Poster: Metabolic Engineering

Abs # 1011: Interactions between the cytoplasmic mevalonate and the plastidial methylerythritol phosphate pathways in tobacco BY-2 cells.

Presenter:	Hemmerlin, Andréa , Andrea.Hemmerlin@ibmp-ulp.u-strasbg.fr
Authors	Hemmerlin, Andréa  (A)   Gerber, Esther  (A)   Hoeffler, Jean-François  (B)   Tritsch, Denis  (B)   Rohmer, Michel  (B)   Bach, Thomas J. (A)
Affiliations:	(A): CNRS (UPR 2357), Institut de Biologie Moléculaire des Plantes, F-67083 Strasbourg (B): Univ. Louis Pasteur/CNRS, Institut Le Bel, 4 rue Blaise Pascal, F-67070 Strasbourg, France

In plants, two pathways are utilized for the synthesis of isopentenyl diphosphate (IPP), the universal precursor for isoprenoid biosynthesis. The key enzyme of the cytosolic mevalonic acid (MVA) pathway is 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGR). Treatment of tobacco Bright Yellow-2 (TBY-2) cells by the HMGR inhibitor mevinolin led to growth reduction and induction of apparent HMGR activity, in parallel to an increase in protein of two HMGR isozymes. Maximum induction was observed at 24 hours. 1-Deoxy-D-xylulose (DX), the dephosphorylated first precursor of the plastidial 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway complemented growth inhibition by mevinolin in the low mM concentration range. Further, DX partially re-established feedback repression of mevinolin-induced HMGR activity. Incorporation studies with [1,1,1,4-²H₄]DX showed that in the presence of mevinolin, sterols, normally derived from MVA, are synthesized using the MEP pathway. Growth inhibition of TBY-2 cells by fosmidomycin, an inhibitor of 1-deoxy-D-xylulose-5-phosphate reductoisomerase, the second enzyme of the MEP pathway, could be partially overcome by MVA. Chemical complementation was further substantiated by incorporation of [2-¹³C]MVA into plastoquinone, representative of plastidial isoprenoids. Best incorporation of precursors was observed in the presence of both inhibitors, thereby avoiding internal isotope dilution. TBY-2 cells expressing various green fluorescent protein (GFP) fusions with a C-terminal domain of a rice calmodulin bearing a CaaX isoprenylation motif were used to visualize their intracellular localization, dependent on either MVA or MEP biosynthesis. They provide useful tools to study in situ exchange between cytoplasmic and plastidial isoprenoid precursors.