Pyruvate orthophosphate dikinase of c(3) seeds and leaves as compared to the enzyme from maize

Pyruvate orthophosphate dikinase (PPDK) was found in various immature seeds of C₃ plants (wheat, pea, green bean, plum, and castor bean), in some C₃ leaves (tobacco, spinach, sunflower, and wheat), and in C₄ (maize) kernels. The enzyme in the C₃ plants cross-reacts with rabbit antiserum against maize PPDK. Based on protein blot analysis, the apparent subunit size of PPDK from wheat seeds and leaves and from sunflower leaves is about 94 kdaltons, the same as that of the enzyme from maize, but is slightly less (about 90 kdaltons) for the enzyme from spinach and tobacco leaves. The amount of this enzyme per mg of soluble protein in C₃ seeds and leaves is much less than in C₄ leaves. PPDK is present in kernels of the C₄ plant, Zea mays in amounts comparable to those in C₄ leaves.

Regulatory properties of the enzyme from C₃ tissues (wheat) are similar to those of the enzyme from C₄ leaves with respect to in vivo light activation and dark inactivation (in leaves) and in vivo cold lability (seeds and leaves).

Following incorporation of ¹⁴CO₂ by illuminated wheat pericarp and adjoining tissue for a few seconds, the labeled metabolites were predominantly products resulting from carboxylation of phosphoenolpyruvate, with lesser labeling of compounds formed by carboxylation of ribulose 1,5-bisphosphate and operation of the reductive pentose phosphate cycle of photosynthesis. PPDK may be involved in mechanisms of amino acid interconversions during seed development.

     

Variation in amounts of pyruvate, orthophosphate dikinase, and some other enzymes of the c(4) pathway in some wheat species

First leaves and flag leaves of the wheat species Triticum aestivum cv Anza (6×), T. boeoticum Boiss (2×) L. were examined for content of pyruvate, orthophosphate dikinase (PPDK), phosphoenolpyruvate carboxylase (PEPC), and ribulose 1,5-bisphosphate carboxylase (RuBPC) by protein blot analyses using antibodies to maize leaf enzymes and by activity assays. In agreement with previous reports, the amount of RuBPC per mesophyll cell was about 3 times more in the hexaploid species, T. aestivum, than in the diploid species, T. boeoticum, both in first leaves and in flag leaves. In contrast, the level of PPDK polypeptide was nearly 3-fold higher per unit leaf area in the first leaf and 63% higher in the flag leaf of this diploid species compared to this hexaploid species. There was no significant difference in the levels of polypeptide and enzyme activity of PEPC between diploid and hexaploid wheat. Despite this significantly greater level of PPDK in the diploid species, the actual amount of PPDK could still supply only a limited amount of the enzyme activity necessary to provide phosphoenolpyruvate (PEP) for any putative intracellular C₄ carbon shuttle providing carbon to RuBPC. Thus, this difference in enzyme amount could not by itself account for the reported high rates of net photosynthesis at high light intensity in T. boeoticum. Together with reported anatomical differences between the diploid and hexaploid species, however, this biochemical difference may be of physiological importance.     

Synthesis and uptake of cytoplasmically synthesized pyruvate, pi dikinase polypeptide by chloroplasts

Polyadenylated RNA was isolated from maize leaves and translated in vitro. In agreement with a previous report by others, we found among the translation products a 110-kilodalton pyruvate orthophosphate dikinase (PPDK) precursor that is about 16 kilodaltons larger than the polypeptide isolated from cells. This maize PPDK precursor polypeptide was taken up from the translation product mixture by intact spinach chloroplasts and yielded a mature PPDK polypeptide (94 kilodaltons). The uptake and processing support the proposal that the extra 16-kilodalton size of the polypeptide from in vitro translation of maize leaf mRNA represents a transit sequence which is cleaved after its entry into chloroplasts. Moreover, these results provide additional evidence that in vivo in maize leaf cells PPDK polypeptide is synthesized in the cytoplasm and is transported into the chloroplasts.

