Molecular and functional characterization of the plastid-localized Phosphoenolpyruvate enolase (ENO1) from Arabidopsis thaliana

Abstract 

The Arabidopsis thaliana gene At1g74030 codes for a putative plastid phosphoenolpyruvate (PEP) enolase (ENO1). The recombinant ENO1 protein exhibited enolase activity and its kinetic properties were determined. ENO1 is localized to plastids and expressed in most heterotrophic tissues including trichomes and non-root-hair cells, but not in the mesophyll of leaves. Two T-DNA insertion eno1 mutants exhibited distorted trichomes and reduced numbers of root hairs as the only visible phenotype. The essential role of ENO1 in PEP provision for anabolic processes within plastids, such as the shikimate pathway, is discussed with respect to plastid transporters, such as the PEP/phosphate translocator.

Abbreviations: BCIP, 5-bromo-4-chloro-3′-indolyl phosphate, BSA, bovine serum albumin, CAM, crassulacean acid metabolism, CaMV, cauliflower mosaic virus, CDS, coding sequence, cue, chlorophyll a/b binding protein underexpressed, DPGA, diphosphoglyceric acid, ENO, enolase, GAPDH, glyceraldehyde 3-phosphate dehydrogenase, GFP, green fluorescent protein, Glc6P, glucose 6-phosphate, GPT, glucose 6-phosphate/phosphate translocator, GUS, β-glucouronidase, IgG, immunoglubulin G, IPTG, isopropyl-β-d-thiogalactopyranoside, LDH, lactate dehydrogenase, MEP, 2-C-methyl-d-erythritol 4-phosphate, NBT, nitroblue tetrazolium chloride, Ni–NTA, Ni–nitrilotriacetic acid, OPPP, oxidative pentose phosphate pathway, PCR, polymerase chain reaction, PEP, phosphoenolpyruvate, PEPCK, phosphoenolpyruvate carboxykinase, PFD, photon flux density, PGA, phosphoglyceric acid, PGK, phosphoglycerate kinase, PGyM, phosphoglycerate mutase, PK, pyruvate kinase, PPDK, pyruvate, orthophosphate dikinase, PPT, phosphoenolpyruvate/phosphate translocator, RT-PCR, Reverse transcriptase polymerase chain reaction, SDS–PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis, TPT, triose phosphate/phosphate translocator, UTR, untranslated region

Keywords: Plant glycolysis, Plastid enolase, Phosphoenolpyruvate, Enzyme kinetics, Trichomes, Phosphate translocator