Expression of cold-tolerant pyruvate,orthophosphate dikinase cDNA,and heterotetramer formation in transgenic maize plants

Maize is a typical C₄ plant of the NADP-malic enzyme type, and its high productivity is supported by the C₄ photosynthetic cycle, which concentrates atmospheric CO₂ in the leaves. The plant exhibits superior photosynthetic ability under high light and high temperature, but under cold conditions the photosynthetic rate is significantly reduced. Pyruvate orthophosphate dikinase (PPDK), a key enzyme of the C₄ pathway in maize, loses its activity below about 12 °C by dissociation of the tetramer and it is considered as one possible cause of the reduction in the photosynthetic rate of maize at low temperatures. To improve the cold stability of the enzyme, we introduced a cold-tolerant PPDK cDNA isolated from Flaveria brownii into maize by Agrobacterium-mediated transformation. We obtained higher levels of expression by using a double intron cassette and a chimeric cDNA made from F. bidentis and F. brownii with a maximum content of 1mg/g fresh weight. In leaves of transgenic maize, PPDK molecules produced from the transgene were detected in cold-tolerant homotetramers or in heterotetramers of intermediate cold susceptibility formed with the internal PPDK. Simultaneous introduction of an antisense gene for maize PPDK generated plants in which the ratio of heterolologous and endogenous PPDK was greatly improved. Arrhenius plot analysis of the enzyme extracted from one such plant revealed that the break point was shifted about 3 °C lower than that of the wild type.     

Cold lability of pyruvate, orthophosphate dikinase in the maize leaf

Cold lability of pyruvate, orthophosphate dikinase was investigated using a homogeneous, purified enzyme preparation from maize (Zea mays L. var. Golden Cross Bantam T51) leaves. Its stability was markedly reduced below about 10 C and the rate of cold inactivation followed first order kinetics at a concentration lower than about 0.1 milligram of enzyme per milliliter. Cold inactivation was little affected by pH in the range which gives good stability for the enzyme at warm temperatures and the enzyme activity was protected strongly by inclusion of substrates (pyruvate and phosphoenolpyruvate) and polyols such as sucrose, sorbitol, and glycerol. Loss of catalytic activity was accompanied by an apparent dissociation of a tetrameric form of the enzyme (9S form) into a new, more slowly sedimenting (5.1S) component. Inclusion of pyruvate at 4 mM in the cold-treated enzyme had no effect on the sedimentation value. A sharp change in activation energy of the dikinase-catalyzed reaction was observed near 12 C and its break point appears to be close to the generally accepted critical low temperature limit for the growth of maize plants.     

Functional evolution of C(4) pyruvate, orthophosphate dikinase

Pyruvate,orthophosphate dikinase (PPDK) plays a controlling role in the PEP-regeneration phase of the C(4) photosynthetic pathway. Earlier studies have fully documented its biochemical properties and its post-translational regulation by the PPDK regulatory protein (PDRP). However, the question of its evolution into the C(4) pathway has, until recently, received little attention. One assumption concerning this evolution is that changes in catalytic and regulatory properties of PPDK were necessary for the enzyme to fulfil its role in the C(4) pathway. In this study, the functional evolution of PPDK from its ancient origins in the Archaea to its ascension as a photosynthetic enzyme in modern C(4) angiosperms is reviewed. This analysis is accompanied by a comparative investigation into key catalytic and regulatory properties of a C(3) PPDK isoform from Arabidopsis and the C(4) PPDK isoform from Zea mays. From these analyses, it is proposed that PPDK first became functionally seated in C(3) plants as an ancillary glycolytic enzyme and that its transition into a C(4) pathway enzyme involved only minor changes in enzyme properties per se.     

