ABSTRACT
In plants, phosphoglycerate kinase (PGK) converts 1,3-bisphosphoglycerate into 3-phosphoglycerate in glycolysis but also participates in the reverse reaction in gluconeogenesis and the Calvin-Benson cycle. In the databases, we found three genes that encode putative PGKs. Arabidopsis (Arabidopsis thaliana) PGK1 was localized exclusively in the chloroplasts of photosynthetic tissues, while PGK2 was expressed in the chloroplast/plastid of photosynthetic and nonphotosynthetic cells. PGK3 was expressed ubiquitously in the cytosol of all studied cell types. Measurements of carbohydrate content and photosynthetic activities in PGK mutants and silenced lines corroborated that PGK1 was the photosynthetic isoform, while PGK2 and PGK3 were the plastidial and cytosolic glycolytic isoforms, respectively. The pgk1.1 knockdown mutant displayed reduced growth, lower photosynthetic capacity, and starch content. The pgk3.2 knockout mutant was characterized by reduced growth but higher starch levels than the wild type. The pgk1.1 pgk3.2 double mutant was bigger than pgk3.2 and displayed an intermediate phenotype between the two single mutants in all measured biochemical and physiological parameters. Expression studies in PGK mutants showed that PGK1 and PGK3 were down-regulated in pgk3.2 and pgk1.1, respectively. These results indicate that the down-regulation of photosynthetic activity could be a plant strategy when glycolysis is impaired to achieve metabolic adjustment and optimize growth. The double mutants of PGK3 and the triose-phosphate transporter (pgk3.2 tpt3) displayed a drastic growth phenotype, but they were viable. This implies that other enzymes or nonspecific chloroplast transporters could provide 3-phosphoglycerate to the cytosol. Our results highlight both the complexity and the plasticity of the plant primary metabolic network.
Subject(s)
Arabidopsis Proteins/metabolism , Arabidopsis/growth & development , Phosphoglycerate Kinase/metabolism , Arabidopsis/genetics , Arabidopsis/metabolism , Arabidopsis Proteins/genetics , Cytosol/metabolism , Gene Expression Regulation, Plant , Glyceric Acids/metabolism , Metabolomics/methods , Multigene Family , Mutation , Phosphoglycerate Kinase/genetics , Plant Components, Aerial/genetics , Plant Components, Aerial/metabolism , Plant Roots/genetics , Plant Roots/metabolism , Plants, Genetically Modified , Plastics/metabolismABSTRACT
The cellular compartmentalization of metabolic processes is an important feature in plants where the same pathways could be simultaneously active in different compartments. Plant glycolysis occurs in the cytosol and plastids of green and non-green cells in which the requirements of energy and precursors may be completely different. Because of this, the relevance of plastidial glycolysis could be very different depending on the cell type. In the associated study, we investigated the function of plastidial glycolysis in photosynthetic and heterotrophic cells by specifically driving the expression of plastidial glyceraldehyde-3-phosphate dehydrogenase (GAPCp) in a glyceraldehyde-3-phosphate dehydrogenase double mutant background (gapcp1gapcp2). We showed that GAPCp is not functionally significant in photosynthetic cells, while it plays a crucial function in heterotrophic cells. We also showed that (i) GAPCp activity expression in root tips is necessary for primary root growth, (ii) its expression in heterotrophic cells of aerial parts and roots is necessary for plant growth and development, and (iii) GAPCp is an important metabolic connector of carbon and nitrogen metabolism through the phosphorylated pathway of serine biosynthesis (PPSB). We discuss here the role that this pathway could play in the control of plant growth and development.
Subject(s)
Arabidopsis/metabolism , Glyceraldehyde-3-Phosphate Dehydrogenases/physiology , Glycolysis , Plastids/metabolism , Arabidopsis/cytology , Arabidopsis/physiology , Carbon/metabolism , Glyceraldehyde-3-Phosphate Dehydrogenases/genetics , Glyceraldehyde-3-Phosphate Dehydrogenases/metabolism , Mutation , Nitrogen/metabolism , Phosphorylation , Photosynthesis , Plant Roots/cytology , Plant Roots/metabolism , Plant Roots/physiology , Serine/biosynthesisABSTRACT
This work contributes to unraveling the role of the phosphorylated pathway of serine (Ser) biosynthesis in Arabidopsis (Arabidopsis thaliana) by functionally characterizing genes coding for the first enzyme of this pathway, 3-phosphoglycerate dehydrogenase (PGDH). We identified two Arabidopsis plastid-localized PGDH genes (3-PGDH and EMBRYO SAC DEVELOPMENT ARREST9 [EDA9]) with a high percentage of amino acid identity with a previously identified PGDH. All three genes displayed a different expression pattern indicating that they are not functionally redundant. pgdh and 3-pgdh mutants presented no drastic visual phenotypes, but eda9 displayed delayed embryo development, leading to aborted embryos that could be classified as early curled cotyledons. The embryo-lethal phenotype of eda9 was complemented with an EDA9 complementary DNA under the control of a 35S promoter (Pro-35S:EDA9). However, this construct, which is poorly expressed in the anther tapetum, did not complement mutant fertility. Microspore development in eda9.1eda9.1 Pro-35S:EDA9 was arrested at the polarized stage. Pollen from these lines lacked tryphine in the interstices of the exine layer, displayed shrunken and collapsed forms, and were unable to germinate when cultured in vitro. A metabolomic analysis of PGDH mutant and overexpressing plants revealed that all three PGDH family genes can regulate Ser homeostasis, with PGDH being quantitatively the most important in the process of Ser biosynthesis at the whole-plant level. By contrast, the essential role of EDA9 could be related to its expression in very specific cell types. We demonstrate the crucial role of EDA9 in embryo and pollen development, suggesting that the phosphorylated pathway of Ser biosynthesis is an important link connecting primary metabolism with development.
