPGI). Esta enzima cataliza la conversión de moléculas de fructosa-6-fosfato del CBC en moléculas de glucosa-6-fosfato, las cuales son metabolizadas en almidón a través de la acción combinada de la fosfoglucomutasa, la ADP-glucosa pirofosforilasa y la almidón sintasa. Las plantas perciben estímulos bióticos mediante el reconocimiento de una gran cantidad de compuestos procedentes de los organismos con los que interactúan. En este sentido cabe destacar que los microorganismos de la rizosfera sintetizan una gran cantidad de sustancias que regulan el desarrollo y el crecimiento de la planta. Además, estos microorganismos emiten una amplia gama de compuestos volátiles (VCs) que actúan como “infoquímicos” en la comunicación entre la planta y el microorganismo. Estudios llevados a cabo por el grupo de investigación en el que he desarrollado este trabajo de tesis doctoral demostraron que los VCs emitidos por una amplia gama de microorganismos (incluyendo patógenos y especies que normalmente no interactúan con la planta) fomentan la acumulación de cantidades excepcionalmente elevadas almidón en la planta. Dada la falta de conocimiento sobre los mecanismos implicados en este fenómeno y teniendo en cuenta a su vez el papel importante que juega el almidón en la interacción de la planta con su entorno, en este trabajo de tesis doctoral investigué las bases moleculares implicadas en la respuesta de la planta a los VCs microbianos, prestando especial atención al papel que juega la pPGI en esta respuesta. El primer capítulo de este trabajo describe la caracterización de dos mutantes (pgi1-2 y pgi1-3) carentes de actividad pPGI. Ambos acumulan en sus hojas un 10- 15% del almidón existente en hojas de plantas salvaje (WT). Contrariamente a lo que pudiera esperarse al tener en cuenta la interpretación clásica de la biosíntesis de almidón, análisis por microscopía revelaron la presencia de gránulos de almidón en cloroplastos de las células del mesófi lo de estos mutantes. Tanto pgi1-2 como pgi1-3 mostraron un crecimiento lento, una reducida capacidad fotosintética y un bajo balance NAD(P)H/NAD(P) con respecto a plantas WT. Estudios hormonómicos mostraron que el contenido de citoquininas (CKs) plastidiales en hojas pgi1 es muy reducido con respecto al existente en hojas WT. Además la aplicación exógena de CKs revirtió el fenotipo de defi ciencia de almidón de hojas de plantas pgi1. Los datos presentados en este trabajo indican que pPGI es un importante determinante de la fotosíntesis, el estado redox de la célula, el crecimiento y la acumulación de almidón en células del mesófi lo como consecuencia de su implicación en la producción de intermediarios de la vía oxidativa de las pentosas fosfato/glicólisis necesarios para la síntesis CKs plastidiales y poder reductor. El capítulo 2 muestra que VCs emitidos por microorganismos fi logenéticamente distantes (incluyendo bacterias y hongos benefi ciosos y patógenos) promueven el crecimiento, la acumulación de niveles excepcionalmente elevados de almidón y la fl oración en varias especies de plantas, incluidos cultivos de interés agronómico. Además, plantas de Arabidopsis expuestas a VCs emitidos por el fi topatógeno Alternaria alternata experimentaron un incremento en la fotosíntesis y un aumento del contenido de CKs. La magnitud de este fenómeno fue reducida en el mutante 35S:AtCKX1 defi ciente en CKs y en el mutante ahk2/3 de señalización de CKs, proporcionando así evidencia de que este tipo de hormonas juega un papel importante en la respuesta de las plantas a VCs microbianos. El análisis transcriptómico de hojas de Arabidopsis expuestas a VCs de A. alternata reveló cambios en la expresión de genes regulados por luz y CKs implicados en la fotosíntesis, la fl oración, el crecimiento y el metabolismo del almidón. Sorprendentemente, una gran cantidad de genes diferencialmente expresados en plantas tratadas con VCs de A. alternata son genes cuya expresión se ve alterada también en plantas expuestas a VCs emitidos por la bacteria benefi ciosa Bacillus subtilis GB03, sugiriendo que las plantas han desarrollado la capacidad de reaccionar a VCs emitidos