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Legends for Supplementary Materials
Figure S1. Schematic representation of the metabolism of stable isotope-labeled iPR in
Arabidopsis.
The possible metabolism of exogenously applied iPR(+15) is illustrated. Adenine and
ribose moieties in red are labeled with 13C and 15N. Arrows on red background indicate
the two-step pathway, and arrows on gray background indicate the single step pathway.
Figure S2. Accumulation levels of cytokinins and their conjugates in log single mutants.
Eleven-day-old Arabidopsis seedlings of wild type (wild) and log single mutants grown
on MGRL-agar medium were harvested, and concentrations of cytokinins and conjugates
were analyzed by LC-MS. Error bars represent the S.D. of three biological replicates.
Data sets marked with single and double asterisk are significantly different from
wild-type as assessed by Student’s t-test at p<0.05 and p<0.01, respectively. FW, fresh
weight.
Figure S3. Expression levels of LOG genes in wild type and log septuple mutant.
(a) PCR for genotyping with genomic DNA prepared from wild type (WT),
log1log2log3log4log5log7log8 septuple mutant (1234578), log1log2log3log4log5log7
(123457), and log1log2log3log4log5log8 (123458) with a pair of gene-specific primers
(gene) and a pair of gene-specific and T-DNA specific primers (T-DNA). Primer
sequences are given in Kuroha et al. (2009).
(b) RT-PCR analysis with total RNA prepared from 2-week-old seedlings of WT and
1234578. The ACT2 gene was used as a control. Cycle numbers of PCR are shown in
parentheses.
(c) Quantitative RT-PCR analysis was performed to quantify the LOG1 expression levels
in 6-day-old seedlings of multiple log mutants containing log1. The expression intensity
of the LOG genes was normalized with respect to that of Ubiquitin 10 gene, and LOG
expression in the WT was defined as 100 arbitrary units. Error bars indicate S.D. of three
experimental replicates. N.D., not detected.
Figure S4. Complementation of the log1log2log3log4log5log7log8 mutant by
introduction of the wild-type LOG7 gene. Six-day-old log1log2log3log4log5log7log8
septuple mutant (a), the septuple mutant transfomed with wild-type LOG7 gene
(pBG-LOG7) at T2 generation (b), and log1log2log3log4log5log8 sextuple mutant (c).
Seven of eight independent T1 transformants showed the recovery of shoot and root
growth. A T2 line that segregated 3:1 (bialaphos resistance : susceptible) was used. About
a quarter of the T2 seeds showed the phenotype of the septuple mutant (black arrow).
Scale bar, 1 cm.
Figure S5. Expression level of CKX genes in log mutants.
Quantitative RT-PCR was performed to quantify CKX expression levels in 6-day-old
seedlings of wild type (WT), log1log2log3log4log7 (12347), log1log2log3log4log5log7
(123457), log1log2log3log4log5log8 (123458), and log1log2log3log4log5log7log8
(1234578). The expression intensity of CKX gene was normalized with respect to that of
Ubiquitin 10 gene, and the expression in the WT was defined as 100 arbitrary units.
Error bars indicates S.D. of three experimental replicates.
Figure S6. Preparation of [1013C,515N]-labeled N6-(Δ2-isopentenyl)adenine riboside.
(a) Synthesis procedure of the [1013C,515N]-labeled N6-(Δ2-isopentenyl)adenine riboside
[iPR(+15)]. N-prenylation of [1013C,515N]-AMP by Tzs and dephoshorylation of iPR
5’-monophosphate [iPRMP(+15)] by alkaline phosphatase (AP) are shown.
(b) Mass spectra of iPR (upper panel) and [1013C,515N]-iPR (lower panel).
Table S1. Quantification of cytokinins in the log1log2log3log4log5log7log8 mutant.
Cytokinin concentrations in whole seedling were determined. Data are means ± SD of
three biological replicates. tZ, trans-zeatin; tZR, tZ riboside; tZRPs, tZR 5’-phosphate;
cZ,
cis-zeatin;
cZR,
cZ
riboside;
cZRPs,
cZR
5’-phosphate;
iP,
N6-(Δ2-isopentenyl)adenine; iPR, iP riboside; iPRPs, iPR 5’-phosphate; tZ7G,
tZ-7-N-glucoside;
tZ9G,
tZ-9-N-glucoside;
iP-9-N-glucoside. n.d., not detected.
iP7G,
iP-7-N-glucoside;
iP9G,