Chloroplast Glyceraldehyde-3-phosphate Dehydrogenase Research Articles

Increasing lipid production in Chlamydomonas reinhardtii through genetic introduction for the overexpression of glyceraldehyde-3-phosphate dehydrogenase.

Microalgae, valued for their sustainability and CO2 fixation capabilities, are emerging as promising sources of biofuels and high-value compounds. This study aimed to boost lipid production in C. reinhardtii by overexpressing chloroplast glyceraldehyde-3-phosphate dehydrogenase (GAPDH), a key enzyme in the Calvin cycle and glycolysis, under the control of a nitrogen-inducible NIT1 promoter, to positively impact overall carbon metabolism. The standout transformant, PNG#7, exhibited significantly increased lipid production under nitrogen starvation, with biomass rising by 44% and 76% on days 4 and 16, respectively. Fatty acid methyl ester (FAME) content in PNG#7 surged by 2.4-fold and 2.1-fold, notably surpassing the wild type (WT) in lipid productivity by 3.4 and 3.7 times on days 4 and 16, respectively. Transcriptome analysis revealed a tenfold increase in transgenic GAPDH expression and significant upregulation of genes involved in fatty acid and triacylglycerol synthesis, especially the gene encoding acyl-carrier protein gene (ACP, Cre13. g577100. t1.2). In contrast, genes related to cellulose synthesis were downregulated. Single Nucleotide Polymorphism (SNP)/Indel analysis indicated substantial DNA modifications, which likely contributed to the observed extensive transcriptomic and phenotypic changes. These findings suggest that overexpressing chloroplast GAPDH, coupled with genetic modifications, effectively enhances lipid synthesis in C. reinhardtii. This study not only underscores the potential of chloroplast GAPDH overexpression in microalgal lipid synthesis but also highlights the expansive potential of metabolic engineering in microalgae for biofuel production.

Six Regulatory Elements Lying in the Promoter Region Imply the Functional Diversity of Chloroplast GAPDH in Duanliella bardawil

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a well-known proverbial protein involved in various functions in vivo. The functional diversity of GAPDH from Dunaliella bardawil (DbGAPDH) may relate to the regulatory elements lying in the promoter at the transcriptional level. Using RT-PCR and RACE reactions, gapdh cDNA was isolated, and the full-length genomic sequence was obtained by LA-PCR-based genome walking. The full-length cDNA sequence was 1645 bp containing an 1128 bp putative open reading frame (ORF), which coded a 375 amino acids-deduced polypeptide whose molecular weight was 40.27 kDa computationally. Protein conserved domain search and structural computation found that DbGAPDH consists of two structural conserved domains highly homologous in most species; multiple sequence alignment discovered two positive charge residues (Lys164 and Arg 233), which play a critical role in the protein-protein interaction between GAPDH, phosphoribulokinase (PRK), and CP12. Phylogenetic analysis demonstrated that DbGAPDH has a closer relationship with analogues from algae and higher plants than with those from other species. In silico analysis of the promoter region revealed six potential regulatory elements might be involved in four hypothesized functions characterized by chloroplast GAPDH: oxygen-, light-, pathogen-, and cold-induced regulation. These results might supply some hints for the functional diversity mechanisms of DbGAPDH, and fresh information for further research to bridge the gap between our knowledge of DNA and protein structure and our understanding of functional biology in GAPDH regulation.

Spontaneous Assembly of Photosynthetic Supramolecular Complexes as Mediated by the Intrinsically Unstructured Protein CP12