por diferentes microorganismos a través de la activación o estimulación de mecanismos altamente conservados. Para entender mejor los mecanismos implicados en las respuestas de las plantas a los VCs emitidos por microorganismos e investigar en qué medida pPGI está implicada en este fenómeno, en el trabajo presentado en el capítulo 3 se caracterizó la respuesta del mutante pgi1-2 a los VCs emitidos por A. alternata. VCs emitidos por este hongo fi topatógeno promovieron el crecimiento, la fotosíntesis y la acumulación de CKs plastidiales en hojas pgi1-2. Contra todo pronóstico, VCs emitidos por A. alternata promovieron la acumulación de niveles excepcionalmente elevados de almidón en hojas pgi1-2. Análisis proteómicos revelaron que los VCs microbianos promueven cambios en la acumulación de proteínas involucradas en la fotosíntesis, el metabolismo del almidón y el crecimiento que pueden explicar las respuestas observadas en plantas pgi1-2. Los datos presentados en este capítulo muestran que las plantas de Arabidopsis son capaces de responder a VCs microbianos mediante la activación o la estimulación de mecanismos independientes de pPGI.Starch is a branched homopolymer of a-1,4-linked glucose subunits with a-1,6-linked glucose at the branching points. Synthesized in plastids, this reserve polysaccharide is the major storage carbohydrate in most higher plants and is an important determinant of plant growth and its relationship with the environment. It is widely accepted that the whole photosynthesis-driven starch biosynthetic process occurring in leaves resides exclusively in the chloroplast. According to this view, starch is considered the endproduct of a metabolic pathway that is linked to the Calvin-Benson cycle (CBC) by means of the plastidic phosphoglucose isomerase (pPGI). This enzyme catalyzes the conversion of fructose-6-phosphate from the CBC into glucose-6-phosphate, which is metabolized into starch by the combined action of phosphoglucomutase, ADP-glucose pyrophosphorylase and starch synthase. Plants are able to perceive biotic stimuli by recognizing a multitude of compounds originating from the interacting organisms. In this regard it is noteworthy that rhizosphere microorganisms synthesize a multitude of substances which regulate morphogenesis and plant growth. Moreover, these microbes emit many volatile compounds (VCs) that play potentially important roles as semiochemicals in the communication between plant and microorganism. Studies carried out by the research group where I have developed this doctoral thesis showed that VCs emitted by a wide range of microorganisms (including plant pathogens and microbes that do not normally interact with plants) promote accumulation of exceptionally high levels of starch in plants. Given the lack of knowledge about the mechanisms involved in this phenomenon and taking into account the important role that plays starch in the interaction of the plant with the environment, in this doctoral thesis, I investigated the molecular basis involved in the plant response to VCs, giving special attention to the role played by pPGI in this response. The First chapter of this work describes the characterization of two mutants (pgi1-2 and pgi1-3) lacking pPGI activity. Starch content in leaves in both mutants was 10-15% of that accumulated by wild type (WT) leaves. Contrary to what might be expected considering the classical view of starch biosynthesis, microscopy analyses revealed the presence of starch granules in the chloroplasts of mesophyll cells of these mutants. Both pgi1-2 and pgi1-3 displayed slow growth, reduced photosynthetic capacity phenotypes and a decrease of the NAD(P)H/NAD(P) ratio with respect to WT leaves. Hormonomic analyses showed that the content of plastidic cytokinins (CKs) in pgi1 leaves is exceedingly lower than in WT leaves. Noteworthy, exogenous application of CKs largely reverted the low starch content phenotype of pgi1 leaves. The data presented in this work show that pPGI is an important determinant of photosynthesis, redox status, growth and starch accumulation in mesophyll cells likely as a consequence