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Glycine decarboxylase is a mitochondrial enzyme complex, which is the site of photorespiratory CO2 and NH3 release. Although the proteins that constitute the complex are located within the mitochondria, because of their intimate association with photosynthesis their expression is controlled by light. Comparisons of the kinetics of mRNA accumulation between the small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase and the H-protein of glycine decarboxylase during the greening of etiolated Arabidopsis thaliana suggest that their expression is controlled in parallel. A genomic clone for the H-protein (gdcH) was isolated from Arabidopsis and sequenced. The upstream region from -856 to +62 was fused to the beta-glucuronidase (GUS) reporter gene, and this construct was transformed into tobacco. This 5' upstream regulatory region appears to control GUS expression in a manner very similar to that of the endogenous H-protein gene. Constructs with deletions in the 5' upstream region were transformed into tobacco. These deletions revealed that light-dependent and tissue-specific expression was largely controlled by a 259-bp region between -376 and -117 bp. This region contains several putative GT boxes with the GGTTAA consensus core sequence. Once these strong light-dependent elements were removed, a second level of control was revealed. In constructs in which the gdcH 5' regulatory region was shortened to -117 bp or less, there was more GUS activity in the roots than in the leaves, and in dark-grown plants than in light-grown plants. This suggests that more proximal control elements may be responsible for the constitutive low levels of gene expression noted in all nonphotosynthetic tissues.

The four component proteins of the glycine decarboxylase multienzyme complex (the P-, H-, T-, and L-proteins) comprise over one-third of the soluble proteins in mitochondria isolated from the leaves of C3 plants. Together with serine hydroxymethyltransferase, glycine decarboxylase converts glycine to serine and is the site of photorespiratory CO2 and NH3 release. The component proteins of the complex are encoded on nuclear genes with N-terminal presequences that target them to the mitochondria. The isolated complex readily dissociates into its component proteins and reassociates into the intact complex in vitro. Because of the intimate association between photosynthesis and photorespiration, the proteins of the complex are present at higher levels in leaves in the light. The expression of these genes is controlled at the transcriptional level and the kinetics of expression are closely related to those of the small subunit of Rubisco. Deletion analysis of fusions between the promoter of the H-protein of the complex and the reporter gene beta-glucuronidase in transgenic tobacco has identified a region responsible for the tissue specificity and light dependence of gene expression. Gel shift experiments show that a nuclear protein in leaves binds to this region. Glycine decarboxylase has proven to be an excellent system for studying problems in plant biochemistry ranging from protein-protein interactions to control of gene expression.

The glycine decarboxylase multienzyme complex is located in the mitochondrial matrix and catalyzes a key reaction of the photorespiratory C-2 cycle of C3 plants. The cDNA encoding the smallest subunit, the 13,900-dalton H-protein, of the glycine decarboxylase complex from pea (Pisum sativum) leaves was cloned, identified, characterized, and sequenced. The 678-nucleotide sequence contained a 495-nucleotide open reading frame capable of encoding the 165-amino-acid H-protein precursor. The N terminus of this protein contains a 34-amino-acid sequence which does not appear in the mature protein. This presequence resembles the amphiphilic helices observed with mitochondrial leader sequences in yeast. The 131-amino-acid mature protein from peas shares substantial homology with the enzyme isolated from chicken liver. The abundance of the H-protein mRNA was about 5-fold greater in light-grown pea seedlings compared to dark-grown seedlings. The amount of H-protein transcript increased within 4 h after the plants were transferred to white light and continued to increase up to 24 h. The time course for the accumulation of the H-protein mRNA was similar to that for the mRNA of the small subunit of ribulose-1,5-bisphosphate carboxylase.

The P, H, and T proteins of the glycine cleavage system have been purified separately from pea leaf mitochondria and demonstrate molecular weights of 98,000, 15,500, and 45,000, respectively, by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The molecular weight of P protein by gel filtration was 210,000, indicating that this enzyme has a native homodimer conformation. Reconstitution assays containing purified P, H, and T proteins and yeast lipoamide dehydrogenase catalyze the oxidation of glycine and demonstrate a strict dependence on pyridoxal phosphate, tetrahydrofolate, NAD+, and dithiothreitol. The released CO2, methylamine-H protein intermediate, and methylenetetrahydrofolate are produced in stoichiometric amounts from glycine during the cleavage reaction. H protein acts as co-substrate with glycine during the decarboxylation reaction, demonstrating an apparent Km value of 2.2 microM. P and H protein alone jointly catalyze the glycine carboxyl-14 CO2 exchange reaction in the presence of pyridoxal phosphate and dithiothreitol. L protein of the glycine cleavage system was immunopurified using monoclonal antibodies. Antigenic and molecular weight similarities of the L protein with the lipoamide dehydrogenase component of the pyruvate dehydrogenase complex were shown suggesting the possibility of common isomers of lipoamide dehydrogenase for the two enzyme complexes.

The rates of mitochondrial glycine oxidation estimated by CO2-release and glycine-bicarbonate exchange activities in fully greened tissues are approximately 10 times greater than those of etiolated pea leaves and potato tuber mitochondria. The release of CO2 from glycine in intact mitochondria isolated from dark-grown and nonphotosynthetic tissues was sensitive to inhibitors of mitochondrial electron transport, glycine transport, and glycine decarboxylase activities. The CO2-release and glycine-bicarbonate exchange activities in crude mitochondrial protein extracts from light-grown versus dark-grown tissues exhibited light/dark ratios of 12 and 21, respectively. This suggests that the differences in capacity to oxidize glycine reside with the glycine decarboxylase enzyme complex itself. The complex is composed of four subunit enzymes, the P, H, T, and L proteins, which can be isolated individually and reconstituted into the active enzyme. The activities of P and T proteins were at least 10 times higher in fully greened pea leaves than in the etiolated tissue, while the H and L protein activities were four times higher in these same tissues. The levels of P and T proteins detected immunochemically were substantially lower in total mitochondrial extracts prepared from leaves of dark-grown pea seedlings. Labeling of whole pea seedlings and in vitro protein synthesis with isolated mitochondria indicated that the entire glycine decarboxylase enzyme complex is cytoplasmically synthesized and therefore encoded by the nucleus. Polypeptides synthesized from total leaf polyadenylated mRNA isolated from leaves of both the dark-grown and light-treated peas indicated the presence of P protein. This implies that translatable messages for this enzyme are present at some level throughout leaf development.