Location of PPDK in C₃ plant leaves was investigated by immunochemical analysis. Intact chloroplasts were isolated from leaves of spinach, wheat, and maize. A protein blot of stromal protein in each case gave rise to bands corresponding to authentic PPDK polypeptide. This result indicates that PPDK is present in chloroplasts of C₃ plant leaves as it is in the case of C₄ plants.

     

High-Level Expression of Cold-Tolerant Pyruvate, Orthophosphate Dikinase from a Genomic Clone with Site-Directed Mutations in Transgenic Maize

Pyruvate, orthophosphate dikinase (PPDK) is a key enzyme in the C₄ photosynthetic pathway of maize. To improve the cold tolerance of the enzyme in maize, we designed two genomic sequence-based constructs in which the carboxy-terminal region of the enzyme was modified to mimic the amino acid sequence of the cold-tolerant PPDK of Flaveria brownii (Asteraceae). A large amount of PPDK was found to have accumulated in the leaves of many of the maize plants transformed with one of these constructs – that which introduced 17 amino acid substitutions without any alteration of the exon-intron structure – although there was a wide range of variation in the amount of PPDK among the separate plants. In contrast, the production was much less in maize transformed with the second construct in which a cDNA fragment for the same carboxy-terminal region was inserted. The specific activity of PPDK in the plants transformed with the gene with the amino acid substitutions was inversely correlated with the amount of enzyme in the leaves. In addition, the activity of the cold-tolerant recombinant enzyme was judged to be regulated by the PPDK regulatory protein, similar to that of the native PPDK. The cold tolerance of PPDK in crude leaf extracts was greatly improved in plants that produced a large amount of the engineered PPDK. The photosynthetic rate at 8°C increased significantly (by 23%, p<0.05), but there was no obvious effect at higher temperatures. These results support the hypothesis that PPDK is one of the limiting factors in the C₄ photosynthesis of maize under cold conditions.     

Cell-specific expression of pyruvate, pi dikinase : in situ mRNA hybridization and immunolocalization labeling of protein in wheat seed

Pyruvate, Pi dikinase (PPDK) is a key enzyme in the C4 photosynthetic pathway. However, its metabolic role in C3 plants remains uncertain. Northern blot analyses of PPDK mRNAs from wheat leaves and seeds probed with maize PPDK cDNA indicates the presence of organ-specific mRNAs. Immunofluorescent labeling of protein in wheat seed demonstrate that the PPDK polypeptide and the ribulose-1, 5-bisphosphate carboxylase small subunit polypeptide are localized predominantly in the aleurone layer and the chlorophyllous pericarp tissue, respectively. This differential distribution of the two polypeptides in wheat seed is paralleled by the differential localization of the their mRNAs as revealed by in situ hybridization. These results suggest a distinct role of cytoplasmic PPDK in seeds, which is different from the well established role in C4 photosynthesis.     

Appearance and accumulation of c(4) carbon pathway enzymes in developing wheat leaves

Soluble protein has been extracted from sections of wheat leaves, from base to tip, and the content of several key enzymes of photosynthetic carbon assimilation in each section has been determined by the protein blot method. In the first leaf, ribulose 1,5-bisphosphate carboxylase (RuBPC) (EC 4.1.1.39) in the basal 0 to 1 centimeter section is about 12% the level in the tip section, whereas phosphoenolpyruvate carboxylase (EC 4.1.1.31) is present in small amounts in the basal section and does not change much in the tip. Pyruvate orthophosphate dikinase (PPDK) (EC 2.7.9.1) first appears in the 4 to 6 centimeter section and increases gradually with development to 10-fold in the tip. Malic enzyme, NADP-dependent (EC 1.3.1.37) also appears in the 4 to 6 centimeter section but remains low to the tip.