of its involvement in the production of oxidative pentose phosphate pathway/glycolysis intermediates necessary for the synthesis of plastidic CKs and reducing power. Chapter 2, shows that VCs emitted by phylogenetically diverse microorganisms (including bacteria and fungi pathogens and nonpathogens) promote growth, accumulation of exceptionally high levels of starch and fl owering of various plant species, including crops of agronomic interest. Moreover, Arabidopsis plants exposed to VCs emitted by the phytopathogen Alternaria alternata, showed enhanced of photosynthesis and accumulated high levels of CKs. The magnitude of this phenomenon was reduced in the mutant 35S:AtCKX1 with CK-defi ciency and the CKs signaling ahk2/3 mutant, providing evidence that CKs play essential roles in this phenomenon. Transcriptomic analyses of Arabidopsis leaves exposed to A. alternata VCs revealed changes in the expression of light- and CK-responsive genes involved in photosynthesis, fl owering, growth and starch metabolism. Notably, many genes differentially expressed in plants treated with fungal VCs were also differentially expressed in plants exposed to VCs emitted by Bacillus subtilis GB03, suggesting that plants react to microbial VCs through activation/stimulation highly conserved regulatory mechanisms. To better elucidate the mechanisms involved in the responses of plants to microbial VCs, and to investigate the extent to which pPGI is involved in this phenomenon, the work of chapter 3 of the work is characterized the response of pgi1-2 mutant to VCs emitted by A. alternata. VCs emitted by this fungal phytopathogen promoted growth, photosynthesis and accumulation of plastidic CKs in pgi1-2 leaves. Notably, VCs emitted by A. alternata promoted accumulation of exceptionally high levels of starch in pgi1-2 leaves thus challenging the “classic” interpretation of transitory starch biosynthesis. Proteomic analyses revealed that microbial VCs promote global changes in the expression of proteins involved in photosynthesis, starch metabolism and growth that account for the observed response in pgi1-2 plants. The data presented in this chapter show that Arabidopsis plants are capable of responding to microbial VCs by activation/stimulation of pPGI independent mechanisms.La autora ha disfrutado de una beca predoctoral FPI del Ministerio de Educación y Ciencia. Este trabajo ha sido financiado por los proyectos BIO2010-182309 y BIO2013-49125-C2-1-P de la Comisión Interministerial de Ciencia y Tecnología.Programa de Doctorado en Biotecnología (RD 99/2011)Bioteknologiako Doktoretza Programa (ED 99/2011

Plastidic Phosphoglucose Isomerase Is an Important Determinant of Starch Accumulation in Mesophyll Cells, Growth, Photosynthetic Capacity, and Biosynthesis of Plastidic Cytokinins in Arabidopsis

Bahaji, Abdellatif et al.Phosphoglucose isomerase (PGI) catalyzes the reversible isomerization of glucose-6-phosphate and fructose-6-phosphate. It is involved in glycolysis and in the regeneration of glucose-6-P molecules in the oxidative pentose phosphate pathway (OPPP). In chloroplasts of illuminated mesophyll cells PGI also connects the Calvin-Benson cycle with the starch biosynthetic pathway. In this work we isolated pgi1-3, a mutant totally lacking pPGI activity as a consequence of aberrant intron splicing of the pPGI encoding gene, PGI1. Starch content in pgi1-3 source leaves was ca. 10-15% of that of wild type (WT) leaves, which was similar to that of leaves of pgi1-2, a T-DNA insertion pPGI null mutant. Starch deficiency of pgi1 leaves could be reverted by the introduction of a sex1 null mutation impeding β-amylolytic starch breakdown. Although previous studies showed that starch granules of pgi1-2 leaves are restricted to both bundle sheath cells adjacent to the mesophyll and stomata guard cells, microscopy analyses carried out in this work revealed the presence of starch granules in the chloroplasts of pgi1-2 and pgi1-3 mesophyll cells. RT-PCR analyses showed high expression levels of plastidic and extra-plastidic β-amylase encoding genes in pgi1 leaves, which was accompanied by increased