Subject(s)
Arabidopsis Proteins/metabolism , Arabidopsis/enzymology , Multigene Family , Phosphoglycerate Dehydrogenase/metabolism , Plastids/enzymology , Amino Acid Sequence , Arabidopsis/genetics , Arabidopsis/metabolism , Arabidopsis Proteins/genetics , Gene Expression Regulation, Enzymologic , Gene Expression Regulation, Plant , Genetic Complementation Test , Isoenzymes/classification , Isoenzymes/genetics , Isoenzymes/metabolism , Metabolomics/methods , Microscopy, Confocal , Molecular Sequence Data , Mutation , Phosphoglycerate Dehydrogenase/classification , Phosphoglycerate Dehydrogenase/genetics , Phosphorylation , Phylogeny , Plant Components, Aerial/enzymology , Plant Components, Aerial/genetics , Plant Components, Aerial/metabolism , Plant Roots/enzymology , Plant Roots/genetics , Plant Roots/metabolism , Plants, Genetically Modified , Pollen/enzymology , Pollen/genetics , Pollen/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Seeds/enzymology , Seeds/genetics , Seeds/metabolism , Sequence Homology, Amino Acid , Serine/genetics , Serine/metabolismABSTRACT
This study characterizes the phosphorylated pathway of Ser biosynthesis (PPSB) in Arabidopsis thaliana by targeting phosphoserine phosphatase (PSP1), the last enzyme of the pathway. Lack of PSP1 activity delayed embryo development, leading to aborted embryos that could be classified as early curled cotyledons. The embryo-lethal phenotype of psp1 mutants could be complemented with PSP1 cDNA under the control of Pro35S (Pro35S:PSP1). However, this construct, which was poorly expressed in the anther tapetum, did not complement mutant fertility. Microspore development in psp1.1/psp1.1 Pro35S:PSP1 arrested at the polarized stage. The tapetum from these lines displayed delayed and irregular development. The expression of PSP1 in the tapetum at critical stages of microspore development suggests that PSP1 activity in this cell layer is essential in pollen development. In addition to embryo death and male sterility, conditional psp1 mutants displayed a short-root phenotype, which was reverted in the presence of Ser. A metabolomic study demonstrated that the PPSB plays a crucial role in plant metabolism by affecting glycolysis, the tricarboxylic acid cycle, and the biosynthesis of amino acids. We provide evidence of the crucial role of the PPSB in embryo, pollen, and root development and suggest that this pathway is an important link connecting primary metabolism with development.
Subject(s)
Arabidopsis Proteins/metabolism , Phosphoric Monoester Hydrolases/metabolism , Plant Roots/metabolism , Pollen/metabolism , Seeds/metabolism , Serine/biosynthesis , Amino Acids/biosynthesis , Arabidopsis/genetics , Arabidopsis/growth & development , Arabidopsis/metabolism , Arabidopsis Proteins/genetics , Biosynthetic Pathways/genetics , Citric Acid Cycle/genetics , Gene Expression Regulation, Developmental , Gene Expression Regulation, Plant , Glycolysis/genetics , Green Fluorescent Proteins/genetics , Green Fluorescent Proteins/metabolism , Immunoblotting , Microscopy, Electron, Transmission , Microscopy, Fluorescence , Mutation , Phosphoric Monoester Hydrolases/genetics , Phosphorylation , Plant Roots/genetics , Plant Roots/growth & development , Plants, Genetically Modified , Pollen/genetics , Pollen/growth & development , Reverse Transcriptase Polymerase Chain Reaction , Seeds/genetics , Seeds/growth & developmentABSTRACT
The phytohormone abscisic acid (ABA) controls the development of plants and plays a crucial role in their response to adverse environmental conditions like salt and water stress. Complex interactions between ABA and sugar signal transduction pathways have been shown. However, the role played by glycolysis in these interactions is not known. In the associated study, we investigated the interactions between plastidial glycolytic glyceraldehyde-3-phosphate dehydrogenase (GAPCp) and ABA signal transduction in Arabidopsis. We followed physiological, genetic and genomic approaches to understand the processes and mechanisms underlying the ABA-glycolysis interactions. Our results indicated that GAPCp deficiency leads to ABA-insensitivity and impaired ABA signal transduction. The gene expression of the transcription factor ABI4, involved in both sugar and ABA signaling, was altered in gapcp double mutants (gapcp1gapcp2), suggesting that the ABA insensitivity of mutants is mediated, at least in part, through this transcriptional regulator. We also suggested that amino acid homeostasis and/or serine metabolism may also be important determinants in the connections of ABA with primary metabolism. These studies provide new insights into the links between plant primary metabolism and ABA signal transduction, and demonstrate the importance of plastidial glycolytic GAPCps in these interactions.