Fixation of ¹⁴CO₂ by wheat leaf base sections resulted in 42% of total incorporation into malate and aspartate, indicating β-carboxylation, whereas in the tip section these labeled compounds were only 8% of the total. Although the amount of PPDK in wheat leaves is only 1 to 3% of that in maize leaves, this C₃ PPDK may have a limited role in photosynthesis leading to formation of C₄ compounds. The possibility of a further role, similar to that in C₄ plants, but for intracellular carbon transport in wheat leaves is discussed. The presence of malic dehydrogenase, NADP-specific (EC 1.1.1.82) in wheat leaf chloroplasts was shown, a necessary though not sufficient condition for such a proposed role. Assuming each of the four enzymes associated with C₄ carbon transport were fully active in vivo during photosynthesis, PPDK would still be rate limiting, even in the leaf tip where its activity is maximal. Possible evolutionary and breeding implications are discussed.

     

Immunological Studies on Pyruvate Orthophosphate Dikinase in C3 Plants

Pyruvate orthophosphate dikinase (PPDK) was detected in someC₃ plants, wheat, barley, rice and tobacco, by protein blottingusing an antibody against maize PPDK, although the amounts weremuch lesser than those of C₄ plants. The PPDK activity in immaturegrains of rice was specifically immunoprecipitated by the anti-(maize)PPDK antibody. The molecular weight of the subunit of PPDK inall tested C₃ plants was similar (ca. 95 kD) to that of maizePPDK, and the fragment patterns of the C₃ PPDKs in peptide mappingwere also similar to that of maize PPDK. These results suggestthat C₃ PPDKs have a primary structure similar to that of maizePPDK. In order to obtain information about the expression of PPDKin C₃ plants, changes in the enzyme activity and in the amountof PPDK protein were investigated during the greening of riceseedlings. PPDK, which was found in the etiolated seedlings,decreased temporarily in an early stage of greening and thenincreased. The mechanism of this variation is discussed. ¹ To whom correspondence should be addressed. (Received December 9, 1986; Accepted March 12, 1987)     

Electrophoretic transfer as a technique for the detection and identification of plant amylolytic enzymes in polyacrylamide gels

Polyadenylated RNA was isolated from leaves and seeds of a C₃ plant (Triticum aestivum L. cv Cheyenne, CI 8885) and from a C₄ plant (Zea mays L. cv Golden bantam). Each polyadenylated RNA preparation was translated in vitro with micrococcal nuclease-treated reticulocyte lysate. When the in vitro translation products were probed with antibodies to pyruvate orthophosphate dikinase (PPDK) (EC 2.7.9.1), two sizes of polypeptide were identified. A 110 kilodalton polypeptide was found in the in vitro translation products of mRNA isolated exclusively from leaves of both wheat and maize. A 94 kilodalton polypeptide, similar to the PPDK polypeptide which can be extracted after in vivo synthesis in maize and wheat leaves and seeds, was found in the in vitro translation products obtained from wheat seeds and maize kernels.

These results indicate that the mRNAs for PPDK polypeptides are organ-specific in both a C₄ and a C₃ plant. Hague et al. (1983 Nucleic Acids Res 11: 4853-4865) proposed that the larger size polypeptide of the in vitro translation polypeptide from maize leaf RNA contains a `transit sequence' which permits entry into the chloroplasts of a polypeptide synthesized in vivo in maize leaf cell cytoplasm. It appears that in wheat leaves also the transit of synthesized PPDK polypeptide through an intracellular membrane may be required, while such a transit sequence seems not to be required within cells of wheat and maize seeds.

     

Pyruvate orthophosphate dikinase gene expression in developing wheat seeds

The amount of pyruvate orthophosphate dikinase (PPDK) (EC 2.7.9.1) protein in wheat (Triticum aestivum L. var Cheyenne) grains was determined at different stages of development by the protein blot method. The variation in PPDK protein with time in developing wheat grains was similar to that of the enzyme's activity reported by Meyer et al. (1982 Plant Physiol 69: 7-10). The variation in levels of PPDK mRNA with seed development was determined by analysis of polypeptides immunoprecipitated by anti-PPDK serum from in vitro translation products of extracted seed RNA. This mRNA variation was similar to that of the in vivo enzyme levels and the correlation is consistent with the regulation of PPDK gene expression by the level of its mRNA.

