Abstract
Plants produce common isoprenoid precursors, isopentenyl diphosphate and dimethylallyl diphosphate (DMAPP), through the methylerythritol phosphate (MEP) pathway in plastids and the mevalonate (MVA) pathway in the cytosol. To assess which pathway contributes to supplying DMAPP to cytokinin (CK) biosynthesis, metabolites from each isoprenoid pathway were selectively labeled with ^<13>C in Arabidopsis seedlings. Efficient ^<13>C-labeling was achieved by blocking the endogenous pathway genetically or chemically during the feed of a ^<13>C-labeled precursor specific to the MEP or MVA pathways. Liquid chromatographymass spectrometry (LC-MS) analysis demonstrated that the prenyl group of trans-zeatin is mainly produced through the MEP pathway in Arabidopsis seedlings. In contrast, the LC-MS data showed that a large fraction of the prenyl group of cis-zeatin derivatives in this system is provided by the MVA pathway. When expressed as fusion proteins with green fluorescent protein (GFP) in Arabidopsis cells, four isopentenyl transferases (IPTs) that utilize AMP/ADP/ATP as substrates were found in plastids in agreement with the idea that the MEP pathway primarily provides DMAPP to the biosynthesis of trans-zeatin. On the other hand, two (putative) GFP-IPTs with tRNA : DMAPP transferase activity were detected in the cytosol and mitochondoria. Because the prenylated adenine moiety of tRNA is usually cis-zeatin-type, but not trans-zeatin-type, a major route for the formation cis-zeatin in Arabidopsis seedlings might involve the transfer of DMAPP from the MVA pathway to tRNA in the cytosol. Distinct origins of DMAPP for the biosynthesis of cis- and trans-zeatin suggest that plants are able to modulate the level of these CK species independently.