Primary structure of pyruvate, orthophosphate dikinase in the dicotyledonous C4 plant Flaveria trinervia

We have isolated and characterized cDNA clones encoding the entire precursor for the leafspecific isoform of pyruvate, orthophosphate dikinase (PPDK) from the dicotyledonous C4 plant Flaveria trinervia. The deduced amino acid sequence reveals a high degree of similarity to the corresponding maize protein indicating a common evolutionary basis. However, no significant similarities are apparent upon comparison of the putative transit peptides. The implications of this divergence are discussed with respect to the evolution of PPDK genes.     

Immunogold localization of photosynthetic enzymes in leaves of various C4 plants, with particular reference to pyruvate orthophosphate dikinase

Cellular localization of photosynthetic enzymes was investigated by immunogold electron microscopy for leaves of nine C4 grasses (three NADP-malic enzyme (NADP-ME)subtype species, three NAD-malic enzyme (NAD-ME) subtype species, and three phosphoenolpyruvate carboxykinase (PCK) subtype species), two C4 sedges (NADP-ME subtype species) and two C4 dicots (an NADP-ME and an NADP/NAD-ME subtype species). In leaves of all species, immunogold labelling was present for phosphoenolpyruvate carboxylase in the cytosol of the mesophyll cells (MC) and for ribulose-1,5-bisphosphate carboxylase/oxygenase in the chloroplasts of the bundle sheath cells (BSC). However, considerable specific variation was found in the intercellular patterns of labelling for pyruvate orthophosphate dikinase (PPDK). In the NADP-ME grasses, two NAD-ME grasses, and the dicots, significant labelling for PPDK was present in the both the BSC and the MC chloroplasts. In the other NAD-ME grass, the PCK grasses, and the sedges, labelling for PPDK was present almost exclusively in the chloroplasts of the MC. These patterns were observed in the leaves of both young seedlings and mature plants. These results indicate that the accumulation of PPDK in leaves of C4 plants is not necessarily restricted to the MC, although the chloroplasts of the MC accumulate more than those of the BSC.Key words: C4 plants, immunolocalization, phosphoenolpyruvate carboxylase, pyruvate orthophosphate dikinase, ribulose-1,5-bisphosphate carboxylase/oxygenase.      

Molecular mechanisms underlying the differential expression of maize pyruvate, orthophosphate dikinase genes.

I describe here the organization of maize C4 chloroplast and non-C4 cytosolic pyruvate, orthophosphate dikinase (PPDK) genes and the molecular mechanisms underlying their differential expression. The maize C4 chloroplast PPDK gene (C4ppdkZm1) appears to have been created by the addition of an exon encoding the chloroplast transit peptide at a site upstream of a cytosolic PPDK gene (cyppdkZm1). A splice acceptor sequence located in the first exon of cyppdkZm1 allows the fusion of the transit peptide to the cyppdkZm1 sequences. A second cyPPDK gene (cyppdkZm2) shares extensive homology with cyppdkZm1 in the coding region and in the 5' flanking region up to the TATA box. By a novel protoplast transient expression method, I show that the light-inducible expression of C4ppdkZm1 is controlled by two expression programs mediated through separate upstream regulatory elements that are active in leaf, but inactive in root and stem. Light-mediated C4ppdkZm1 expression in maize is apparently uncoupled from leaf development and partially associated with chloroplast development. For cyppdkZm1 expression, distinct upstream elements and a specific TATA promoter element, located in the first intron of C4ppdkZm1, are required. The low expression of cyppdkZm2 can be attributed to an absence of upstream positive elements and weak activity of the TATA promoter element.     

