Cristina Echevarría

PROTEIN TURNOVER AS A COMPONENT IN THE LIGHT/DARK REGULATION OF PHOSPHOENOLPYRUVATE CARBOXYLASE PROTEIN-SERINE KINASE ACTIVITY IN C4 PLANTS

Maize leaf phosphoenolpyruvate carboxylase [PEPC; orthophosphate:oxaloacetate carboxy-lyase (phosphorylating), EC 4.1.1.31] protein-serine kinase (PEPC-PK) phosphorylates serine-15 of its target enzyme, thus leading to an increase in catalytic activity and a concomitant decrease in malate sensitivity of this cytoplasmic C4 photosynthesis enzyme in the light. We have recently demonstrated that the PEPC-PK activity in maize leaves is slowly, but strikingly, increased in the light and decreased in darkness. In this report, we provide evidence that cycloheximide, an inhibitor of cytoplasmic protein synthesis, when fed to detached leaves of C4 monocots (maize, sorghum) and dicots (Portulaca oleracea) in the dark or light, completely prevents the in vivo light activation of PEPC-PK activity regardless of whether the protein kinase activity is assessed in vivo or in vitro. In contrast, chloramphenicol, an inhibitor of protein synthesis in chloroplasts, has no effect on the light activation of maize PEPC-PK. Similarly, treatment with cycloheximide did not influence the light activation of other photosynthesis-related enzymes in maize, including cytoplasmic sucrose-phosphate synthase and chloroplast stromal NADPH-malate dehydrogenase and pyruvate, Pi dikinase. These and related results, in which detached maize leaves were treated simultaneously with cycloheximide and microcystin-LR, a potent in vivo and in vitro inhibitor of the PEPC type 2A protein phosphatase, indicate that short-term protein turnover of the PEPC-PK itself or some other essential component(s) (e.g., a putative protein that modifies this kinase activity) is one of the primary levels in the complex and unique regulatory cascade effecting the reversible light activation/seryl phosphorylation of PEPC in the mesophyll cytoplasm of C4 plants.

Reversible light activation of the phosphoenolpyruvate carboxylase protein-serine kinase in maize leaves

C 4‐leaf phosphoenolpyruvate carboxylase (PEPC; EC 4.1, 1.31) undergoes reversible, light‐induced increases in its activity—seryl phosphorylation‐status in vivo. We now report that the PEPC‐protein kinase activity in desalted crude extracts of light‐adapted maize leaves is several‐fold greater than that from the corresponding dark tissue when in vitro phosphorylation assays are performed with either endogeneous or purified dark‐form maize PEPC as substrate, both in the absence or presence of okadaic acid, a potent inhibitor of the PEPC type 2A protein phosphatase(s). These and related results indicate that the PEPC protein‐serine kinase(s) per se is reversibly light activated in vivo by either covalent modification, protein turnover, less likely, a tight‐binding effector.

In Vivo and in Vitro Phosphorylation of the Phosphoenolpyruvate Carboxylase from Wheat Seeds during Germination

Phosphoenolpyruvate carboxylase (PEPC) activity was detected in the aleurone endosperm of wheat (Triticum aestivum cv Chinese Spring) seeds, and specific anti-Sorghum C4 PEPC polyclonal anti-bodies cross-reacted with 103- and 100-kD polypeptides present in dry seeds and seeds that had imbibed; in addition, a new, 108-kD polypeptide was detected 6 h after imbibition. The use of specific anti-phosphorylation-site immunoglobulin G (APS-IgG) identified the presence of a phosphorylation motif equivalent to that found in other plant PEPCs studied so far. The binding of this APS-IgG to the target protein promoted changes in the properties of seed PEPC similar to those produced by phosphorylation, as previously shown for the recombinant Sorghum leaf C4 PEPC. In desalted seed extracts, an endogenous PEPC kinase activity catalyzed a bona fide phosphorylation of the target protein, as deduced from the immunoinhibition of the in vitro phosphorylation reaction by the APS- IgG. In addition, the major, 103-kD PEPC polypeptide was also shown to be radiolabeled in situ 48 h after imbibition in [32P]orthophosphate. The ratio between optimal (pH 8) and suboptimal (pH 7.3 or 7.1) PEPC activity decreased during germination, thereby suggesting a change in catalytic rate related to an in vivo phosphorylation process. These collective data document that the components needed for the regulatory phosphorylation of PEPC are present and functional during germination of wheat seeds.