The highest level of PPDK in developing wheat seeds occurs later than the highest levels of both ribulose bisphosphate carboxylase (EC 4.1.1.39) and of chlorophyll, which are located in the green pericarp tissue. PPDK was located in both endosperm and pericarp tissue of the seeds. The tissue location and developmental profile of seed PPDK are consistent with a metabolic role of providing phosphoenolpyruvate as a substrate for recapturing respiratory CO₂ in the seed, and possibly for amino acid interconversions during development.

     

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.     

In vitro phosphorylation of maize leaf phosphoenolpyruvate carboxylase

Autoradiography of total soluble maize (Zea mays) leaf proteins incubated with ³²P-labeled adenylates and separated by denaturing electrophoresis revealed that many polypeptides were phosphorylated in vitro by endogenous protein kinase(s). The most intense band was at 94 to 100 kilodaltons and was observed when using either [γ-³²P]ATP or [β-³²P]ADP as the phosphate donor. This band was comprised of the subunits of both pyruvate, Pi dikinase (PPDK) and phosphoenolpyruvate carboxylase (PEPCase). PPDK activity was previously shown to be dark/light-regulated via a novel ADP-dependent phosphorylation/Pi-dependent dephosphorylation of a threonyl residue. The identity of the acid-stable 94 to 100 kilodalton band phosphorylated by ATP was established unequivocally as PEPCase by two-dimensional gel electrophoresis and immunoblotting. The phosphorylated amino acid was a serine residue, as determined by two-dimensional thin-layer electrophoresis. While the in vitro phosphorylation of PEPCase from illuminated maize leaves by an endogenous protein kinase resulted in a partial inactivation (~25%) of the enzyme when assayed at pH 7 and subsaturating levels of PEP, effector modulation by l-malate and glucose-6-phosphate was relatively unaffected. Changes in the aggregation state of maize PEPCase (homotetrameric native structure) were studied by nondenaturing electrophoresis and immunoblotting. Enzyme from leaves of illuminated plants dissociated upon dilution, whereas the protein from darkened tissue did not dissociate, thus indicating a physical difference between the enzyme from light- versus dark-adapted maize plants.     

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.     

Appearance and accumulation of c(4) carbon pathway enzymes in developing maize leaves and differentiating maize a188 callus

Regenerating maize A188 tissue cultures were examined for the presence of enzymes involved in C₄ photosynthesis, for cell morphology, and for ¹⁴C labeling kinetics to study the implementation of this pathway during plant development. For comparison, sections of maize seedling leaves were examined. Protein blot analysis using antibodies to leaf enzymes showed a different profile of these enzymes during the early stages of shoot regeneration from callus from the closely-coordinated profile observed in seedling leaves. Pyruvate orthophosphate dikinase (PPDK) (EC 2.7.9.1) and phosphoenolpyruvate carboxylase (PEPC) (EC 4.1.1.31) were found in nonchlorophyllous callus while ribulose 1,5-bisphosphate carboxylase (RuBPC, EC 4.1.1.39) and malic enzyme, NADP-specific (ME-NADP) (EC 1.3.1.37) were not detectable until later.

Enzyme activity assays showed the presence of ME-NADP as well as PEPC and PPDK in nonchlorophyllous callus. However, the activities of ME-NADP and PEPC had properties similar to those of the enzymes from C₃ leaves and from etiolated C₄ leaf tissues, but differing from the corresponding enzymes in the mature leaf.

Immunoprecipitation of in vitro translation products of poly(A)RNA extracted from embryoid-forming callus showed both the 110 kilodalton precursor to chloroplast PPDK and the 94 kilodalton polypeptide. Therefore, the chloroplast tye of PPDK mRNA is present prior to the appearance of leaf morphology.

