Knockdown of glycine decarboxylase complex alters photorespiratory carbon isotope fractionation in Oryza sativa leaves.

The influence of reduced glycine decarboxylase complex (GDC) activity on leaf atmosphere CO2 and 13CO2 exchange was tested in transgenic Oryza sativa with the GDC H-subunit knocked down in leaf mesophyll cells. Leaf measurements on transgenic gdch knockdown and wild-type plants were carried out in the light under photorespiratory and low photorespiratory conditions (i.e. 18.4 kPa and 1.84 kPa atmospheric O2 partial pressure, respectively), and in the dark. Under approximately current ambient O2 partial pressure (18.4 kPa pO2), the gdch knockdown plants showed an expected photorespiratory-deficient phenotype, with lower leaf net CO2 assimilation rates (A) than the wild-type. Additionally, under these conditions, the gdch knockdown plants had greater leaf net discrimination against 13CO2 (Δo) than the wild-type. This difference in Δo was in part due to lower 13C photorespiratory fractionation (f) ascribed to alternative decarboxylation of photorespiratory intermediates. Furthermore, the leaf dark respiration rate (Rd) was enhanced and the 13CO2 composition of respired CO2 (δ13CRd) showed a tendency to be more depleted in the gdch knockdown plants. These changes in Rd and δ13CRd were due to the amount and carbon isotopic composition of substrates available for dark respiration. These results demonstrate that impairment of the photorespiratory pathway affects leaf 13CO2 exchange, particularly the 13C decarboxylation fractionation associated with photorespiration.

Measurement of Enzyme Activities.

The determination of enzyme activities in organ or organellar extracts is an important means of investigating metabolic networks and allows testing the success of enzyme-targeted genetic engineering. It also delivers information on intrinsic enzyme parameters such as kinetic properties or impact of effector molecules. This chapter provides protocols on how to assess activities of the enzymes of the core photorespiratory pathway, from 2-phosphoglycolate phosphatase to glycerate 3-kinase.

Photosynthesis in C3-C4 intermediate Moricandia species.

Evolution of C4 photosynthesis is not distributed evenly in the plant kingdom. Particularly interesting is the situation in the Brassicaceae, because the family contains no C4 species, but several C3-C4 intermediates, mainly in the genus Moricandia Investigation of leaf anatomy, gas exchange parameters, the metabolome, and the transcriptome of two C3-C4 intermediate Moricandia species, M. arvensis and M. suffruticosa, and their close C3 relative M. moricandioides enabled us to unravel the specific C3-C4 characteristics in these Moricandia lines. Reduced CO2 compensation points in these lines were accompanied by anatomical adjustments, such as centripetal concentration of organelles in the bundle sheath, and metabolic adjustments, such as the balancing of C and N metabolism between mesophyll and bundle sheath cells by multiple pathways. Evolution from C3 to C3-C4 intermediacy was probably facilitated first by loss of one copy of the glycine decarboxylase P-protein, followed by dominant activity of a bundle sheath-specific element in its promoter. In contrast to recent models, installation of the C3-C4 pathway was not accompanied by enhanced activity of the C4 cycle. Our results indicate that metabolic limitations connected to N metabolism or anatomical limitations connected to vein density could have constrained evolution of C4 in Moricandia.

The genetic basis of classic nonketotic hyperglycinemia due to mutations in GLDC and AMT.

PURPOSE: The study's purpose was to delineate the genetic mutations that cause classic nonketotic hyperglycinemia (NKH). METHODS: Genetic results, parental phase, ethnic origin, and gender data were collected from subjects suspected to have classic NKH. Mutations were compared with those in the existing literature and to the population frequency from the Exome Aggregation Consortium (ExAC) database. RESULTS: In 578 families, genetic analyses identified 410 unique mutations, including 246 novel mutations. 80% of subjects had mutations in GLDC. Missense mutations were noted in 52% of all GLDC alleles, most private. Missense mutations were 1.5 times as likely to be pathogenic in the carboxy terminal of GLDC than in the amino-terminal part. Intragenic copy-number variations (CNVs) in GLDC were noted in 140 subjects, with biallelic CNVs present in 39 subjects. The position and frequency of the breakpoint for CNVs correlated with intron size and presence of Alu elements. Missense mutations, most often recurring, were the most common type of disease-causing mutation in AMT. Sequencing and CNV analysis identified biallelic pathogenic mutations in 98% of subjects. Based on genotype, 15% of subjects had an attenuated phenotype. The frequency of NKH is estimated at 1:76,000. CONCLUSION: The 484 unique mutations now known in classic NKH provide a valuable overview for the development of genotype-based therapies.Genet Med 19 1, 104-111.

Targeted Knockdown of GDCH in Rice Leads to a Photorespiratory-Deficient Phenotype Useful as a Building Block for C4 Rice.