CP12 is a protein of 8.7 kDa that contributes to Calvin cycle regulation by acting as a scaffold element in the formation of a supramolecular complex with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and phosphoribulokinase (PRK) in photosynthetic organisms. NMR studies of recombinant CP12 (isoform 2) of Arabidopsis thaliana show that CP12-2 is poorly structured. CP12-2 is monomeric in solution and contains four cysteines, which can form two intramolecular disulfides with midpoint redox potentials of -326 and -352 mV, respectively, at pH 7.9. Site-specific mutants indicate that the C-terminal disulfide is involved in the interaction between CP12-2 and GAPDH (isoform A(4)), whereas the N-terminal disulfide is involved in the interaction between this binary complex and PRK. In the presence of NAD, oxidized CP12-2 interacts with A(4)-GAPDH (K(D) = 0.18 microm) to form a binary complex of 170 kDa with (A(4)-GAPDH)-(CP12-2)(2) stoichiometry, as determined by isothermal titration calorimetry and multiangle light scattering analysis. PRK is a dimer and by interacting with this binary complex (K(D) = 0.17 microm) leads to a 498-kDa ternary complex constituted by two binary complexes and two PRK dimers, i.e. ((A(4)-GAPDH)-(CP12-2)(2)-(PRK))(2). Thermodynamic parameters indicate that assembly of both binary and ternary complexes is exoergonic although penalized by a decrease in entropy that suggests an induced folding of CP12-2 upon binding to partner proteins. The redox dependence of events leading to supramolecular complexes is consistent with a role of CP12 in coordinating the reversible inactivation of chloroplast enzymes A(4)-GAPDH and PRK during darkness in photosynthetic tissues.

Characterization of native and recombinant A4 glyceraldehyde 3-phosphate dehydrogenase. Kinetic evidence for confromation changes upon association with the small protein CP12.

A4 glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was purified from the green alga Chlamydomonas reinhardtii and was also overexpressed in Escherichia coli. Both purified A4 tetramers of recombinant and native GAPDH were characterized for the first time. The pH optimum for both native and recombinant enzymes was close to 7.8. The pKs of the residues involved in catalysis indicate that a cysteine and a histidine may take part in catalysis by chloroplast GAPDH, as is the case for glycolytic GAPDH. Native and recombinant GAPDH show Michaelis-Menten kinetics with respect to their cofactors, NADH and NADPH, with greater specificity for NADPH. The kinetic parameters are similar to those of the heterotetrameric A2B2 spinach chloroplast GAPDH. Native C. reinhardtii and recombinant GAPDHs exhibit a cooperative behavior towards the substrate 1,3-bisphosphoglycerate (BPGA). This positive cooperativity is specific to the C. reinhardtii enzyme, as higher plant A2B2 GAPDHs show Michaelis-Menten kinetics. Native GAPDH has twofold lower catalytic constant and K0.5 for BPGA than recombinant GAPDH. Mass spectrometry analysis of native GAPDH shows that it is a complex of GAPDH and the small protein CP12. In vitro reconstitution assays indicate that the kinetic differences are the result conformation changes of GAPDH upon association with CP12.

Crystal structure of the non-regulatory A4 isoform of spinach chloroplast glyceraldehyde-3-phosphate dehydrogenase complexed with NADP

Here, we report the first crystal structure of a photosynthetic glyceraldehyde-3-phosphate dehydrogenase (GAPDH) complexed with NADP. The enzyme, purified from spinach chloroplasts, is constituted of a single type of subunit (A) arranged in homotetramers. It shows non-regulated NADP-dependent and NAD-dependent activities, with a preference for NADP. The structure has been solved to 3.0 Å resolution by molecular replacement. The crystals belong to space group C222 with three monomers in the asymmetric unit. One of the three monomers generates a tetramer using the space group 222 point symmetry and a very similar tetramer is generated by the other two monomers, related by a non-crystallographic symmetry, using a crystallographic 2-fold axis.The protein reveals a large structural homology with known GAPDHs both in the cofactor-binding domain and in regions of the catalytic domain. Like all other GAPDHs investigated so far, the A4-GAPDH belongs to the Rossmann fold family of dehydrogenases. However, unlike most dehydrogenases of this family, the adenosine 2′-phosphate group of NADP does not form a salt-bridge with any positively charged residue in its surroundings, being instead set in place by hydrogen bonds with a threonine residue belonging to the Rossmann fold and a serine residue located in the S-loop of a symmetry-related monomer. While increasing our knowledge of an important photosynthetic enzyme, these results contribute to a general understanding of NADP versus NAD recognition in pyridine nucleotide-dependent enzymes.Although the overall structure of A4-GAPDH is similar to that of the cytosolic GAPDH from bacteria and eukaryotes, the chloroplast tetramer is peculiar, in that it can actually be considered a dimer of dimers, since monomers are bound in pairs by a disulphide bridge formed across Cys200 residues. This bridge is not found in other cytosolic or chloroplast GAPDHs from animals, bacteria, or plants other than spinach.