Analysis of the labeled products of ¹⁴CO₂ fixation by nonchlorophyllous calli indicated β-carboxylation to give acids of the tricarboxylic acid cycle, but no incorporation into phosphoglycerate. With greening of the callus, some incorporation into phosphoglycerate and sugar phosphates occurred, and this increased in shoots as they developed, although with older shoots the increase in β-carboxylation products was even greater. Analysis of enzyme levels in young leaf sections by protein blot and of ¹⁴C-labeling patterns in the present study are in general agreement with enzyme activity determinations of previous studies, providing additional information about PPDK levels, and supporting the model proposed for developing young leaves.

These results suggest that maize leaves begin to express C₄ enzymes during ontogeny through several stages from greening and cell differentiation as seen in the callus and then shoot formation, and finally acquire capacity for full C₄ photosynthesis during leaf development concomitant with the development of Kranz anatomy and accumulation of large amounts of enzymes involved in carbon metabolism.

     

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     

Stabilization of pyruvate, pi dikinase regulatory protein in maize leaf extracts

The objective of this study was to determine the biochemical basis for genetic variability in pyruvate,Pi dikinase (PPDK) activity among inbred lines of maize (Zea mays L.). Although in vitro PPDK activity varied more than 5-fold among eight maize inbreds, immunochemical determinations of the proportion of leaf soluble protein as PPDK revealed no significant differences among the inbreds. Genetic differences in the stability of PPDK activity in crude homogenates over 5 hours were not evident, but PPDK from some inbreds could not be activated in vitro. In vitro PPDK activation in crude homogenates could be restored by addition of casein (1% w/v) to homogenization media, and to a lesser extent, by gentle homogenization in a mortar. The major effect of casein appeared to be on processes other than proteolysis, as casein exerted its effects during tissue homogenization, rather than later. During homogenization, PPDK did not lose its ability to undergo in vitro activation; instead, it was instability of the regulatory protein responsible for PPDK activation that was the cause of the lack of PPDK activation in homogenates prepared without casein.     

Further analysis of maize C(4) pyruvate,orthophosphate dikinase phosphorylation by its bifunctional regulatory protein using selective substitutions of the regulatory Thr-456 and catalytic His-458 residues

In C(4) plants such as maize, pyruvate,orthophosphate dikinase (PPDK) catalyzes the regeneration of the initial carboxylation substrate during C(4) photosynthesis. The primary catalytic residue, His-458 (maize C(4) PPDK), is involved in the ultimate transfer of the beta-phosphate from ATP to pyruvate. C(4) PPDK activity undergoes light-dark regulation in vivo by reversible phosphorylation of a nearby active-site residue (Thr-456) by a single bifunctional regulatory protein (RP). Using site-directed mutagenesis of maize recombinant C(4) dikinase, we made substitutions at the catalytic His residue (H458N) and at this regulatory target Thr (T456E, T456Y, T456F). Each of these affinity-purified mutant enzymes was assayed for changes in dikinase activity. As expected, substituting His-458 with Asn results in a catalytically incompetent enzyme. Substitutions of the Thr-456 residue with Tyr and Phe reduced activity by about 94 and 99%, respectively. Insertion of Glu at this position completely abolished activity, presumably by the introduction of negative charge proximal to the catalytic His. Furthermore, neither the T456Y nor inactive H458N mutant enzyme was phosphorylated in vitro by RP. The inability of the former to serve as a phosphorylation substrate indicates that RP is functionally a member of the Ser/Thr family of protein kinases rather than a "dual-specificity" Ser-Thr/Tyr kinase, since our previous work showed that RP effectively phosphorylated Ser inserted at position 456. The inability of RP to phosphorylate its native target Thr residue when Asn is substituted for His-458 documents that RP requires the His-P catalytic intermediate form of PPDK as its protein substrate. For these latter studies, synthetic phosphopeptide-directed antibodies specific for the Thr(456)-P form of maize C(4) PPDK were developed and characterized.