Identification of phosphoenolpyruvate carboxylase isoforms in leaf, stem and roots of the obligate CAM plant Vanilla planifolia Salib. (Orchidaceae): a physiological and molecular approach

Abstract

This study provides the first comparative analysis of phosphoenolpyruvate carboxylase isoforms (PEPc; EC 4.1.1.31) in an obligate crassulacean acid metabolism (CAM) plant, Vanilla planifolia Salisb. (Orchidaceae). Nocturnal CO2 fixation and malate accumulation by the leaves and the green stem show that these organs perform CAM. The chloroplast-containing aerial roots, however, exhibit C3 photosynthesis. The catalytic activity of PEPc was highest in the leaves compared with the stem and aerial roots. The Km (PEP) and Ki (malate) were similar in the PEPc extracted from leaf and aerial roots, and significant higher in stem. cDNA was obtained from those tissues and also from the soil-grown roots, and various cDNA clones were detected and amplified by means of RT-PCR and RACE-PCR. The amino-acid sequences of the PEPc isoforms deduced from the cDNA showed a great degree of homology, and Southern blot analysis suggests that the encoding genes form a small multigene family of at least two members. One PEPc isoform (PpcV1) is assumed to be related to CAM because, as shown by northern blot analysis, it is mainly expressed in the CAM-performing organs, i.e. in the leaves and the stem. A further isoform (PpcV2) was identified in the soil-grown roots and aerial roots, but northern blots show that to some extent PpcV2 is also expressed in the leaf and the stem tissues. Thus, it is assumed that PpcV2 encodes the housekeeping isoform of PEPc. Altogether, the present study provides support in favour of the view that isoforms of PEPc are related to specific functions.