Xanthophyll cycle, light energy dissipation and electron transport in transgenic tobacco with reduced carbon assimilation capacity

The effects of reduced CO2 assimilation capacity on the leaf pigment composition and the dissipation of light energy were studied using transgenic tobacco (Nicotiana tabacum L. cv. W38). Two plant types were used: anti-SSu plants with reduced amounts of Rubisco and anti-GAPDH plants with reduced activity of chloroplast glycer-aldehyde 3-phosphate dehydrogenase. A moderate reduction in the photosynthetic capacity increased the de-epoxidation state of the xanthophyll-cycle pigments. In contrast, there was no large effect on the leaf pigment composition and the ratio of the xanthophyll cycle pigments to chlorophyll, and total carotenoids increased only in the most severe transgenic plants. The light induction of photosynthesis, fluorescence quenching and de-epoxida ion of the xanthophyll cycle pigments were also followed in wild-type and anti-SSu plants. Anti-SSu plants maintained high nonphotochemical quenching and increased xanthophyll de-epoxidation in the light but the reduction state of QA remained high. For both wild-type and anti-SSu plants, the electron transport rate estimated from chlorophyll a fluorescence appeared to be much higher than that required to support the observed rate of CO2 assimilation and photorespiration during the early phase of photosynthetic induction. However, the two estimates converged with the onset of steady-state photosynthesis.

Genetic and biochemical evidence for distinct key functions of two highly divergent GAPDH genes in catabolic and anabolic carbon flow of the cyanobacterium Synechocystis sp. PCC 6803.

Cyanobacterial genomes harbour two separate highly divergent glyceraldehyde-3-phosphate dehydrogenase (GAPDH) genes, gap1 and gap2, which are closely related at the sequence level to the nuclear genes encoding cytosolic and chloroplast GAPDH of higher plants, respectively. Genes gap1 and gap2 of the unicellular cyanobacterium Synechocystis sp. PCC 6803 were cloned and sequenced and subsequently inactivated by insertional mutagenesis to understand their metabolic functions. We obtained homozygous gap1- mutants which have lost the capacity to grow on glucose under dim light while growth on organic acids as well as photosynthetic growth under CO2 and high light is not impaired. Homozygous gap2- mutants show the reciprocal phenotype. Under dim light they only grow on glucose but not on organic acids nor do they survive under photosynthetic conditions. Measurements of the anabolic activities (reduction of 1,3-bisphosphoglycerate) in extracts from wild type and mutant cells show that Gap2 is a major enzyme with dual cosubstrate specificity for NAD and NADP, while Gap1 displays a minor NAD-specific GAPDH activity. However, if measured in the catabolic direction (oxidation of glyceraldehyde-3-phosphate) Gap2 activity is very low and increases three- to fivefold after gel filtration of extracts over Sephadex G25. Our results suggest that enzymes Gap1 and Gap2, although coexpressed in cyanobacterial wild-type cells, play distinct key roles in catabolic and anabolic carbon flow, respectively. While Gap2 operates in the photosynthetic Calvin cycle and in non-photosynthetic gluconeogenesis, Gap1 seems to be essential only for glycolytic glucose breakdown, conditions under which the catabolic activity of Gap2 seems to be repressed by a specific low-molecular-weight inhibitor.

Cis-acting elements essential for light regulation of the nuclear gene encoding the A subunit of chloroplast glyceraldehyde 3-phosphate dehydrogenase in Arabidopsis thaliana.