The pyruvate, orthophosphate dikinase regulatory proteins of Arabidopsis are both bifunctional and interact with the catalytic and nucleotide-binding domains of pyruvate, orthophosphate dikinase

Pyruvate orthophosphate dikinase (PPDK) is a key enzyme in C(4) photosynthesis and is also found in C(3) plants. It is post-translationally modified by the PPDK regulatory protein (RP) that possesses both kinase and phosphotransferase activities. Phosphorylation and dephosphorylation of PPDK lead to inactivation and activation respectively. Arabidopsis thaliana contains two genes that encode chloroplastic (RP1) and cytosolic (RP2) isoforms of RP, and although RP1 has both kinase and phosphotransferase activities, to date RP2 has only been shown to act as a kinase. Here we demonstrate that RP2 is able to catalyse the dephosphorylation of PPDK, although at a slower rate than RP1 under the conditions of our assay. From yeast two-hybrid analysis we propose that RP1 binds to the central catalytic domain of PPDK, and that additional regions towards the carboxy and amino termini are required for a stable interaction between RP2 and PPDK. For 21 highly conserved amino acids in RP1, mutation of 15 of these reduced kinase and phosphotransferase activity, while mutation of six residues had no impact on either activity. We found no mutant in which only one activity was abolished. However, in some chimaeric fusions that comprised the amino and carboxy termini of RP1 and RP2 respectively, the kinase reaction was severely compromised but phosphotransferase activity remained unaffected. These findings are consistent with the findings that both RP1 and RP2 modulate reversibly the activity of PPDK, and possess one bifunctional active site or two separate sites in close proximity.     

Effect of adenine nucleotides on the reaction catalyzed by pyruvate,orthophosphate dikinase in maize

The effect of adenine nucleotides in pyruvate, orthophosphate dikinase (EC 2.7.9.1, ATP, pyruvate, orthophosphate phosphotransferase)_was studied with the enzyme furified from maize, and with the enzyme obtained from mesophyll chloroplast extracts during assay in the direction of pyruvate conversion to phosphoenolpyruvate. (1) In studies with the purified enzyme, the relationship of initial velocity to ATP concentrations follows Michaelis-Menten kinetics, and the K_m value for ATP was 22.8 μM (± 5.1 μM, n = 5). (2) AMP was a competitive inhibitor with respect to ATP, and its K_i value was 35.8 μM (± μM, n = 4). There was no inhibition of catalysis by ADP up to a concentration of 460 μM. (3) The theoretical response of the enzyme to change in the adenylate energy charge was calculated from the kinetic constants for ATP and AMP. The experimentally obtained values were similar to the theoretical response when varying energy charge was generated by addition of appropriate amounts of ATP, ADP and AMP in assays with the purified enzyme. The response of the enzyme to energy charge at different pH values (pH 7.0, 7.5, and 8.0) was similar, although the activity of the enzyme at pH 7.0 was about 40% of that at pH 8.0. (4) When mesophyll chloroplast extracts of maize, which contain high levels of adenylate kinase, were used as the source of the enzyme and the adenylate energy charge was generated by addition of different concentrations of ATP and AMP, the influence on catalysis was similar to that with the purified enzyme. (5) The data show that the effect of varying energy chage on the activity of the dikinase is not typical of a U-type enzyme, in contrast to phosphoglycerate kinase (EC 2.7.2.3, ATP: 3-phospho-D-glycerate 1-phosphotransferase), which is more strongly regulated. (6) Evidence is presented for competition between the dikinase and phosphoglycerate kinase for ATP in mesophyll chloroplast extracts of maize. (7) When the effect of adenylate energy charge on the state of activation and the direct effect on catalysis of the dikanase are combined, the total capacity for catalysis is very dependent on the energy charge.     

Sequence of the phosphothreonyl regulatory site peptide from inactive maize leaf pyruvate, orthophosphate dikinase