Regulatory phosphorylation of C4 phosphoenolpyruvate carboxylase from Sorghum: An immunological study using specific anti-phosphorylation site-antibodies

A peptide containing the N-terminal phosphorylation site (Ser-8) of Sorghum C4-phospho enolpyruvate carboxylase (PEPC) was synthesized, purified and used to raise an antiserum in rabbits. Affinity-purified IgGs prevented PEPC phosphorylation in a reconstituted in vitro assay and reacted with both the phosphorylated and dephosphorylated forms of either native or denatured PEPC in immunoblotting experiments. Saturation of dephospho-PEPC with these specific IgGs resulted in a marked alteration of its functional and regulatory properties that mimicked phosphorylation of Ser-8. A series of recombinant C4 PEPCs mutated in the N-terminal phosphorylation domain and a C3-like PEPC isozyme from Sorghum behaved similarly to their C4 counterpart with respect to these phosphorylation-site antibodies.

The phosphorylation of Sorghum leaf phosphoenolpyruyate carboxylase is a Ca++-calmodulin dependent process

Regulation of the in vitro phosphorylation process of the photosynthetic form (G form) of Sorghum leaf Phosphoenolpyruvate carboxylase (PEPC: EC 4.1.1.31) was studied. Results established that: 1) PEPC was efficiently phosphorylated on seryl residues in crude leaf extract 2) Pyruvate, orthophosphate dikinase (EC 2.7.9.1.) which has been supposed to interfere with the process, was found not to be significantly phosphorylated in our experimental conditions 3) KF, as well as both Ca++ and Mg++ ions increased the radioactive signal detected 4) addition of EDTA or EGTA nullified it and Ca++ alone was found to reverse the inhibitory effect exerted by both chelators 5) addition of anti-Calmodulin antibodies to the medium also abolished the PEPC phosphorylation. Present data demonstrated that the post-translational modification of the C4-plant photosynthetic PEPC is a Ca++/Calmodulin dependent process.

Properties of the in vivo Nitrate Reductase Assay in Maize, Soybean, and Spinach Leaves

Summary The effect of different parameters on the in vivo nitrate reductase activity in leaves of maize, soybean, and spinach has been examined. Leaf sections infiltrated under vacuum with an assay medium containing CO 2 tension and NO - 3 and incubated under air, excreted CO 2 tension and NO - 2 in the dark but not in the light. Nevertheless, addition of 3-(3′,4′-dichlorophenyl)-1,1-dimethylurea, a potent inhibitor of the photosynthetic electron transport, promoted an immediate excretion of CO 2 tension and NO - 2 in the light. Under those conditions, subsequent addition of ascorbate-reduced dichlorophenolindophenol or N,N,N′,N′-tetramethylphenylenediamine, which can supply electrons to photosystem I, abolished the excretion of CO 2 tension and NO - 2 by the leaf sections. These results support the view that, in green tissues, the reducing equivalents photogenerated in the chloroplasts are more directly used for NO - 2 reduction than for NO - 2 reduction. Incubation of leaf discs under O 2 -atmosphere severely impaired NO - 2 accumulation. Under strict anaerobic conditions, the rate of NO - 2 production was only slightly enhanced as compared with that observed in the assay under air. Exogenous NO - 3 stimulated the activity, more markedly in soybean than in the other two species. Concentrations of exogenous NO - 3 between 50 and 100 mM were required for maximal activities in all the three species. The external pH optimum for in vivo NO - 3 reduction was 7.5 for maize and spinach and 7.0 for soybean. Inclusion of 1% (v/v) n -propanol in the infiltration-incubation medium enhanced the activity about 15%. However, 7% (v/v) n -propanol caused a drastic decrease in the rate of NO - 2 accumulation which could be greatly overcome by inclusion in the medium of NADH or reduced flavin mononucleotide.

