Jean-Noël Pierre

Metabolite control of Sorghum C4 phosphoenolpyruvate carboxylase catalytic activity and phosphorylation state

Kinetic analyses were performed on the nonphosphorylated and in vitro phosphorylated forms of recombinant Sorghum C4 phospho enolpyruvate carboxylase (C4 PEPC). The native enzyme was purified by immunoaffinity chromatography and its integrity demonstrated by Western blot analyses using anti N- and C-terminus antibodies. At suboptimal pH (7.1 to 7.3) and PEP concentration (2.5 mM), phosphorylation, positive metabolite effectors e.g., glucose-6-phosphate, glycine and dihydroxyacetone-phosphate, or an increase in pH strongly activated the enzyme and lowered the inhibitory effect of L-malate. C4 PEPC phosphorylation strengthened the effect of the positive effectors thereby decreasing further the enzymes sensitivity to this inhibitor. L-malate also decreased the phosphorylation rate of C4 PEPC, a process antagonized by positive metabolite effectors. This was shown both in vitro, in a reconstituted phosphorylation assay containing the catalytic subunit of a cAMP-dependent protein kinase or the Sorghum leaf PEPC-PK and in situ, during induction of C4 PEPC phosphorylation in mesophyll cell protoplasts.

In-situ evidence for the involvement of calcium and bundle-sheath-derived photosynthetic metabolites in the C4 phosphoenolpyruvate-carboxylase kinase signal-transduction chain

Regulation of the light activation of C4 phosphoenolpyruvate-carboxylase (PEPC) protein kinase (PEPC-PK) and the ensuing phosphorylation of its cytosolic target protein were studied in intact mesophyll cells (MC) and protoplasts (MP) isolated from dark-adapted leaves of Digitaria sanguinalis [L.] Scop, (hairy crabgrass). The apparent in-situ phosphorylation state of PEPC (EC 4.1.1.31) was assessed by the sensitivity of its activity in desalted MC- and MP-extracts to l-malate under suboptimal assay conditions, while the activity-state of PEPC-PK was determined by in-vitro 32P-labeling of purified maize or recombinant sorghum PEPC by these extracts. In-situ pretreatment of intact MC at pH 8.0 by illumination and calcium addition led to significant decreases in PEPC malate sensitivity and increases in PEPC-kinase activity that were negated by the addition of EGTA to the external cell medium. Similarly, in-situ pretreatment of MP with light plus NH4Cl at pH 7.6 led to significant decreases in malate sensitivity which did not occur when a Ca2+ ionophore and EGTA were included in the suspension medium. In contrast, neither EGTA nor exogenous Ca2+ had a major direct effect on the in-vitro activity of PEPC-PK extracted from Digitaria MC and MP. Preincubation of intact MC with 5 mM 3-phosphoglycerate or pyruvate at pH 8.0 in the dark led to significant decreases in PEPC malate sensitivity and increases in PEPC-PK activity which were not observed with various other exogenous metabolites. These collective in-situ experiments with isolated C4 MC and MP (i) support our earlier hypothesis that alkalization of cytosolic pH is involved in the PEPC-PK signal-transduction cascade (see J.-N. Pierre et al., Eur J Biochem, 1992,210: 531–537), (ii) suggest that intracellular calcium is involved in the PEPC-kinase signal-transduction chain, but at a step upstream of PEPC-PK per se, and (iii) provide direct evidence that the bundle-sheath-derived, C4-pathway intermediates 3-PGA and/or pyruvate also play a role in this signal-transduction cascade which ultimately effects the up-regulation of PEPC in the C4 mesophyll cytosol.

In Situ C4 Phosphoenolpyruvate Carboxylase Activity and Kinetic Properties in Isolated Digitaria Sanguinalis Mesophyll Cells

Isolated mesophyll cells from darkened leaves of the C4 plant Digitaria sanguinalis keep functional plasmodesmata that allow the free exchange of low molecular mass compounds with the surrounding medium. This cell suspension system has been used to measure C4 PEPC activity in situ using a spectrophotometric assay. Compared to the extracted enzyme assayed in vitro, the essentially non-phosphorylated ‘in-cell’ C4 PEPC showed altered functional and regulatory properties. While the S0.5 for PEP at pH 7.3 was only modestly changed (0.4–0.6 mM), the response to pH was shifted towards the acidic range, being close to the maximal value at pH 7.3. Using expected physiological concentrations of the metabolites, at pH 7.3, the IC50 for malate showed a five-fold increase, from 1.5 to 8 mM, and was increased further to 22 mM in the presence of the allosteric activator glucose-6-phosphate (4 mM). Thiol compounds like DTT, mercaptoethanol and reduced glutathione weakened the in-situ sensitivity of C4 PEPC to malate. However, none of them had any effect on this process in vitro. This was not due to thioredoxin-mediated or phoshorylation-dependent processes. Since glutathione is a physiological compound that is present mostly in the reduced state in the cell cytosol, a possible contribution of this thiol to the protection of the enzyme against malate in situ is proposed.

