The pyruvate, orthophosphate dikinase regulatory proteins of Arabidopsis possess a novel, unprecedented Ser/Thr protein kinase primary structure

Pyruvate, orthophosphate dikinase (PPDK) is a ubiquitous, low-abundance metabolic enzyme of undetermined function in C3 plants. Its activity in C3 chloroplasts is light-regulated via reversible phosphorylation of an active-site Thr residue by the PPDK regulatory protein (RP), a most unusual bifunctional protein kinase (PK)/protein phosphatase (PP). In this paper we document the molecular cloning and functional analysis of the two unique C3 RPs in Arabidopsis thaliana . The first of these, AtRP1 , encodes a typical chloroplast-targeted, bifunctional C4-like RP. The second RP gene, AtRP2 , encodes a monofunctional polypeptide that possesses in vitro RP-like PK activity but lacks PP activity, and is localized in the cytosol. Notably, the deduced primary structures of these two highly homologous polypeptides are devoid of any canonical subdomain structure that unifies all known eukaryotic and prokaryotic Ser/Thr PKs into one of three superfamilies, despite the direct demonstration that AtRP1 is functionally a member of this group. Instead, these C3 RPs and the related C4 plant homologues encode a conserved, centrally positioned, approximately 260-residue sequence currently described as the ' d omain of u nknown f unction 299' (DUF 299). We propose that vascular plant RPs form a unique protein kinase family now designated as the DUF 299 gene family.     

Maize recombinant C4-pyruvate,orthophosphate dikinase: Expression in Escherichia coli, partial purification, and characterization of the phosphorylatable protein

The gene for C₄-pyruvate,orthophosphate dikinase (PPDK) from maize (Zea mays) was cloned into an Escherichia coli expression vector and recombinant PPDK produced in E. coli cells. Recombinant enzyme was found to be expressed in high amounts (5.3 U purified enzyme-activity liter^-1 of induced cells) as a predominantly soluble and active protein. Biochemical analysis of partially purified recombinant PPDK showed this enzyme to be equivalent to enzyme extracted from illuminated maize leaves with respect to (i) molecular mass, (ii) specific activity, (iii) substrate requirements, and (iv) phosphorylation/inactivation by its bifunctional regulatory protein.Abbreviations DTT- dithiothreitol - FPLC- fast-protein liquid chromatography - HAP- hydroxyapatite - IPTG- isopropyl--thiogalactoside - MOPS- 3-(N-morpholino)propanesulfonic acid - PCR- polymerase chain reaction - PEP- phosphoenolpyruvate - PMSF- phenylmethylsufonyl fluoride - PPDK- pyruvate,orthophosphate dikinase - RP- regulatory protein     

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.     

Cold stability of pyruvate,orthophosphate dikinase of Flaveria brownii

The nucleotide sequences of the complementary DNA of pyruvate, P_i dikinase (PPDK) from Flaveria bidentis, a C₄ plant which possesses a cold-sensitive form of PPDK, and Flaveria brownii, a C₄-like plant which possesses a cold-tolerant form of PPDK, were determined. PPDK was isolated from the leaves of both Flaveria species and purified and the N-terminal amino acid sequences characterised. Together with a maize PPDK cDNA, cDNA inserts which code for the mature form of PPDK of F. bidentis and of F. brownii were expressed in bacteria and the cold sensitivity of the expressed PPDK studied. The cold sensitivity of the PPDK expressed in bacteria mimics the cold sensitivity of PPDK found in vivo in all three plant species. This study indicates that the cold sensitivity of plant PPDK is controlled by the primary structure of the enzyme.     

Effects of glycerol on the in vitro stability and regulatory activation/inactivation of pyruvate,orthophosphate dikinase of Zea mays L.

Glycerol stabilizes the activity of pyruvate, orthophosphate dikinase extracted from darkened or illuminated maize leaves. It serves as a better protectant of activity than dithiothreitol for the active day-form and the glycerol concentration needed for full protection is inversely related to the level of protein. The night-form of the enzyme is also protected by glycerol not only against inactivation, but also against partial reactivation in storage. Glycerol does not prevent the P_i-dependent activation nor the ADP-dependent inactivation of pyruvate, orthophosphate dikinase, but the rates of both processes are substantially decreased. The ability of the inactive night-form for P_i-dependent activation is also sustained by glycerol for at least 2 h at 20°C, apparently through stabilization of the labile regulatory protein.Abbreviations BSA bovine serum albumin - G-6-P glucose-6-phosphate - MDH malate dehydrogenase - PCMB p-chloromercuribenzoate - PEP phosphoenolpyruvate - PEPCase phosphoenol-pyruvate carboxylase - PPDK pyruvate, orthophosphate dikinase - PVP polyvinylpyrrolidone     

