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Kinetic characterization of phosphoenolpyruvate carboxylase extracted from whole-leaf and from guard-cell protoplasts of Vicia faba L. (C3 plant) with respect to tissue pre-illumination

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Summary

Whole leaves and guard-cell protoplasts of the C3 plant Vicia faba L. (broad bean) were separately extracted following a period of illumination or following a period of darkness. Kinetic parameters of phosphoenolpyruvate carboxylase (PEPC, EC 4.1.1.31), Vmax and Km (PEP · Mg), were determined as a function of assay pH (7.0 or 8.1), the presence of 5 mm glucose-6-Pfree (Glc-6-P, an activator), and the presence of 5 mm malatefree (an inhibitor). On the basis of these parameters, guard-cell PEPC was distinguished from that of whole leaf, indicating either that guard cells contain a unique isoenzyme of PEPC or a different complement of isoenzymes or - and less likely - that the obligatorily different methodologies for the leaf (intact organ) and the guard-cell (protoplast) enzymes altered them specifically.

The values of Vmax were relatively unchanged, regardless of assay conditions or tissue pretreatment. The values obtained for whole-leaf PEPC Vmax were restricted to a small range (52.4 ± 5.9 (SD) to 64.4 ± 4.8 (SD) μmol · g fresh mass-1 · h-1; the high value coincided with the presence of Glc-6-P, and the low value was obtained in the presence of malate. Guard-cell PEPC Vmax was also restricted to a small range: 7.48 ± 0.89 (SD) pmol · guard-cell pair-1 · h-1 (pH 8.1, light, +Glc-6-P) to 5.79 ± 0.60 (SD) pmol · guard-cell pair-1 · h-1 (pH 7.0, dark, +malate). Depending on effectors, and particularly pH, large changes in Km (PEP · Mg) were calculated (whole-leaf PEPC: 0.03 to 3.84 mm; guard-cell PEPC: 0.06 to 3.43 mm). For both extracts, the low values were obtained at pH 8.1, +Glc-6-P, and the high values at pH 7.0, +malate. Although the ranges of Km values were broadly similar, the PEPCs reacted differently to individual changes in assay components. In very general terms, whole-leaf PEPC was relatively more efficient at pH 8.1, whereas at pH 7.0, the enzymes behaved more similarly.

An effect of in vivo pre-illumination on guard-cell PEPC was not detected. A leaf pre-illumination effect on whole-leaf PEPC was highly statistically significant when assayed under control conditions at pH 7.0. The effect was small - typically a 26% decrease in Km (PEP · Mg) this typical decrease was less than the range of values in replicate experiments. Such a small pre-illumination effect (even if real) could, therefore, easily go undetected. Whether such a small change could have physiological relevance is an open question. Neither with the whole-leaf PEPC nor with the guard-cell PEPC was the IC50 (malate) or A0.5 (Glc-6-P) determined for any condition. These kinetic parameters are a focus of present work.

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  1. Xue-Chen Wang

    Present address: College of Biology, Beijing Agricultural University, 100094, Beijing, People's Republic of China

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  1. Department of Biological Science, Biology Unit I, Florida State University, 32306-3050, Tallahassee, FL, USA

    Xue-Chen Wang, William H. Outlaw Jr, Jean André De Bedout & Zhirong Du

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Wang, XC., Outlaw, W.H., De Bedout, J.A. et al. Kinetic characterization of phosphoenolpyruvate carboxylase extracted from whole-leaf and from guard-cell protoplasts of Vicia faba L. (C3 plant) with respect to tissue pre-illumination. Histochem J 26, 152–160 (1994). https://doi.org/10.1007/BF00157964

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