Supporting Information Legends
Figure S1. Phenotypes of the phr1 mutant at the 3-DAG seedling stage under the
Pi-sufficient and deficient conditions.
Areal parts (a) and roots (b) of 3-DAG wild-type (wt) and phr1 mutant seedlings grown
under the Pi-sufficient (1 mM Pi) and deficient (3 M Pi) conditions are shown. Bar = 1
mm (a) and 0.5 mm (b).
Figure S2. Relative levels of functional transcripts from the pip5k3 and pip5k4
mutant genes.
(a) The structures of the PIP5K3 and PIP5K4 genes are shown schematically, with
boxes and lines corresponding to coding and non-coding regions, respectively. Vertical
arrows indicate the sites of T-DNA insertions in the pip5k3 (SALK_000024) and pip5k4
(SALK_001138) mutants. Horizontal arrows indicate the positions of the primers used
in RT-PCR. (b) Functional transcripts, which contain the regions bracketed by the
horizontal arrows in (a), from the wild-type (PIP5K3 and PIP5K4) and mutant (pip5k3
and pip5k4) genes were quantified by RT-PCR using total RNA prepared from 7-DAG
wild-type and mutant roots grown under the Pi-sufficient condition. The relative
transcript levels compared with that of the UBC21 gene (mean + S.D.), and the ratios of
mutant levels to wild-type levels are shown.
Figure S3. Conserved cis-elements in the upstream intergenic regions of
Brassicaceae PIP5K3 orthologs.
(a) Sequence information of Arabidopsis thliana, Arabidopsis lyrata, Capsella rubella,
Brassica rapa, Thellungiella parvula, and Thellungiella halophila was obtained from
GenBank at National Center for Biotechnology Information (NCBI: Bethesda, USA).
Intergenic sequences between the termination codon of the upstream-neighboring gene
and the initiation codon of PIP5K3 orthologs are aligned. Conserved nucleotides and
cis-elements (RHE and P1BS) are marked in grey and black, respectively. (b)
Consensus sequences of RHE and P1BS are shown.
Figure S4. Root hair lengths of 3-DAG and 7-DAG seedlings of the pip5k3-3,
pip5k3-4, pip5k3-3pip5k4, and pip5k3-4pip5k4 mutants grown under the
Pi-sufficient and deficient conditions.
(a) The structure of the PIP5K3 gene and the T-DNA insertion loci of the pip5k3-3
(SALK_000024) and pip5k3-4 (SALK_026683) mutants are shown schematically.
Boxes and lines correspond to coding and non-coding regions, respectively. Vertical
arrows indicate the sites of T-DNA insertions. (b) Root hairs of 3-DAG and 7-DAG
wild-type (wt), pip5k3-3, pip5k3-4, pip5k3-3pip5k4, and pip5k3-4pip5k4 seedlings
(three or more for each) were measured. Root hair lengths (mean + S.D., n = 50) under
the 1 mM (white box) and 3 M (gray box) Pi conditions are shown. Asterisks indicate
significant increases in the length compared with that under the 1 mM Pi condition (p <
0.05, ANOVA and Tukey’s HSD test). Significant differences (p < 0.05, ANOVA and
Tukey’s NSD test) were detected neither between pip5k3-3 and pip5k3-4 nor between
pip5k3-3pip5k4 and pip5k3-4pip5k4 in any conditions.
(c) Roots of 3-DAG wild-type
(wt) and mutant seedlings grown under the 1 mM and 3 M Pi conditions are shown.
Bar = 0.2 mm.
Figure S5. Expression of PIP5K genes in roots of 7-DAG seedlings grown under
the Pi-sufficient and deficient conditions.
Transcript levels of PIP5K genes and the IPS1 gene in roots of 7-DAG wild-type
seedlings were determined by RT-PCR. Relative transcript levels (mean + S.D., n = 3)
under the 1 mM (white box) and 3 M (gray box) Pi conditions are shown with the
mean value of PIP5K3 under the 1 mM Pi condition arbitrarily set as 1. Asterisks
indicate significant increases in the level compared with that of the 1 mM Pi condition
(p < 0.02, Student’s t-test).
Figure S6. Wild-type and pip5k3pip5k4 double mutant seedlings grown under the
Pi-sufficient and deficient conditions.
Total plants of 8-DAG wild-type and pip5k3pip5k4 double mutant seedlings grown
under the 1 mM and 3 M Pi conditions are shown. Bar = 5 mm.
Table S1. Primer sets used in this study
The sequences of PCR primers used in this study for DNA construction (a) and RT-PCR
(b) are shown. 5’-extra sequences containing restriction sites for cloning are underlined
in (a).