Light Regulation of the Photosynthetic Phosphoenolpyruvate Carboxylase (PEPC) in Hydrilla verticillata

摘要

The submersed monocot, Hydrilla verticillata (L.f.) Royle, is a facultative C₄ NADP-malic enzyme (NADP-ME) plant in which the C₄ and Calvin cycles co-exist in the same cell. Futile cycling is avoided by an intracellular separation of carboxylases between the cytosol and chloroplasts. Of the two sequenced H. verticillata phosphoenolpyruvate carboxylase (PEPC) isoforms, hvpepc3 and hvpepc4, transcript expression of the latter was substantially up-regulated during C₄ induction, especially in the light. Western blots revealed two PEPC-specific bands in C₃ and C₄ leaf extracts; the lower band dominated in the C₄ and underwent post-translational phosphorylation in the light as determined by immunological studies. This band probably represents the photosynthetic isoform, HVPEPC4, despite the lack of the C₄ signature serine (Flaveria residue 774; Hydrilla 779). In C₄ leaves, PEPC activity increased 14-fold, was enhanced by leaf exposure to light, and showed allosteric regulation. Glucose-6-phosphate acted as a positive effector, but malate was inhibitory, with I₅₀ values of 0.4 and 0.2 mM in the light and dark, respectively, similar to those of other C₄ PEPC isoforms. In contrast, in C₃ leaves, transcript expression of both isoforms was weak, with little evidence of diel regulation, and the PEPC proteins showed essentially no indication of phosphorylation. PEPC activity in C₃ leaves was low, light independent and followed Michaelis–Menten kinetics. It was tolerant to malate, with 10-fold higher I₅₀ values than the PEPC from C₄ leaves. These data suggest that hvpepc4 encodes the C₄ photosynthetic PEPC, and hvpepc3 encodes an anaplerotic form.