The glycine decarboxylase complex (GDC) plays a critical role in the photorespiratory C2 cycle of C3 species by recovering carbon following the oxygenation reaction of ribulose-1,5-bisphosphate carboxylase/oxygenase. Loss of GDC from mesophyll cells (MCs) is considered a key early step in the evolution of C4 photosynthesis. To assess the impact of preferentially reducing GDC in rice MCs, we decreased the abundance of OsGDCH (Os10g37180) using an artificial microRNA (amiRNA) driven by a promoter that preferentially drives expression in MCs. GDC H- and P-proteins were undetectable in leaves of gdch lines. Plants exhibited a photorespiratory-deficient phenotype with stunted growth, accelerated leaf senescence, reduced chlorophyll, soluble protein and sugars, and increased glycine accumulation in leaves. Gas exchange measurements indicated an impaired ability to regenerate ribulose 1,5-bisphosphate in photorespiratory conditions. In addition, MCs of gdch lines exhibited a significant reduction in chloroplast area and coverage of the cell wall when grown in air, traits that occur during the later stages of C4 evolution. The presence of these two traits important for C4 photosynthesis and the non-lethal, down-regulation of the photorespiratory C2 cycle positively contribute to efforts to produce a C4 rice prototype.

OsmC and incomplete glycine decarboxylase complex mediate reductive detoxification of peroxides in hydrogenosomes of Trichomonas vaginalis.

Osmotically inducible protein (OsmC) and organic hydroperoxide resistance protein (Ohr) are small, thiol-dependent peroxidases that comprise a family of prokaryotic protective proteins central to the defense against deleterious effects of organic hydroperoxides, which are reactive molecules that are formed during interactions between the host immune system and pathogens. Trichomonas vaginalis, a sexually transmitted parasite of humans, possesses OsmC homologues in its hydrogenosomes, anaerobic mitochondrial organelles that harbor enzymes and pathways that are sensitive to oxidative damage. The glycine decarboxylase complex (GDC), which consists of four proteins (i.e., L, H, P and T), is in eukaryotes exclusively mitochondrial enzymatic system that catalyzes oxidative decarboxylation and deamination of glycine. However, trichomonad hydrogenosomes contain only the L and H proteins, whose physiological functions are unknown. Here, we found that the hydrogenosomal L and H proteins constitute a lipoate-dependent redox system that delivers electrons from reduced nicotinamide adenine dinucleotide (NADH) to OsmC for the reductive detoxification of peroxides. Our searches of genome databases revealed that, in addition to prokaryotes, homologues of OsmC/Ohr family proteins with predicted mitochondrial localization are present in various eukaryotic lineages. Therefore, we propose that the novel OsmC-GDC-based redox system may not be limited to T. vaginalis.

Validation of a modified method for Bxb1 mycobacteriophage integrase-mediated recombination in Plasmodium falciparum by localization of the H-protein of the glycine cleavage complex to the mitochondrion.

The glycine cleavage complex (GCV) is a potential source of the one carbon donor 5,10-methylene-tetrahydrofolate (5,10-CH(2)-THF) in the malaria parasite Plasmodium falciparum. One carbon (C1) donor units are necessary for amino acid and nucleotide biosynthesis, and for the initiation of mitochondrial and plastid translation. In other organisms, GCV activity is closely coordinated with the activity of serine hydroxymethyltransferase (SHMT) enzymes. P. falciparum contains cytosolic and mitochondrial SHMT isoforms, and thus, the subcellular location of the GCV is an important indicator of its role in malaria metabolism. To determine the subcellular localization of the GCV, we used a modified version of the published method for mycobacteriophage integrase-mediated recombination in P. falciparum to generate cell lines containing one of the component proteins of the GCV, the H-protein, fused to GFP. Here, we demonstrate that this modification results in rapid generation of chromosomally integrated transgenic parasites, and we show that the H-protein localizes to the mitochondrion.

Stage-specific expression of the glycine cleavage complex subunits in Leishmania infantum.

The mitochondrial glycine cleavage complex (GCC) is an important part of cellular metabolism due to its role in the maintenance and balance of activated one-carbon units for a wide range of biosynthetic processes. In the protozoan parasite Leishmania, little is known about these metabolic processes. However, the importance of amino acid catabolism, especially for the clinically relevant amastigote form of this parasite, is becoming increasingly clear. Using a bioinformatics approach, we have identified orthologs of the genes encoding the four loosely associated GCC subunits (GCVP, GCVT, GCVH, and GCVL) in the visceral species Leishmania infantum. We report here that all GCC genes are expressed in L. infantum and that several are enriched in the intracellular amastigote stage. To further assess the regulation of GCC components throughout the life cycle of Leishmania, we focused on the T-protein component GCVT. GCVT is encoded by two almost identical tandemly arranged gene copies that have very divergent 3'UTRs. Using two different reporter gene systems, we demonstrate that the divergent GCVT 3'UTRs are responsible for the differential regulation of GCVT-1 and GCVT-2 isogenes at the protein level in both developmental forms of L. infantum. The GCVT-1 3'UTR is responsive to heat stress, resulting in higher expression of GCVT-1 in promastigotes, whereas the GCVT-2 3'UTR harbors a SIDER2 retroposon, which contributes to the amastigote-specific expression of GCVT-2 protein. Interestingly, our data indicate that expression of most GCC genes is inducible upon excess glycine and that this regulation is not conferred by 5'- or 3'-untranslated regions. Altogether, these data suggest a complex and multilayered regulation of the GCC both at the mRNA and protein levels throughout the L. infantum life cycle.