Assaying for pyruvate,orthophosphate dikinase activity: Necessary precautions with phosphoenolpyruvate carboxylase as coupling enzyme

Phosphoenolpyruvate carboxylase (EC 4.1.1.31), used as a coupling enzyme in the assay of the pyruvate, orthophosphate dikinase (EC 2.7.9.1) forward reaction, is a serious limiting factor for the overall rate when added at a level of 0.2–0.3 unit/ml of assay medium. Nonlimiting assay conditions are obtained by either increasing the level of the coupling enzyme to 3 units/ml or adding 6mM glucose-6-phosphate as an activator/stabilizer of phosphoenolpyruvate carboxylase.Abbreviations G-6-P glucose-6-phosphate - LDH lactate dehydrogenase - MDH malate dehydrogenase - PEP phosphoenolpyruvate - PEPCase phosphoenolpyruvate carboxylase - PVP polyvinylpyrrolidone - PPDK pyruvate, orthophosphate dikinase - U unit of enzyme activity (mol/min)     

Cytosolic pyruvate,orthophosphate dikinase functions in nitrogen remobilization during leaf senescence and limits individual seed growth and nitrogen content

The protein content of seeds determines their nutritive value, downstream processing properties and market value. Up to 95% of seed protein is derived from amino acids that are exported to the seed after degradation of existing protein in leaves, but the pathways responsible for this nitrogen metabolism are poorly defined. The enzyme pyruvate,orthophosphate dikinase (PPDK) interconverts pyruvate and phosphoenolpyruvate, and is found in both plastids and the cytosol in plants. PPDK plays a cardinal role in C₄ photosynthesis, but its role in the leaves of C₃ species has remained unclear. We demonstrate that both the cytosolic and chloroplastic isoforms of PPDK are up‐regulated in naturally senescing leaves. Cytosolic PPDK accumulates preferentially in the veins, while chloroplastic PPDK also accumulates in mesophyll cells. Analysis of microarrays and labelling patterns after feeding ¹³C‐labelled pyruvate indicated that PPDK functions in a pathway that generates the transport amino acid glutamine, which is then loaded into the phloem. In Arabidopsis thaliana, over‐expression of PPDK during senescence can significantly accelerate nitrogen remobilization from leaves, and thereby increase rosette growth rate and the weight and nitrogen content of seeds. This indicates an important role for cytosolic PPDK in the leaves of C₃ plants, and allows us to propose a metabolic pathway that is responsible for production of transport amino acids during natural leaf senescence. Given that increased seed size and nitrogen content are desirable agronomic traits, and that efficient remobilization of nitrogen within the plant reduces the demand for fertiliser applications, PPDK and the pathway in which it operates are targets for crop improvement.     

Elevated pyruvate,orthophosphate dikinase (PPDK) activity alters carbon metabolism in C3 transgenic potatoes with a C4 maize PPDK gene

Changes in carbon metabolism and δ¹³C value of transgenic potato plants with a maize pyruvate,orthophosphate dikinase (PPDK; EC 2.7.9.1) gene are reported. PPDK catalyzes the formation of phospho enol pyruvate (PEP), the initial acceptor of CO₂ in the C₄ photosynthetic pathway. PPDK activities in the leases of transgenic potatoes were up to 5.4‐fold higher than those of control potato plants (wild‐type and treated control plants). In the transgenic potato plants, PPDK activity in leaves was negatively correlated with pyruvate content (r²= 0.81), and was positively correlated with malate content (r²= 0.88). A significant increase in the δ¹³C value was observed in the transgenic potato plants, suggesting a certain contribution of PEP carboxylase as the initial acceptor of atmospheric CO₂. These data suggest that elevated PPDK activity may alter carbon metabolism and lead to a partial operation of C₄‐type carbon metabolism. However, since parameters associated with CO₂ gas exchange were not affected, the altered carbon metabolism had only a small effect on the total photosynthetic characteristics of the transgenic plants.     