We report the characterization of cis-acting elements involved in light regulation of the nuclear gene (GapA) that encodes the A subunit of glyceraldehyde 3-phosphate dehydrogenase in Arabidopsis thaliana. Our previous deletion analyses indicate that the -277 to -195 upstream region of GapA is essential for light induction of the beta-glucuronidase reporter gene in transgenic tobacco (Nicotiana tabacum) plants. This region contains three direct repeats with the consensus sequence 5'-CAAATGAA(A/G)A-3' (Gap boxes). Our results show that 2-bp substitutions of the last four nucleotides (AA or GA) of the Gap boxes by CC abolish light induction of the beta-glucuronidase reporter gene in vivo and affect binding of the Gap box binding factor in vitro. We have also identified an additional cis-acting element, AE (Activation Element) box, that is involved in regulation of GapA. A combination of a Gap box trimer and an AE box dimer can confer light responsiveness of the cauliflower mosaic virus 35S promoter containing the -92 to +6 upstream sequence, whereas oligomers of Gap boxes or AE boxes alone cannot confer light responsiveness on the same promoter. These results suggest that Gap boxes and AE boxes function together as the light-responsive element of GapA.

A nuclear gene of eubacterial origin in Euglena gracilis reflects cryptic endosymbioses during protist evolution.

Genes for glycolytic and Calvin-cycle glyceraldehyde-3-phosphate dehydrogenase (GAPDH) of higher eukaryotes derive from ancient gene duplications which occurred in eubacterial genomes; both were transferred to the nucleus during the course of endosymbiosis. We have cloned cDNAs encoding chloroplast and cytosolic GAPDH from the early-branching photosynthetic protist Euglena gracilis and have determined the structure of its nuclear gene for cytosolic GAPDH. The gene contains four introns which possess unusual secondary structures, do not obey the GT-AG rule, and are flanked by 2- to 3-bp direct repeats. A gene phylogeny for these sequences in the context of eubacterial homologues indicates that euglenozoa, like higher eukaryotes, have obtained their GAPDH genes from eubacteria via endosymbiotic (organelle-to-nucleus) gene transfer. The data further suggest that the early-branching protists Giardia lamblia and Entamoeba histolytica--which lack mitochondria--and portions of the trypanosome lineage have acquired GAPDH genes from eubacterial donors which did not ultimately give rise to contemporary membrane-bound organelles. Evidence that "cryptic" (possibly ephemeral) endosymbioses during evolution may have entailed successful gene transfer is preserved in protist nuclear gene sequences.

Identification of a Light-Responsive Region of the Nuclear Gene Encoding the B Subunit of Chloroplast Glyceraldehyde 3-Phosphate Dehydrogenase from Arabidopsis thaliana

We report here the identification of a cis-acting region involved in light regulation of the nuclear gene (GapB) encoding the B subunit of chloroplast glyceraldehyde 3-phosphate dehydrogenase from Arabidopsis thaliana. Our results show that a 664-bp GapB promoter fragment is sufficient to confer light induction and organ-specific expression of the Escherichia coli beta-glucuronidase reporter gene (Gus) in transgenic tobacco (Nicotiana tabacum) plants. Deletion analysis indicates that the -261 to -173 upstream region of the GapB gene is essential for light induction. This region contains four direct repeats with the consensus sequence 5'-ATGAA(A/G)A-3' (Gap boxes). Deletion of all four repeats abolishes light induction completely. In addition, we have linked a 109-bp (-263 to -152) GapB upstream fragment containing the four direct repeats in two orientations to the -92 to +6 upstream sequence of the cauliflower mosaic virus 35S basal promoter. The resulting chimeric promoters are able to confer light induction and to enhance leaf-specific expression of the Gus reporter gene in transgenic tobacco plants. Based on these results we conclude that Gap boxes are essential for light regulation and organ-specific expression of the GapB gene in A. thaliana. Using gel mobility shift assays we have also identified a nuclear factor from tobacco that interacts with GapA and GapB DNA fragments containing these Gap boxes. Competition assays indicate that Gap boxes are the binding sites for this factor. Although this binding activity is present in nuclear extracts from leaves and roots of light-grown or dark-treated tobacco plants, the activity is less abundant in nuclear extracts prepared from leaves of dark-treated plants or from roots of greenhouse-grown plants. In addition, our data show that this binding factor is distinct from the GT-1 factor, which binds to Box II and Box III within the light-responsive element of the RbcS-3A gene of pea.