The unique phosphoenolpyruvate carboxylase kinase

Abstract This paper deals with the contribution of P. Gadal’s group to the study of the phosphoenolpyruvate carboxylase protein kinase. It traces the important steps from the discovery up to the present time leading to characterize a new protein kinase which is specific for plants and displays original properties.

Regulation of Phosphoenolpyruvate Carboxylase Phosphorylation by Metabolites and Abscisic Acid during the Development and Germination of Barley Seeds

During barley (Hordeum vulgare) seed development, phosphoenolpyruvate carboxylase (PEPC) activity increased and PEPC-specific antibodies revealed housekeeping (103-kD) and inducible (108-kD) subunits. Bacterial-type PEPC fragments were immunologically detected in denatured protein extracts from dry and imbibed conditions; however, on nondenaturing gels, the activity of the recently reported octameric PEPC (in castor [Ricinus communis] oil seeds) was not detected. The phosphorylation state of the PEPC, as judged by l-malate 50% inhibition of initial activity values, phosphoprotein chromatography, and immunodetection of the phosphorylated N terminus, was found to be high between 8 and 18 d postanthesis (DPA) and during imbibition. In contrast, the enzyme appeared to be in a low phosphorylation state from 20 DPA up to dry seed. The time course of 32/36-kD, Ca2+-independent PEPC kinase activity exhibited a substantial increase after 30 DPA that did not coincide with the PEPC phosphorylation profile. This kinase was found to be inhibited by l-malate and not by putative protein inhibitors, and the PEPC phosphorylation status correlated with high glucose-6-phosphate to malate ratios, thereby suggesting an in vivo metabolic control of the kinase. PEPC phosphorylation was also regulated by photosynthate supply at 11 DPA. In addition, when fed exogenously to imbibing seeds, abscisic acid significantly increased PEPC kinase activity. This was further enhanced by the cytosolic protein synthesis inhibitor cycloheximide but blocked by protease inhibitors, thereby suggesting that the phytohormone acts on the stability of the kinase. We propose that a similar abscisic acid-dependent effect may contribute to produce the increase in PEPC kinase activity during desiccation stages.

Isolation and characterisation of a wheat phosphoenolpyruvate carboxylase gene. Modelling of the encoded protein

Abstract A phosphoenolpyruvate carboxylase gene (referred to as Ppc1 ) was isolated from a wheat genomic library and sequenced in both strands. The deduced protein sequence is encoded by 10 exons and shows extensive similarities to that of other plant PEPCs sequenced so far, with respect to the predicted motives involved in the structure (β-strands and α-helices) and function (active site, inhibitor site, N- and C-terminus) of the enzyme. Modelling of the 3D-structure of the wheat PEPC monomer using the Swiss-Model programme and the Escherichia coli PEPC monomer 3D-structure as a reference revealed that these motives and corresponding key residues share very similar positions in the folded plant and bacterial polypeptides. Interestingly, the 5′-flanking sequence of Ppc1 gene contains various putative regulatory cis -elements including an ABRE box. Relative RT-PCR analysis showed a ubiquitous expression of Ppc1 gene in developing and germinating wheat seeds as well as in developing seedlings.

Reversible inactivation of maize leaf nitrate reductase

Abstract Preincubation of maize leaves crude extracts with NADH resulted in a progressive accumulation of nitrite which mimicked a rapid and lineal activation of nitrate reductase. Nevertheless, in partially purified preparations it was found that preincubation at pH 8.8 with NADH promoted a gradual inactivation of nitrate reductase. At pH 7.5, the enzyme was not inactivated by the presence of NADH alone, but, with the simultaneous presence of a low concentration of cyanide, a fast inactivation took place. The NADH-cyanide-inactivated nitrate reductase remained inactive after removing the excess of NADH and cyanide by filtration through Sephadex G-25. However, it could be readily reactivated by incubation with ferricyanide or by a short exposure to light in the presence of FAD. Prolonged irradiation caused a progressive inactivation of the photoreactivated enzyme.