ABSTRACT
Plants produce the 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 pathways contribute DMAPP for cytokinin 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 chromatography-mass spectrometry analysis demonstrated that the prenyl group of trans-zeatin (tZ) and isopentenyladenine is mainly produced through the MEP pathway. In comparison, a large fraction of the prenyl group of cis-zeatin (cZ) derivatives was provided by the MVA pathway. When expressed as fusion proteins with green fluorescent protein in Arabidopsis cells, four adenosine phosphate-isopentenyltransferases (AtIPT1, AtIPT3, AtIPT5, and AtIPT8) were found in plastids, in agreement with the idea that the MEP pathway primarily provides DMAPP to tZ and isopentenyladenine. On the other hand, AtIPT2, a tRNA isopentenyltransferase, was detected in the cytosol. Because the prenylated adenine moiety of tRNA is usually of the cZ type, the formation of cZ in Arabidopsis seedlings might involve the transfer of DMAPP from the MVA pathway to tRNA. Distinct origins of large proportions of DMAPP for tZ and cZ biosynthesis suggest that plants are able to separately modulate the level of these cytokinin species.
Subject(s)
Arabidopsis/metabolism , Terpenes/metabolism , Zeatin/chemistry , Zeatin/metabolism , Adenine Nucleotides/metabolism , Carbon Isotopes , Cytokinins/metabolism , Erythritol/metabolism , Green Fluorescent Proteins , Luminescent Proteins/genetics , Mevalonic Acid/pharmacokinetics , Pentosephosphates/pharmacokinetics , Plastids/metabolism , Seedlings/metabolism , Transferases/metabolismABSTRACT
The expulsion mechanism of xylitol 5-phosphate in Streptococcus mutans ATCC 25175 was studied using resting cells incubated in the presence of 14C-xylitol. The expulsion appeared to be a two-step process: xylitol 5-phosphate was first hydrolyzed to xylitol and inorganic phosphate, and the xylitol was subsequently expelled from the cells. The dephosphorylation step appeared to be energy-requiring and it was most likely associated with a phosphatase which was active on xylitol 5-phosphate. Two to three successive cultivations of the cells in the presence of 6% xylitol increased this enzyme activity 4.3-fold. These results are in accordance with the presence of an energy-dependent xylitol 5-phosphate cycle in S. mutans, which is regulated by exogenous xylitol.