The evolutionary origin of red algae as deduced from the nuclear genes encoding cytosolic and chloroplast glyceraldehyde-3-phosphate dehydrogenases from Chondrus crispus

Algae are a heterogeneous group of photosynthetic eukaryotes traditionally separated into three major subdivisions: rhodophytes, chlorophytes, and chromophytes. The evolutionary origin of rhodophytes or red algae and their links to other photosynthetic and nonphotosynthetic eukaryotes have been a matter of much controversy and speculation. Here we present the first cDNAs of nuclear protein genes from red algae: Those encoding cytosolic and chloroplast glyceraldehyde-3-phosphate dehydrogenases (GAPDH) from Chondrus crispus. A phylogenetic analysis including GAPDH gene sequences from a number of eukaryotic taxa, cyanobacteria, and purple bacteria suggests that chloroplasts and rhodoplasts together form a monophyletic group of cyanobacterial descent and that rhodophytes separated from chlorophytes at about the same time as animals and fungi. The composite GAPDH tree further demonstrates that chloroplast and cytosolic GAPDH genes are closely related to their homologs in cyanobacteria and purple bacteria, respectively, the presumptive ancestors of chloroplasts and mitochondria, thereby firmly establishing the endosymbiotic origin of these nuclear genes and their fixation in eukaryotic cells before the rhodophyte/chlorophyte separation. The present data are in conflict with phylogenetic inferences based on plastid-encoded rbcL sequences supporting a polyphyletic origin of rhodoplasts and chloroplasts. Comparison of rbcL to GAPDH phylogenies suggests that rbcL trees may be misleading because they are composed of branches representing ancient duplicated (paralogous) genes.

Activation of spinach chloroplast glyceraldehyde 3-phosphate dehydrogenase: effect of glycerate 1,3-bisphosphate

Spinach (Spinacia oleracea L.) chloroplast NAD(P)-dependent glyceraldehyde 3-phosphate dehydrogenase (NAD(P)-GAPDH; EC 1.2.1.13) was purified. The association state of the protein was monitored by fast protein liquid chromatography-Superose 12 gel filtration. Protein chromatographed in the presence of NADP+ and dithiothreitol consisted of highly NADPH-active protomers of 160 kDa; otherwise, it always consisted of a 600-kDa oligomer (regulatory form) favoured by the addition of NAD+ in buffers and with low NADPH-dependent activity (ratio of activities with NADPH versus NADH of 0.2–0.4). Glycerate 1,3-bisphosphate (BPGA) was prepared enzymatically using rabbit-muscle NAD-GAPDH, and purified. Among known modulators of spinach NAD(P)-GAPDH, BPGA is the most effective on a molar basis in stimulating NADPH-activity of “dark” chloroplast extracts and purified NAD(P)-GAPDH (activation constant, K a= 12 μM). It also causes the enzyme to dissociate into 160-kDa protomers. The K m of BPGA both with NADPH or NADH as coenzyme is 4–7 μM. NAD+ and NADH are inhibitory to the activation process induced by BPGA. This compound, together with NADP(H) and ATP belongs to a group of substrate-modifiers of the NADPH-activity and conformational state of spinach NAD(P)-GAPDH, all characterized by K a values three- to tenfold higher than the K m. Since NADP(H) is largely converted to NAD(H) in darkened chloroplasts Heineke et al. 1991, Plant Physiol. 95, 1131–1137, it is proposed that NAD+ promotes NAD(P)-GAPDH association into a regulatory conformer with low NADPH-activity during dark deactivation. The process is reversed in the light by BPGA and other substrate-modifiers whose concentration increases during photosynthesis, in addition to reduced thioredoxin.