The glycine decarboxylase complex multienzyme family in Populus.

In plants, the glycine decarboxylase complex (GDC) cooperates with serine hydroxymethyltransferase (SHMT) to mediate photorespiratory glycine-serine interconversion. GDC is also postulated to be an integral component of one-carbon (C1) metabolism in heterotrophic tissues, although molecular evidence in plants is scarce. An initial report of a xylem-specific isoform of GDC component H-protein, PtgdcH1, in aspen (Populus tremuloides Michx.) provided molecular evidence consistent with an important role for GDC in plant C1 metabolism. PtgdcH1 is phylogenetically distinct from the leaf-abundant photorespiratory PtgdcH3, but both isoforms restored GDC activity in a yeast H-protein knockout mutant, suggesting their functional equivalence. The Populus genome contains eight transcriptionally active GDC genes, encoding four H-proteins, two T-proteins, and single P- and L-proteins. The two Populus T-protein isoforms, PtgdcT1 and PtgdcT2, exhibited differential expression in leaves and xylem, similar to PtgdcH3 and PtgdcH1. In silico identification of AC elements in the promoters of xylem-abundant PtgdcH1 and PtgdcT2, as well as many lignin biosynthetic genes of Populus is consistent with a prominent role for GDC in methyl-intensive lignification during wood formation. The AC element is absent from Arabidopsis GDC promoters, and GDC expression has not been linked to secondary growth in this herbaceous annual. Taken together, the results suggest that the association of distinct H-protein and T-protein isoforms with photorespiration and C1 metabolism is a distinguishing feature of Populus, and may signify molecular adaptation of GDC to cope with the C1 demands of lignification in woody perennials.

Proteins of the glycine decarboxylase complex in the hydrogenosome of Trichomonas vaginalis.

Trichomonas vaginalis is a unicellular eukaryote that lacks mitochondria and contains a specialized organelle, the hydrogenosome, involved in carbohydrate metabolism and iron-sulfur cluster assembly. We report the identification of two glycine cleavage H proteins and a dihydrolipoamide dehydrogenase (L protein) of the glycine decarboxylase complex in T. vaginalis with predicted N-terminal hydrogenosomal presequences. Immunofluorescence analyses reveal that both H and L proteins are localized in hydrogenosomes, providing the first evidence for amino acid metabolism in this organelle. All three proteins were expressed in Escherichia coli and purified to homogeneity. The experimental Km of L protein for the two H proteins were 2.6 microM and 3.7 microM, consistent with both H proteins serving as substrates of L protein. Analyses using purified hydrogenosomes showed that endogenous H proteins exist as monomers and endogenous L protein as a homodimer in their native states. Phylogenetic analyses of L proteins revealed that the T. vaginalis homologue shares a common ancestry with dihydrolipoamide dehydrogenases from the firmicute bacteria, indicating its acquisition via a horizontal gene transfer event independent of the origins of mitochondria and hydrogenosomes.

The plant-like C2 glycolate cycle and the bacterial-like glycerate pathway cooperate in phosphoglycolate metabolism in cyanobacteria.

The occurrence of a photorespiratory 2-phosphoglycolate metabolism in cyanobacteria is not clear. In the genome of the cyanobacterium Synechocystis sp. strain PCC 6803, we have identified open reading frames encoding enzymes homologous to those forming the plant-like C2 cycle and the bacterial-type glycerate pathway. To study the route and importance of 2-phosphoglycolate metabolism, the identified genes were systematically inactivated by mutagenesis. With a few exceptions, most of these genes could be inactivated without leading to a high-CO(2)-requiring phenotype. Biochemical characterization of recombinant proteins verified that Synechocystis harbors an active serine hydroxymethyltransferase, and, contrary to higher plants, expresses a glycolate dehydrogenase instead of an oxidase to convert glycolate to glyoxylate. The mutation of this enzymatic step, located prior to the branching of phosphoglycolate metabolism into the plant-like C2 cycle and the bacterial-like glycerate pathway, resulted in glycolate accumulation and a growth depression already at high CO(2). Similar growth inhibitions were found for a single mutant in the plant-type C2 cycle and more pronounced for a double mutant affected in both the C2 cycle and the glycerate pathway after cultivation at low CO(2). These results suggested that cyanobacteria metabolize phosphoglycolate by the cooperative action of the C2 cycle and the glycerate pathway. When exposed to low CO(2), glycine decarboxylase knockout mutants accumulated far more glycine and lysine than wild-type cells or mutants with inactivated glycerate pathway. This finding and the growth data imply a dominant, although not exclusive, role of the C2 route in cyanobacterial phosphoglycolate metabolism.