Distinct Isoprenoid Origins of cis- and trans-Zeatin Biosyntheses in Arabidopsis*

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 13C in Arabidopsis seedlings. Efficient 13C labeling was achieved by blocking the endogenous pathway genetically or chemically during the feed of a 13C 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.

[1]  R. Amasino,et al.  T-DNA of Agrobacterium tumefaciens encodes an enzyme of cytokinin biosynthesis. , 1984, Proceedings of the National Academy of Sciences of the United States of America.

[2]  Takanori Hirano,et al.  Engineered GFP as a vital reporter in plants , 1996, Current Biology.

[3]  G. Sandberg,et al.  An alternative cytokinin biosynthesis pathway. , 2000, Proceedings of the National Academy of Sciences of the United States of America.

[4]  M. Zenk,et al.  The Tzs protein from Agrobacterium tumefaciens C58 produces zeatin riboside 5′‐phosphate from 4‐hydroxy‐3‐methyl‐2‐(E)‐butenyl diphosphate and AMP , 2002, FEBS letters.

[5]  Y. Kamiya,et al.  Contribution of the Mevalonate and Methylerythritol Phosphate Pathways to the Biosynthesis of Gibberellins inArabidopsis * , 2002, The Journal of Biological Chemistry.

[6]  E. Nambara,et al.  A Unique Short-Chain Dehydrogenase/Reductase in Arabidopsis Glucose Signaling and Abscisic Acid Biosynthesis and Functions , 2002, The Plant Cell Online.

[7]  D. Inzé,et al.  Zeatin is indispensable for the G2‐M transition in tobacco BY‐2 cells , 1998, FEBS letters.

[8]  M. Rohmer.  The discovery of a mevalonate-independent pathway for isoprenoid biosynthesis in bacteria, algae and higher plants. , 1999, Natural product reports.

[9]  J. Giner.  NEW AND EFFICIENT SYNTHETIC ROUTES TO 1-DEOXY-D-XYLULOSE , 1998 .

[10]  H. Ohigashi,et al.  Biosynthesis of Abscisic Acid by the Non-mevalonate Pathway in Plants, and by the Mevalonate Pathway in Fungi , 2000, Bioscience, biotechnology, and biochemistry.

[11]  A. Golovko,et al.  Identification of a tRNA isopentenyltransferase gene from Arabidopsis thaliana , 2002, Plant Molecular Biology.

[12]  M. Mok,et al.  CYTOKININ METABOLISM AND ACTION. , 2003, Annual review of plant physiology and plant molecular biology.

[13]  C. Chen,et al.  Metabolism of Cytokinin : DEPHOSPHORYLATION OF CYTOKININ RIBONUCLEOTIDE BY 5'-NUCLEOTIDASES FROM WHEAT GERM CYTOSOL. , 1981, Plant physiology.

[14]  C. Chen,et al.  Modification of cytokinins by cauliflower microsomal enzymes. , 1984, Plant physiology.

[15]  C. Chen,et al.  Metabolism of cytokinin: deribosylation of cytokinin ribonucleoside by adenosine nucleosidase from wheat germ cells. , 1981, Plant physiology.

[16]  M. Shimizu,et al.  Non-invasive quantitative detection and applications of non-toxic, S65T-type green fluorescent protein in living plants. , 1999, The Plant journal : for cell and molecular biology.

[17]  U. Stahl,et al.  Immunoaffinity co-purification of cytokinins and analysis by high-performance liquid chromatography with ultraviolet-spectrum detection , 1993, Planta.

[18]  N. Bassil,et al.  Partial Purification of a cis-trans-Isomerase of Zeatin from Immature Seed of Phaseolus vulgaris L , 1993, Plant physiology.

[19]  D. Crowell,et al.  Inhibition of growth of cultured tobacco cells at low concentrations of lovastatin is reversed by cytokinin. , 1992, Plant physiology.

[20]  M. Rohmer.  Mevalonate-independent methylerythritol phosphate pathway for isoprenoid biosynthesis. Elucidation and distribution , 2003 .