Light activation and molecular-mass changes of NAD(P)-glyceraldehyde 3-phosphate dehydrogenase of spinach and maize leaves

Light modulation of chloroplast glyceraldehyde 3-phosphate dehydrogenase (NAD(P)-GAPDH; EC 1.2.1.13) has been investigated. Complete activation of NADPH-dependent activity is achieved at 25 W.m−2 photosynthetically active radiation in spinach (Spinacia oleracea L.) and 100 W.m−2 in maize (Zea mays L.) leaves. Light activation is stronger in spinach (fivefold on average) than in maize (twofold), which shows higher “dark” activity. The NADH dependent activity does not change appreciably. Several substrate activators can simulate in vitro the light effect with recovery of latent NADPH-dependent activity of spinach enzyme, but they are almost inactive with maize enzyme. A mixture of activators has been devised to fully activate the spinach enzyme under most conditions. The NAD(P)-GAPDH protein can be resolved by rapid gel filtration (fast protein liquid chromatography) into three conformers which have different molecular masses according to the light conditions. Enzyme from darkened leaves or chloroplasts, or dichlorophenyl-1,1-dimethylurea-treated chloroplasts is mainly a 600-kDa regulatory form with low NADPH-dependent activity relative to NADH-activity. Enzyme from spinach leaves or chloroplasts during photosynthesis is mainly a 300-kDa oligomer, which along with the 600-kDa form also occurs in leaves of darkened maize. The conformer of illuminated maize leaves is mainly a 160-kDa species. Results are consistent with a model of NAD(P)-GAPDH freely interconvertible between protomers of the 160-kDa (or 300-kDa intermediate) form with high NADPH-activity, produced in the light by the action of thioredoxin and activating metabolites (spinach only), and a regulatory 600-kDa conformer with lower NADPH-activity produced in darkness or when photosynthesis is inhibited. This behavior is reminiscent of the in-vitro properties of purified enzyme; therefore, it seems unlikely that NAD(P)-GAPDH in the chloroplast is part of a stable multienzyme complex or is bound to membranes.

Light‐induced circadian rhythms in NADP+‐glyceraldehyde‐3‐phosphate dehydrogenase mRNA in corn seedlings

Abstract Chloroplast GAPDH (NADP+‐glyceraldehyde‐3‐phosphate dehydrogenase) (EC 1.2.1.13) is one of the photosynthetic enzymes which make up the set of reactions responsible for carbon fixation in plants. We here report that fully expanded green corn leaves showed diurnal variations in GAPDH mRNA level as well as enzyme activity in response to light:dark cycles. In continuous bright light the mRNA, but not the enzyme activity rhythm persisted. This is the first time a circadian rhythm in GAPDH mRNA level is reported; the existence of light‐driven rhythms in higher plants is thus further established.

Cloning and sequence analysis of cDNAs encoding the cytosolic precursors of subunits GapA and GapB of chloroplast glyceraldehyde-3-phosphate dehydrogenase from pea and spinach

Chloroplast glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is composed of two different subunits, GapA and GapB. cDNA clones containing the entire coding sequences of the cytosolic precursors for GapA from pea and for GapB from pea and spinach have been identified, sequenced and the derived amino acid sequences have been compared to the corresponding sequences from tobacco, maize and mustard. These comparisons show that GapB differs from GapA in about 20% of its amino acid residues and by the presence of a flexible and negatively charged C-terminal extension, possibly responsible for the observed association of the enzyme with chloroplast envelopes in vitro. This C-terminal extension (29 or 30 residues) may be susceptible to proteolytic cleavage thereby leading to a conversion of chloroplast GAPDH isoenzyme I into isoenzyme II. Evolutionary rate comparisons at the amino acid sequence level show that chloroplast GapA and GapB evolve roughly two-fold slower than their cytosolic counterpart GapC. GapA and GapB transit peptides evolve about 10 times faster than the corresponding mature subunits. They are relatively long (68 and 83 residues for pea GapA and spinach GapB respectively) and share a similar amino acid framework with other chloroplast transit peptides.

