Supplemental Data
Supplemental Figure S1.
Root developmental phenotype of weak MIZ1OE seedlings.
(A) Time course of primary root elongation. (B) Lateral root density of 10-day-old
seedlings. n = 25-29. Asterisks indicate statistically significant differences determined
by Student t-test (P < 0.05). Error bars represent mean S. E.
Supplemental Figure S2.
Morphological analyses of stage III to emerged LRP in the wild type, miz1-1 and OE7.
Scales represent 10 μm.
Supplemental Figure S3.
Effect of osmotic stress on lateral root formation in miz1 and MIZ1OE roots.
Wild type, miz1 and MIZ1OE were germinated on control or 60 mM of mannitol
containing medium. Lateral root density was determined 10 days after germination. n =
14-22. *, Statistically significant differences between control and mannitol treated
samples were determined using a Student’s t-test (P < 0.05). Error bars represent mean
S. E.
Supplemental Figure S4.
GFP-fused MIZ1 protein complements the root ahydrotropic phenotype of miz1-1.
(A) Schematic model of the GFP-fused MIZ1 protein. (B) Root hydrotropic curvatures
of the wild type, miz1-1 and transgenic line of miz1-1:proMIZ1：MIZ1-GFP. n = 24.
Error bars mean S. D.
Supplemental Figure S5.
Ethylene treatment did not rescue lateral root formation in MIZ1OE seedlings.
Four-day-old wild type, miz1-1 and MIZ1OE seedlings were transferred and grown for 4
days on medium containing 100 nM NAA (A) or1 μM ACC (B). From left to right; wild
type, miz1-1, OE7 and OE29, respectively. Scales represent 5 mm.
Supplemental Figure S6.
DR5-GFP expression in MIZ1OE plants.
(A-B) Confocal images of auxin responsive reporter, DR5-GFP, in MIZ1OE LRP (A),
and root tip (B). DIC images represent stage I LRP. Yellow arrowheads indicate the
occurrence of cell divisions. Scale bars represent 10 μm in A and 20 μm in B. (C)
Quantification of DR5-GFP expression in stage II LRP and root cap of wild type and
OE7 seedlings by image analysis. Error bars mean S. E. n = 12-16 (LRP) or 18 (root tip).
*, Statistically significant differences were determined using a Student’s t-test (P <
0.05).
Supplemental Figure S7.
Semiquantitative RT-PCR analysis of MIZ1 gene expression in the wild type, miz2, OE7
and miz2 OE7. Amplifications were performed at 25 and 30 cycles, and subjected to
agarose gel electrophoresis and were further visualized by ethidium bromide staining.
For control, cDNA fragments for ACT2 were amplified.
Supplemental Figure S8.
Relationships between MIZ1 localization and GNOM function.
(A-B) MIZ1-GFP localization in LRP of miz2 mutant in the absence of BAP (A) or
presence of 10 μM of BAP (B). Yellow arrowheads indicate MIZ1-GFP localization in
LRP. (C) Intercellular localization of MIZ1-GFP in the wild type or miz2. Scales
indicate 10 μm in A-B and 5 μm in C.
Supplemental Figure S9.
Free IAA contents in root of the wild type, miz2 and miz2 OE7. Error bars mean S. E. n
= 3-8
Supplemental Figure S10.
PIN2-GFP localization in BFA-treated wild type seedlings and mock-treated miz2
seedlings. (A) Wild type:PIN2-GFP seedlings germinated in the presence of 5 μM of
BFA. (B) miz2:PIN2-GFP seedlings. Scales indicate 5 μm.
Supplemental Figure S11.
Localization of endocytosis and endosome markers in MIZ1OE seedlings. (A) Uptakes
of endocytotic tracer FM4-64. Images were acquired 20 min after treatment. (B)
Aggregation of FM4-64 following 60 min of soaking in 50 μM BFA. (C-D)
Localization of GNOM-GFP (C) and ARA7-GFP (D) in the wild type and OE7 root
cells. FM4-64 was treated for 60 min (C) or 30 min (D). Scales indicate 5 μm.
Supplemental Figure S12.
Schematic model of MIZ1-GNOM-dependent pathway for lateral root development.
Cytokinin disrupts establishment of auxin gradient by down-regulating of expression of
PIN genes, and thereby inhibiting LRP development (Laplaze et al., 2007). Cytokinin
also induces MIZ1 expression in LRP. MIZ1 or MIZ1-like genes will negatively control
auxin accumulation and lateral root development downstream of cytokinin.
GNOM-dependent vesicle trafficking is required for both of PIN polar localization and
action of MIZ1.