[21]  H. Sakakibara,et al.  Identification of Genes Encoding Adenylate Isopentenyltransferase, a Cytokinin Biosynthesis Enzyme, inArabidopsis thaliana * , 2001, The Journal of Biological Chemistry.

[22]  W. Eisenreich,et al.  Studies on the nonmevalonate pathway to terpenes: The role of the GcpE (IspG) protein , 2001, Proceedings of the National Academy of Sciences of the United States of America.

[23]  H. Sakakibara,et al.  A method for separation and determination of cytokinin nucleotides from plant tissues , 2003, Journal of Plant Research.

[24]  T. Kakimoto.  Identification of plant cytokinin biosynthetic enzymes as dimethylallyl diphosphate:ATP/ADP isopentenyltransferases. , 2001, Plant & cell physiology.

[25]  A. Hemmerlin,et al.  Cross-talk between the Cytosolic Mevalonate and the Plastidial Methylerythritol Phosphate Pathways in Tobacco Bright Yellow-2 Cells* , 2003, Journal of Biological Chemistry.

[26]  Hartmut K. Lichtenthaler,et al.  THE 1-DEOXY-D-XYLULOSE-5-PHOSPHATE PATHWAY OF ISOPRENOID BIOSYNTHESIS IN PLANTS. , 1999, Annual review of plant physiology and plant molecular biology.

[27]  G. Heijne,et al.  ChloroP, a neural network‐based method for predicting chloroplast transit peptides and their cleavage sites , 1999, Protein science : a publication of the Protein Society.

[28]  A. Hemmerlin,et al.  Mevalonate-derived isopentenyl diphosphate is the biosynthetic precursor of ubiquinone prenyl side chain in tobacco BY-2 cells. , 1998, The Biochemical journal.

[29]  Kazuo Shinozaki,et al.  Identification of CRE1 as a cytokinin receptor from Arabidopsis , 2001, Nature.

[30]  G. Sandberg,et al.  The Arabidopsis AtIPT8/PGA22 Gene Encodes an Isopentenyl Transferase That Is Involved in De Novo Cytokinin Biosynthesis1 , 2003, Plant Physiology.

[31]  T. Mizuno,et al.  The Arabidopsis AHK4 histidine kinase is a cytokinin-binding receptor that transduces cytokinin signals across the membrane. , 2001, Plant & cell physiology.

[32]  A. Piaggesi,et al.  Cytokinin biosynthesis in endosperm of Sechium edule Sw. , 1997 .

[33]  Chong-maw Chen,et al.  Biosynthesis of N6-(Δ2-isopentenyl)adenosine in the transfer ribonucleic acid of cultured tobacco pith tissue , 1969 .

[34]  Yoshimasa Tanaka,et al.  5′-AMP is a direct precursor of cytokinin in Dictyostelium discoideum , 1978, Nature.

[35]  H. Simon,et al.  Messung von radioaktiven und stabilen Isotopen , 1974 .

[36]  S. Clough,et al.  Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. , 1998, The Plant journal : for cell and molecular biology.

[37]  P. Dobrev,et al.  Fast and efficient separation of cytokinins from auxin and abscisic acid and their purification using mixed-mode solid-phase extraction . , 2002, Journal of chromatography. A.

[38]  S. G. Rogers,et al.  Identification of a cloned cytokinin biosynthetic gene. , 1984, Proceedings of the National Academy of Sciences of the United States of America.

[39]  H. Kawaide,et al.  Analysis of the expression of CLA1, a gene that encodes the 1-deoxyxylulose 5-phosphate synthase of the 2-C-methyl-D-erythritol-4-phosphate pathway in Arabidopsis. , 2000, Plant physiology.

[40]  C. Helliwell,et al.  A plastid envelope location of Arabidopsis ent-kaurene oxidase links the plastid and endoplasmic reticulum steps of the gibberellin biosynthesis pathway. , 2001, The Plant journal : for cell and molecular biology.