Endosymbiotic origin and codon bias of the nuclear gene for chloroplast glyceraldehyde-3-phosphate dehydrogenase from maize.

The nuclei of plant cells harbor genes for two types of glyceraldehyde-3-phosphate dehydrogenases (GAPDH) displaying a sequence divergence corresponding to the prokaryote/eukaryote separation. This strongly supports the endosymbiotic theory of chloroplast evolution and in particular the gene transfer hypothesis suggesting that the gene for the chloroplast enzyme, initially located in the genome of the endosymbiotic chloroplast progenitor, was transferred during the course of evolution into the nuclear genome of the endosymbiotic host. Codon usage in the gene for chloroplast GAPDH of maize is radically different from that employed by present-day chloroplasts and from that of the cytosolic (glycolytic) enzyme from the same cell. This reveals the presence of subcellular selective pressures which appear to be involved in the optimization of gene expression in the economically important graminaceous monocots.

Evidence in favor of the symbiotic origin of chloroplasts: Primary structure and evolution of tobacco glyceraldehyde-3-phosphate dehydrogenases

We report nucleotide sequences of cDNAs for the nuclear genes encoding chloroplast (GapA and GapB) and cytosolic (GapC) glyceraldehyde-3-phosphate dehydrogenases (GAPDH) from N. tabacum. Comparison of nucleotide sequences indicates that the GapA and GapB genes evolved following duplication of an ancestral gene about 450 million years ago. However, the divergence of GapA B and GapC occurred much earlier in evolution than the divergence of GapC and GAPDH genes of animals and fungi, suggesting that chloroplast and cytosolic GAPDHs evolved from different lineages. Comparison of amino acid sequences shows that the chloroplast GAPDHs are related to GAPDHs found in thermophilic bacteria, while the cytosolic GAPDH is related to the GAPDH found in mesophilic prokaryotes. These results strongly support the symbiotic origin of chloroplasts.

Prokaryotic features of a nucleus-encoded enzyme. cDNA sequences for chloroplast and cytosolic glyceraldehyde-3-phosphate dehydrogenases from mustard (Sinapis alba).

Two cDNA clones, encoding cytosolic and chloroplast glyceraldehyde-3-phosphate dehydrogenases (GAPDH) from mustard (Sinapis alba), have been identified and sequenced. Comparison of the deduced amino acid sequences with one another and with the GAPDH sequences from animals, yeast and bacteria demonstrates that nucleus-encoded subunit A of chloroplast GAPDH is distinct from its cytosolic counterpart and the other eukaryotic sequences and relatively similar to the GAPDHs of thermophilic bacteria. These results are compatible with the hypothesis that the nuclear gene for subunit A of chloroplast GAPDH is of prokaryotic origin. They are in puzzling contrast with a previous publication demonstrating that Escherichia coli GAPDH is relatively similar to the eukaryotic enzymes [Eur. J. Biochem. 150, 61-66 (1985)].

Modes of control by the circadian oscillator and the hourglass mechanism of the activities of cytoplasmic and chloroplast glyceraldehyde 3-phosphate dehydrogenases in Lemna gibba G3

Modes of control by the circadian oscillator and the hourglass mechanism of the activities of cytoplasmic and chloroplast glyceraldehyde 3-phosphate dehydrogenases in Lemna gibba G3

Mutually inverse rhythmic and sigmoidal changes in activity of cytoplasmic and chloroplast glyceraldehyde 3-phosphate dehydrogenases in Lemna gibba G3

Two major timing devices probably control the temporal changes in activity of cytoplasmic NAD- and chloroplast NADP-GPDs in the long-day plant Lemna gibba G3; one is an oscillator and the other an hourglass. The former is reset by a light-on signal and responsible for diurnal changes in activities of GPDs in continuous light, while the latter is reset by a light-off signal to control sigmoidal changes in activities of GPDs in continuous darkness. The activities of Cyt-NAD-GPD and Chl-NADP-GPD always change mutually inversely.