β-amylase activity. Both pgi1-2 and pgi1-3 mutants displayed slow growth and reduced photosynthetic capacity phenotypes even under continuous light conditions. Metabolic analyses revealed that the adenylate energy charge and the NAD(P)H/NAD(P) ratios in pgi1 leaves were lower than those of WT leaves. These analyses also revealed that the content of plastidic 2-C-methyl-D-erythritol 4-phosphate (MEP)-pathway derived cytokinins (CKs) in pgi1 leaves were exceedingly lower than in WT leaves. Noteworthy, exogenous application of CKs largely reverted the low starch content phenotype of pgi1 leaves. The overall data show that pPGI is an important determinant of photosynthesis, energy status, growth and starch accumulation in mesophyll cells likely as a consequence of its involvement in the production of OPPP/glycolysis intermediates necessary for the synthesis of plastidic MEP-pathway derived hormones such as CKs.This work was partially supported by the Comisión Interministerial de Ciencia y Tecnología and Fondo Europeo de Desarrollo Regional (Spain) [grant numbers BIO2010-18239 and BIO2013-C2-1-P] and by the Government of Navarra [grant number IIM010491.RI1], the Ministry of Education, Youth and Sports of the Czech Republich [Grant L01204 from the National Program of Sustainability] and the European Social Fund and the state budget of the Czech Republic [project POST-UP, reg. No. CZ.1.07/2.3.00/30.0004]. AMS-L acknowledges a predoctoral fellowship from the Spanish Ministry of Science and Innovation. MB acknowledges a post-doctoral fellowship from the Public University of Navarra.Peer reviewe

Plastidic Phosphoglucose Isomerase Is an Important Determinant of Starch Accumulation in Mesophyll Cells, Growth, Photosynthetic Capacity, and Biosynthesis of Plastidic Cytokinins in Arabidopsis

<div><p>Phosphoglucose isomerase (PGI) catalyzes the reversible isomerization of glucose-6-phosphate and fructose-6-phosphate. It is involved in glycolysis and in the regeneration of glucose-6-P molecules in the oxidative pentose phosphate pathway (OPPP). In chloroplasts of illuminated mesophyll cells PGI also connects the Calvin-Benson cycle with the starch biosynthetic pathway. In this work we isolated <i>pgi1-3</i>, a mutant totally lacking pPGI activity as a consequence of aberrant intron splicing of the pPGI encoding gene, <i>PGI1</i>. Starch content in <i>pgi1-3</i> source leaves was ca. 10-15% of that of wild type (WT) leaves, which was similar to that of leaves of <i>pgi1-2</i>, a T-DNA insertion pPGI null mutant. Starch deficiency of <i>pgi1</i> leaves could be reverted by the introduction of a <i>sex1</i> null mutation impeding β-amylolytic starch breakdown. Although previous studies showed that starch granules of <i>pgi1-2</i> leaves are restricted to both bundle sheath cells adjacent to the mesophyll and stomata guard cells, microscopy analyses carried out in this work revealed the presence of starch granules in the chloroplasts of <i>pgi1-2</i> and <i>pgi1-3</i> mesophyll cells. RT-PCR analyses showed high expression levels of plastidic and extra-plastidic β-amylase encoding genes in <i>pgi1</i> leaves, which was accompanied by increased β-amylase activity. Both <i>pgi1-2</i> and <i>pgi1-3</i> mutants displayed slow growth and reduced photosynthetic capacity phenotypes even under continuous light conditions. Metabolic analyses revealed that the adenylate energy charge and the NAD(P)H/NAD(P) ratios in <i>pgi1</i> leaves were lower than those of WT leaves. These analyses also revealed that the content of plastidic 2-C-methyl-D-erythritol 4-phosphate (MEP)-pathway derived cytokinins (CKs) in <i>pgi1</i> leaves were exceedingly lower than in WT leaves. Noteworthy, exogenous application of CKs largely reverted the low starch content phenotype of <i>pgi1</i> leaves. The overall data show that pPGI is an important determinant of photosynthesis, energy status, growth and starch accumulation in mesophyll cells likely as a consequence of its involvement in the production of OPPP/glycolysis intermediates necessary for the synthesis of plastidic MEP-pathway derived hormones such as CKs.</p></div