The regulatory site peptide sequence of phosphorylated inactive pyruvate, orthophosphate dikinase from maize leaf tissue was determined by automated Edman degradation analysis of 32P-labeled peptides purified by reversed-phase high performance liquid chromatography. The overlapping phosphopeptides were products of a digestion of the [beta-32P]ADP-inactivated dikinase with either trypsin or Pronase E. The sequence is Thr-Glu-Arg-Gly-Gly-Met-Thr(P)-Ser-His-Ala-Ala-Val-Val-Ala-Arg. The phosphothreonine residue, which appeared as either an anomalous proline or an unidentifiable phenylthiohydantoin derivative during sequencing, was verified by two-dimensional phosphoamino acid analysis of the phosphopeptides and by resequencing the tryptic peptide after dephosphorylation with exogenous alkaline phosphatase. This sequence, starting at position 4, is completely homologous to the previously published sequence of the tryptic dodecapeptide harboring the catalytically essential (phospho)histidyl residue in the active-site domain of the dikinase from the nonphotosynthetic bacterium, Bacteroides symbiosus (Goss, N.H., Evans, C.T., and Wood, H.G. (1980) Biochemistry 19, 5805-5809). These comparative results indicate that the regulatory phosphothreonine causing complete inactivation of maize leaf dikinase is separated from the critical active-site (phospho)histidine by just one intervening residue in the primary sequence.     

Studies on the dark/light regulation of maize leaf pyruvate, orthophosphate dikinase by reversible phosphorylation

In maize leaves, pyruvate, orthophosphate dikinase (PPDK) is deactivated in the dark and reactivated in the light. Studies in vitro using purified PPDK and a partially purified regulatory protein from maize confirmed previous reports correlating deactivation/reactivation with the reversible phosphorylation/dephosphorylation of a threonyl residue. By monitoring the stability of the exogenous 32P-labeled adenylate substrates during deactivation, we have firmly established ADP as the specific phosphate donor. In isolated maize leaf mesophyll protoplasts preilluminated with 32Pi, we observed a three- to fivefold higher PPDK activity in situ in the light, and a corresponding three- to fivefold higher level of phosphorylation of the 94-kDa PPDK protomer in the dark. HPLC-based phosphoamino acid analysis of PPDK purified from maize leaves of both light- and dark-adapted plants revealed the presence of P-serine. The inactive enzyme from dark-adapted plants (inactivated in vivo) also contained P-threonine. Total phosphate content of PPDK purified from leaves of light-adapted plants was approximately 0.5 mol/mol protomer, and 1.5 mol/mol protomer from leaves of dark-adapted plants. Since the difference between enzyme purified from light-adapted (active PPDK) and dark-adapted (inactive PPDK) plants is the presence of P-threonine in the latter, this suggests an inactivation stoichiometry in vivo of 1 mol P-threonine/mol 94-kDa protomer. These complementary studies with maize leaf PPDK in vitro, in situ, and in vivo provide convincing evidence for the dark/light regulation of this key C4-photosynthesis enzyme by reversible phosphorylation.     

A rapid screening method to detect specific inhibitors of pyruvate orthophosphate dikinase as leads for C4 plant-selective herbicides

Plants using the C(4) photosynthetic pathway are highly represented among the world's worst weeds, with only 4 C(4) species being agriculturally productive (maize, sorghum, millet, and sugar cane). With the C(4) acid cycle operating as a biochemical appendage of C(3) photosynthesis, the additional enzymes involved in C(4) photosynthesis represent an attractive target for the development of weed-specific herbicides. The rate-limiting enzyme of this metabolic pathway is pyruvate orthophosphate dikinase (PPDK). PPDK, coupled with phosphoenolpyruvate carboxylase and nicotinamide adenine dinucleotide-malate dehydrogenase, was used to develop a microplate-based assay to detect inhibitors of enzymes of the C(4) acid cycle. The resulting assay had a Z' factor of 0.61, making it a high-quality assay able to reliably identify active test samples. Organic extracts of 6679 marine macroscopic organisms were tested within the assay, and 343 were identified that inhibited the 3 enzyme-coupled reaction. A high confirmation rate was achieved, with 95% of these hit extracts proving active again upon retesting. Sequential addition of phosphoenolpyruvate and oxaloacetate to the assay facilitated identification of 83 extracts that specifically inhibited PPDK.