Light and temperature dependence of the rate and degree of activation of pyruvate,Pi dikinase in vivo in maize

Activation of pyruvate,Pi dikinase by light was studied in leaf discs of maize which were illuminated for 1 h at light intensities ranging from approximately 3% to 50% of full sunlight and at temperatures of 10, 22.5, and 35°C. At the highest light intensity the degree of activation was similar and relatively independent of temperature between 10 and 35°C. Under low light the degree of activation was high at 10°C but decreased rapidly with increasing temperature. There was a similar effect of light and temperature on the activation of NADP-malate dehydrogenase.At low temperature, the rate of activation of pyruvate,Pi dikinase was relatively low and independent of the light intensity used and the rate of inactivation in the dark was extremely low. At high temperature, the rate of activation was high and dependent on the light intensity used while the rate of dark inactivation was also relatively high. The degree of activation is discussed in relation to the possible influence of light and temperature on the turnover between the active and inactive forms of pyruvate,Pi dikinase during illumination.This research was supported by the Japan-U.S. Cooperative Research Program (The Japan Society for the Promotion of Science, NFS Grant INT 78-17245), NSF Grant PCM 77-09284, by the Japanese Ministry of Education and by the College of Agriculture and Life Sciences, University of Wisconsin, Madison, Wisconsin.     

Posttranslational regulation of pyruvate, orthophosphate dikinase in developing rice (Oryza sativa) seeds

Pyruvate, orthophosphate dikinase (PPDK; E.C.2.7.9.1) is most well known as a photosynthetic enzyme in C₄ plants. The enzyme is also ubiquitous in C₃ plant tissues, although a precise non-photosynthetic C₃ function(s) is yet to be validated, owing largely to its low abundance in most C₃ organs. The single C₃ organ type where PPDK is in high abundance, and, therefore, where its function is most amenable to elucidation, are the developing seeds of graminaceous cereals. In this report, we suggest a non-photosynthetic function for C₃ PPDK by characterizing its abundance and posttranslational regulation in developing Oryza sativa (rice) seeds. Using primarily an immunoblot-based approach, we show that PPDK is a massively expressed protein during the early syncitial-endosperm/-cellularization stage of seed development. As seed development progresses from this early stage, the enzyme undergoes a rapid, posttranslational down-regulation in activity and amount via regulatory threonyl-phosphorylation (PPDK inactivation) and protein degradation. Immunoblot analysis of separated seed tissue fractions (pericarp, embryo + aleurone, seed embryo) revealed that regulatory phosphorylation of PPDK occurs in the non-green seed embryo and green outer pericarp layer, but not in the endosperm + aleurone layer. The modestly abundant pool of inactive PPDK (phosphorylated + dephosphorylated) that was found to persist in mature rice seeds was shown to remain largely unchanged (inactive) upon seed germination, suggesting that PPDK in rice seeds function in developmental rather than in post-developmental processes. These and related observations lead us to postulate a putative function for the enzyme that aligns its PEP to pyruvate-forming reaction with biosynthetic processes that are specific to early cereal seed development.     

Expression of C3 and C4 photosynthetic characteristics in the amphibious plant Eleocharis vivipara: structure and analysis of the expression of isogenes for pyruvate,orthophosphate dikinase

Eleocharis vivipara, a unique leafless amphibious sedge, adopts the C4 mode of photosynthesis under terrestrial conditions and the C3 mode under submerged aquatic conditions. To analyze the molecular basis of these responses to the contrasting environments, we isolated and characterized two full-length cDNAs for a key C4 enzyme, pyruvate, orthophosphate dikinase (PPDK; EC 2.7.9.1). The isogenes for PPDK, designated ppdk1 and ppdk2, were highly homologous to one another but not identical. The PPDK1 protein, deduced from the nucleotide sequence of the cDNA, contained an extra domain at the amino terminus which, presumably, serves as a chloroplast transit peptide, while PPDK2 lacked this extra domain. It seems likely, therefore, that the ppdk1 and ppdk2 genes encode a chloroplastic and a cytosolic PPDK, respectively. Genomic Southern blot analysis revealed the existence of a small family of genes for PPDK in the genome of E. vivipara. Northern blot analysis indicate that both chloroplastic and cytosolic genes for PPDK are expressed simultaneously in the culms, a photosynthetic organ, of E. vivipara and that the pattern of expression of these genes differs between the growth forms.     

