Volume 6, Issue 5, Pages 1661-1672 (September 2013) Adenine Phosphoribosyl Transferase 1 is a Key Enzyme Catalyzing Cytokinin Conversion from Nucleobases to Nucleotides in Arabidopsis  Xinyan Zhang, Yutao Chen, Xin Lin, Xinyu Hong, Ying Zhu, Wenyang Li, Wenrong He, Fengying An, Hongwei Guo  Molecular Plant  Volume 6, Issue 5, Pages 1661-1672 (September 2013) DOI: 10.1093/mp/sst071 Copyright © 2013 The Authors. All rights reserved. Terms and Conditions

Figure 1 A Schematic Model of the Interconversions among Cytokinin (CK) Nucleotides, Nucleosides, and Nucleobases. The CK bases are active cytokinins while the CK nucleotides and nucleosides are inactive cytokinin precursors. R represents an isopentenyl side chain (iP-type) or a hydroxylated isopentenyl side chain (zeatin-type) attached to N6 of adenine backbone. Solid arrows indicate those well-characterized steps while broken arrows indicate those proposed or uncharacterized steps. In this study, we provide multiple lines of evidence to verify that the APRT (Adenine phosphoribosyltransferase) family proteins, particularly APT1, catalyze the conversion from CK bases to nucleotides, which functions in the opposite way to LONELY GUY (LOG) enzymes. IPTs, isopentenyl transferases; CKXs, cytokinin oxidases; ADKs, adenosine kinases. Molecular Plant 2013 6, 1661-1672DOI: (10.1093/mp/sst071) Copyright © 2013 The Authors. All rights reserved. Terms and Conditions

Figure 2 Isolation of the c9 Mutant that Shows Resistance to High-Dose Cytokinins. (A) Six-day-old light-grown seedlings under 200-μM kinetin treatment. Bar = 2 mm. (B) Root lengths of wild-type (Col-0), ein2-9, and c9 seedlings grown in light on MS medium supplemented with different concentrations of zeatin (Z) and zeatin riboside (ZR) for 6 d. Mean ± SD, n > 20. Asterisks indicate significant difference between ein2-9 and c9 at ** p < 0.01 and *** p < 0.001 by Student’s t-test. Molecular Plant 2013 6, 1661-1672DOI: (10.1093/mp/sst071) Copyright © 2013 The Authors. All rights reserved. Terms and Conditions

Figure 3 Characterization of the apt1 Mutants and APT1-Overexpressing Transgenic Plants. (A) Schematic diagram of Arabidopsis APT1 gene structure and mutations. (B) Five-day-old light-grown seedlings of wild-type (Col-0), apt1-1, and apt1-4 under 200-µM kinetin treatment. c9 was backcrossed with Col-0 twice to exclude ein2-9 and the backcrossed line c9 BC2 (apt1-4) was assayed. Bar = 1 mm. (C) Five-day-old light-grown wild-type, apt1-4, and APT1 overexpression in apt1-4 (35S: APT1/apt1-4) under 200-µM kinetin treatment. Bar = 1 mm. (D) Response of etiolated seedlings to different concentrations of zeatin. Root (F) lengths of wild-type, apt1-4, and APT1ox/apt1-4 were measured 3 d after germination. Mean ± SD, n > 20. Molecular Plant 2013 6, 1661-1672DOI: (10.1093/mp/sst071) Copyright © 2013 The Authors. All rights reserved. Terms and Conditions

Figure 4 Mutation in APT1 Leads to Delayed Leaf Senescence and Elevated Expression of Cytokinin-Responsive Genes that Are Suppressed by AHK2/3 Mutations. (A, B) The fourth true leaves were detached from wild-type, apt1-1, ahk2-5 ahk3-7 double mutant, and apt1-1 ahk2-5 ahk3-7 triple mutant, and shown from left to right in order. Bar = 5 mm. Leaf pictures were taken after dark treatment for 5 d (A), and chlorophyll contents were measured before and after dark treatment (B). Numbers indicate the ratios of the average chlorophyll contents after the dark treatment to those before dark treatment for each genotype. (C, D) Relative expression levels of cytokinin-responsive genes ARR6 (C) and ARR7 (D). RNA was extracted from rosette leaves of 3-week-old adult plants of Col-0, apt1-1, ahk2-5 ahk3-7, and apt1-1 ahk2-5 ahk3-7, which was followed by quantitative RT–PCR analysis. Molecular Plant 2013 6, 1661-1672DOI: (10.1093/mp/sst071) Copyright © 2013 The Authors. All rights reserved. Terms and Conditions

Figure 5 Relative Enzymatic Activity of Arabidopsis APT1, a Yeast APRT (ScAPT), and Six APT1 Mutant Proteins. The wild-type APT1 protein, ScAPT (A) and six APT1 mutant proteins (B) were expressed in E. coli, purified by Ni affinity columns, and analyzed using zeatin and adenine as substrates, respectively. The relative activity was normalized with that of APT1. Molecular Plant 2013 6, 1661-1672DOI: (10.1093/mp/sst071) Copyright © 2013 The Authors. All rights reserved. Terms and Conditions

Figure 6 APT1 Plays a Predominant Role in the Arabidopsis APRT Family. (A) The phylogenic tree of the Arabidopsis APRT family generated by Mega 4.1 (beta) software (Tamura et al., 2007). An APRT homolog from Oryza sativa (OsAPT, EMBL accession: AY238894) was used as an out-group. (B) Relative enzymatic activity of the APT proteins. These APT proteins were expressed in E. coli, purified by Ni affinity columns, and analyzed using zeatin and adenine as substrates. An attempt to express APT3 in E. coli failed. The relative activity was normalized with that of APT1. (C) Five-day-old seedlings of various apt mutants and their combinations grown on MS medium supplemented with (bottom panel) or without (top panel) 200 μM kinetin. Bar = 1 mm. (D) High-dose cytokinin response of seedlings with transgenic overexpression of APT1 homologs. Arabidopsis APT2, 3, 4, 5, and yeast ScAPT were overexpressed in Col-0. Transgenic lines were grown on MS medium with 200 μM kinetin for 5 d in light. Col-0, apt1-1, and APT1ox/apt1-1 were used as controls. Bar = 2 mm. Molecular Plant 2013 6, 1661-1672DOI: (10.1093/mp/sst071) Copyright © 2013 The Authors. All rights reserved. Terms and Conditions

Figure 7 The apt1 Mutant Accumulates Excessive Level of Active Cytokinins. (A) Content of different cytokinins (iP, iPR, iP9G, iP7G) in 3-week-old light-grown wild-type (Col-0) and apt1-1. Statistical analysis on the iP and iPR levels comparing apt1-1 and wild-type (Col-0) was performed using Student’s t-test (p = 0.002 for iP and p = 0.012 for iPR), and asterisks indicate significant difference at ** p < 0.01 and * p < 0.05. (B) Five-day-old seedlings grown on the half-strength MS medium. Bar = 1.5 mm. (C) Quantification of primary root length of seedlings in (B). Mean ± SD, n > 20. Asterisks indicate significant difference at *** p < 0.001 by Student’s t-test. Molecular Plant 2013 6, 1661-1672DOI: (10.1093/mp/sst071) Copyright © 2013 The Authors. All rights reserved. Terms and Conditions