Role of the phosphoinositide pathway in the light-dependent C4 phosphoenolpyruvate carboxylase phosphorylation cascade in Digitaria sanguinalis protoplasts.

Stimulus-response coupling in animal cells frequently involves the hydrolysis of PtdIns(4,5)P(2) which is catalysed by phosphoinositide-specific phospholipase C (PI-PLC). There is an increasing body of evidence for PI-PLC-based signalling in plant cells; however, the physiological role of this system remains poorly documented in plants. Our data provide the first evidence that a PI-PLC-based signalling system is a committed step in the transduction chain controlling the phosphorylation state of C(4) phosphoenolpyruvate carboxylase (PEPC), the regulation of which is central to the assimilation of atmospheric CO(2) in C(4) plants.

The Regulatory Phosphorylation of C4 Phosphoenolpyruvate Carboxylase: a Cardinal Event in C4 Photosynthesis

Phosphoenolpyruvate (PEP) carboxylase (EC 4.1.1.31, PEPC) is a widely distributed enzyme in plant tissues (embryophytes), green algae (chlorobionts) and microorganisms, but it is not present in animals (O’Leary, 1982; Andreo et al., 1987). It catalyses the exergonic β-carboxylation of PEP (ΔG = −7kcal/mol) in the presence of a bivalent cation, generally Mg2+:

Reversible Phosphorylation in the Regulation of Photosynthetic Phosphoenolpyruvate Carboxylase in C4 Plants

C4 species have a specific isoform of phosphoenolpyruvate Carboxylase (PEPC) that catalyzes primary CO2 fixation in the C4 photosynthesis pathway. It has long been known that the enzyme in the cytosol of the mesophyll cells is subject to allosteric control by opposing photosynthesis-related metabolites. The discovery of a phosphorylation process acting on C4 PEPC, via a complex light-signal transduction cascade, has revitalized interest in this enzyme and the ensuing wealth of data has highlighted one of a few signaling cascades known so far in the regulation of plant metabolism. The cascade depends upon a cross-talk between the two neighboring photosynthetic cell types, involves classical second messengers like pH, Inosital-1,4 5-trisphosphate (Ins(1,4,5) P3) and calcium, and upregulates the activity of a Ca2+-independent, C4 PEPC-specific protein-serine/threonine kinase, which finally phosphorylates PEPC. The final activity of C4 PEPC and the resulting carbon flux to bundle sheath cells are dependent on the mutual interaction between metabolite and covalent control mechanisms acting on this enzyme.

Light-Dependent Regulatory Mechanisms Acting on Photosynthetic Phosphoenolpyruvate Carboxylase in C 4 Plants

Phosphoenolpyruvate carboxylase (EC 4.1.1.31; PEPC) catalyzes the β-carboxylation of PEP by HCO3− in the presence of a divalent cation. PEPC isozymes are widely distributed in plant tissues in which they are involved in a variety of physiological contexts. In C4 plants, a specific isoform (C4 PEPC) plays a key role in the primary CO2 fixation in the photosynthesis pathway. C4 PEPC is subject to a light-dependent transcriptional control resulting in the accumulation of high amounts of the corresponding protein in the mesophyll cell cytosol during greening of the etiolated C3 leaf. In vitro studies have shown that the enzyme activity is regulated by photosynthesis-related metabolites, e.g., feedback inhibition by L-malate and allosteric activation by sugar-P. During the last decade, a posttranslational process, i.e., reversible phosphorylation, acting on C4 PEPC has been established and shown to modulate both its functional and regulatory properties. The main purpose of the work reported here was to explore the light-dependent mechanisms which control the biosynthesis and covalent modification of C4 PEPC.

Molecular basis of plant adaptation to light. Example of two enzymes of the C4 photosynthesis cycle

Abstract The light-dependent mechanisms responsible for the regulation of two enzymes of the C4 photosynthetic cycle, phosphoenolpyruvate carboxylase and NADP-dependent malate dehydrogenase in Sorghum are presented as an illustration of adaptative mechanisms of plants to light. Emphasis is put on the organization of the signaltransduction chains which upregulate both enzymes at transcriptional and post-translational levels. For both enzymes, small gene families include a photosynthesis-related gene, the expression of which is light-triggered. The light-controlled phosphorylation of C4 PEPC implies cross-talk between photosynthetic cell types, cytosolic pH and calcium changes, and the upregulation of a specific protein-serine kinase. This post-translational modification strongly influences its regulation by photosynthesis-related metabolites. NADP-MDH activity is controlled by a chloroplastic redox cascade involving thioredoxin as the ultimate relay. The significance of these regulatory circuits is discussed in relation with the flexibility of the metabolism in the context of the C4 photosynthesis.