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.

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.

The function of glycine decarboxylase complex is optimized to maintain high photorespiratory flux via buffering of its reaction products.

Oxidation of glycine in photorespiratory pathway is the major flux through mitochondria of C3 plants in the light. It sustains increased intramitochondrial concentrations of NADH and NADPH, which are required to engage the internal rotenone-insensitive NAD(P)H dehydrogenases and the alternative oxidase. We discuss here possible mechanisms of high photorespiratory flux maintenance in mitochondria and suggest that it is fulfilled under conditions where the concentrations of glycine decarboxylase reaction products NADH and CO2 achieve an equilibrium provided by malate dehydrogenase and carbonic anhydrase, respectively. This results in the removal of these products from the glycine decarboxylase multienzyme active sites and in the maintenance of their concentrations at levels sufficiently low to prevent substrate inhibition of the reaction.

Mutations in genes encoding the glycine cleavage system predispose to neural tube defects in mice and humans.

Neural tube defects (NTDs), including spina bifida and anencephaly, are common birth defects of the central nervous system. The complex multigenic causation of human NTDs, together with the large number of possible candidate genes, has hampered efforts to delineate their molecular basis. Function of folate one-carbon metabolism (FOCM) has been implicated as a key determinant of susceptibility to NTDs. The glycine cleavage system (GCS) is a multi-enzyme component of mitochondrial folate metabolism, and GCS-encoding genes therefore represent candidates for involvement in NTDs. To investigate this possibility, we sequenced the coding regions of the GCS genes: AMT, GCSH and GLDC in NTD patients and controls. Two unique non-synonymous changes were identified in the AMT gene that were absent from controls. We also identified a splice acceptor site mutation and five different non-synonymous variants in GLDC, which were found to significantly impair enzymatic activity and represent putative causative mutations. In order to functionally test the requirement for GCS activity in neural tube closure, we generated mice that lack GCS activity, through mutation of AMT. Homozygous Amt(-/-) mice developed NTDs at high frequency. Although these NTDs were not preventable by supplemental folic acid, there was a partial rescue by methionine. Overall, our findings suggest that loss-of-function mutations in GCS genes predispose to NTDs in mice and humans. These data highlight the importance of adequate function of mitochondrial folate metabolism in neural tube closure.

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.

Ozone-induced changes in photosynthesis and photorespiration of hybrid poplar in relation to the developmental stage of the leaves.

Young poplar trees (Populus tremula Michx. x Populus alba L. clone INRA 717-1B4) were subjected to 120 ppb of ozone for 35 days in phytotronic chambers. Treated trees displayed precocious leaf senescence and visible symptoms of injury (dark brown/black upper surface stippling) exclusively observed on fully expanded leaves. In these leaves, ozone reduced parameters related to photochemistry (Chl content and maximum rate of photosynthetic electron transport) and photosynthetic CO(2) fixation [net CO(2) assimilation, Rubisco (ribulose-1,5-bisphosphate carboxylase oxygenase) activity and maximum velocity of Rubisco for carboxylation]. In fully expanded leaves, the rate of photorespiration as estimated from Chl fluorescence was markedly impaired by the ozone treatment together with the activity of photorespiratory enzymes (Rubisco and glycolate oxidase). Immunoblot analysis revealed a decrease in the content of serine hydroxymethyltransferase in treated mature leaves, while the content of the H subunit of the glycine decarboxylase complex was not modified. Leaves in the early period of expansion were exempt from visible symptoms of injury and remained unaffected as regards all measured parameters. Leaves reaching full expansion under ozone exposure showed potential responses of protection (stimulation of mitochondrial respiration and transitory stomatal closure). Our data underline the major role of leaf phenology in ozone sensitivity of photosynthetic processes and reveal a marked ozone-induced inhibition of photorespiration.

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.