Estimation of pyruvate,phosphate dikinase activity in maize leaf tissue by (phosphoenolpyruvate plus pyrophosphate)-dependent phosphorylation of AMP

Crude extracts of maize leaf tissue catalysed the phosphorylation of AMP by ³²PPi in the presence of phosphoenolpyruvate (PEP). The reaction was enhanced by F^? and NH₄⁺. The optimum concentrations of AMP, PEP and PPi were 0.3, 10 and 1 mM, respectively. Under these conditions, ca75% of the AMP phosphorylated by ³²PPi was present as ATP and ca25 % as ADP. The activity was reversibly cold labile. The specific activity of crude extracts in the presence of F^? was proportional to enzyme concentration only at protein concentrations < 25,μg/ml. Partially purified pyruvate, phosphate dikinase (PPD) from maize leaf quantitatively phosphorylated AMP to ATP in a (PEP plus PPi)-dependent reaction with the concomitant production of 0.9 mol of pyruvate per mol of AMP phosphorylated. It was concluded that (PEP plus PPi)-dependent phosphorylation of AMP provides a reliable method for estimating PPD activity in crude extracts of maize. Crude maize extracts also catalysed ³²Pi-ATP and ³²PPi-ATP exchange but these activities were not specific for PPD.     

Diurnal changes in phosphoenolpyruvate carboxylase and pyruvate, orthophosphate dikinase properties in the natural environment: interplay of light and temperature in a C4 thermophile

Activities of phosphoenolpyruvate (PEP) carboxylase (EC 4.1.1.31) were measured in leaf extracts of field grown Amaranthus paniculatus L. (C₄) during a natural diurnal irradiance and temperature pattern. Enzyme assays were run at both fixed (30°C) and the corresponding leaf temperature at the time of harvest. Light activation of PEP carboxylase (PEPCase) at fixed assay temperatures was expressed as a decrease in S_0–5 (PEP) after a threshold (> 330 μmol m^–2 s^–1) photon fluence rate was surpassed at noon. Earlier in the morning, increase in apparent enzyme affinity for PEP was observed when the assay was run at leaf temperature, indicating a physiologically meaningfull effect of temperature on S^0.5 (PEP). The 3.3-fold increase in PEPCase activity at low PEP and fixed assay temperature between the minimal and maximal irradiance and temperature hours of the day, became 12.8-, 11.5- and 7.4-fold when assays were run at the corresponding leaf temperature during three diurnal cycles with respective temperature differences (max minus min) of 9.0, 8.3 and 7.4°C. The extent of malate inhibition was the same for both day and night forms of PEPCase assayed at 35°C, but increased considerably with night enzyme at 25°C. The results indicate that light increases the apparent affinity of PEPCase for PEP and that at lower temperatures malate becomes more inhibitory. Pyruvate orthophosphate dikinase activity started to increase immediately after sunrise and the 10-fold increase at fixed temperature became 14.8-, 14.2- and 13.1-fold when assays were run at the above leaf temperatures. This indicates that the light effect predominates with pyruvate, orthophosphate dikinase, while with phosphoenolpyravate carboxylase, light and temperature co-operate to increase the day enzyme activities.     

Expression of photosynthetic genes from the C4 plant, maize, in tobacco.

Summary Previous results from this laboratory have demonstrated the presence of genes for phosphoenolpyruvate carboxylase and pyruvate, orthophosphate dikinase in C₃ plants. The structure and light-enhanced expression of these genes is very similar to that of the genes found in the C₄ plant, maize. In order to investigate whether or not the regulation of these genes is similar in C₃ and C₄ plants, we have constructed chimeric genes using -glucuronidase as a reporter gene under the control of the maize promoters of the genes for phosphoenolpyruvate carboxylase, pyruvate, orthophosphate dikinase, and the small subunit of ribulose bisphosphate carboxylase (RuBisCO). The chimeric genes were introduced into tobacco, a C₃ plant. These genes were expressed primarily in leaf and stem tissue and the expression was enhanced by light. Thus, as in C₄ plants, the genes are expressed in a tissue-specific and light-inducible manner in the C₃ plant. Since the expression of these genes is restricted to specific cells in leaf tissue of C₄ plants, we also investigated the spatial pattern of expression of the chimeric genes using histochemical analysis of -glucuronidase activity. High level expression of all of these genes was found in mesophyll cells. This included the small subunit of RuBisCO, which is not expressed in mesophyll cells but in bundle sheath cells in C₄ plants. This report describes similarities between C₃ and C₄ plants in regulating the expression of these genes.