SUPPORTING INFORMATION LEGENDS
SUPPORTING FIGURE S1. The MUS open reading frame is 3,586 base pairs
long and lacks introns.
(a) Schematic diagram of the MUS gene (At1g75640) showing locations of the mus
alleles. The two alleles that harbor point mutations are indicated with vertical black
lines. These mutations result in a G (glycine) to R (arginine) transition in mus-1,
and a W (tryptophan) to stop transition in mus-2. The location of the T-DNA
insertion is marked by a white triangle. (b) Relative mRNA levels in the three mus
alleles.
SUPPORTING FIGURE S2. mus does not disrupt organelle distribution.
(a-f) Red fluorescence corresponds to Propidium Iodide (PI) staining of cell walls.
Scale bars = 5 µm. (a-c) Green fluorescence represents the vacuolar membrane
marker 35Spro:delta-TIP:GFP (Cutler et al., 1999). Both WT (a) and mus (b-c)
stomata contain several vacuoles. (d-f) Green fluorescence corresponds to
35Spro:TUA:GFP localization. Each micrograph depicts the mid-plane of a stoma,
where 35Spro:TUA:GFP marks perinuclear MTs surrounding the nucleus. Nuclei
in both WT (d) and mus (e-f) stomata are centrally located near the ventral wall.
Chloroplasts (red autofluorescence) are also visible along both the dorsal and
ventral walls.
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SUPPORTING FIGURE S3. Association between the direction of MT growth
and the degree of symmetry disruption in mus-1 stomata.
Cell walls visualized utilizing Propidium iodide (PI) staining. Images were
converted to gray scale for clarity. Y-axis represents direction of MT growth
(expressed as the number of inbound (blue) and outbound (red) comets). The Xaxis indicates the degree of disruption of morphological symmetry in stomata of
different backgrounds. On the X-axis the phenotypic severity increases from left to
right, with the left most micrograph representing a normal (wild-type) stoma and the
right most micrograph representing the most severely disrupted stomatal
phenotypes observed in mus-1. Note that higher levels of symmetry disruption are
associated with an increase in the number of inbound comets (blue squares), and
a decrease in the number of outbound comets (red triangles).
SUPPORTING FIGURE S4. MUS maintains bilateral symmetry via acting after
symmetric division but before pore formation.
(a, c, d, f) Red fluorescence corresponds to cell walls visualized by PI staining. (b,
e) Green fluorescence of microtubules derives from 35Spro:TUA:GFP expression.
Scale bars = 5 µm. (a) Recently divided WT stomata prior to pore formation.
Formation of a new cell wall normally generates two symmetrical young GCs. The
young newly formed wall is visualized through propidium iodide. (d) Recently
divided mus-1 stomata prior to pore formation. As in WT, the new wall in young
mus-1 stomata form two symmetrically arranged GCs. (b-c) Radial MT arrays in
young WT stomata are symmetrically arranged around the future pore site. (c)
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Micrograph of b, with identical levels of intensity and fluorescence, but with green
channel removed in order to make the young GC wall visible. (e) MT arrays are
not symmetrically arranged around the future pore site in mus, and instead appear
to concentrate at the center of each GC. (f) Micrograph of (e), with identical levels
of intensity and fluorescence, but with green channel removed. All scale bars = 5
µm.
SUPPORTING FIGURE S5. MUS orthologs are present in diverse plant
lineages.
An unrooted, maximum likelihood phylogeny shows that MUS orthologs are
present in plant lineages ranging from mosses and liverworts to dicots and
monocots. Each ortholog’s gene ID, or clone name, is contained in brackets
adjacent to the corresponding ortholog.
SUPPORTING FIGURE S6. MUS localization patterns remain consistent
throughout multiple plant organs.
(a-j) Red fluorescence corresponds to cell walls visualized by PI staining (except in
(h). Green fluorescence represents MUSpro:MUS:tripleGFP localization. (a-c, f-g)
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Scale bars = 15 µm. (d, f, i) Scale bars = 30µm. (a-b) Stem. (c) Sepal. (d-e)
Petal. (f-h) Anther. (h) Micrograph of (g), with identical levels of intensity and
fluorescence, but with red channel removed. (i) Stigma (j) Style. Arrowheads
signify GMCs, while asterisks signify mature stomata.
SUPPORTING FIGURE S7. MUS localizes to the cell membrane.
Red fluorescence corresponds to FM4-64 cell membrane (PM) staining; green
fluorescence derives from MUSpro:MUS:tripleGFP expression. (a) Meristemoid
(left) and a GMC (lower right) shown in face view (paradermal optical section) that
resulted from two asymmetric divisions. At the left, MUS expression is localized in
the new cell wall of the meristemoid, but not in the newly formed pore wall in the
GMC (lower right). The brightness in all micrographs was increased to demonstrate
overlaps in localization. (b-c) Both micrographs depict different planes of section
of the same newly divided GMC shown in (a). White dashes in (b) indicate where
the plane of section was obtained. In (c), the GMC is viewed in an optical section
perpendicular to the plane of the epidermis in (b). Yellow fluorescence indicates
overlap between FM4-64 stain and MUSpro:MUS:tripleGFP localization. (d-e)
Both micrographs depict different planes of section of the same meristemoid
shown in micrograph (a) (at the left). White dashes in (d) indicate the location that
the plane of section e was obtained from. In (e), the meristemoid is viewed in a
plane of section that is anticlinal to the plane of the epidermis in (b). Yellow
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fluorescence indicates the overlap between FM-464 & MUSpro:MUS:tripleGFP
localization (a). Scale bar = 10 µm and b. Scale bar = 5µm.
SUPPORTING FIGURE S8. MUS does not localize to the cortical Endoplasmic
Reticulum.
Green fluorescence represents MUSpro:MUS:tripleGFP, while red fluorescence
corresponds to Hexyl Rhodamine staining of the cortical Endoplasmic Reticulum
(ER). The first column of micrographs represents both MUS and ER signal, the
second column corresponds to only MUS signal, and the third column represents
only ER signal. Scale bars = 5 µm. (a) Meristemoid. (b) Guard Mother Cell. (c)
Mature Stoma.
SUPPORTING FIGURE S9. mus displays additive phenotypes with several
other mutants known to disrupt stomatal development.
(a) Wild type Col-0 gl1 stoma exhibiting normal bilateral symmetry. (b) mus-1
stoma displaying disrupted bilateral symmetry and morphogenesis. (c) Stomatal
cluster in flp-1 with two bilaterally symmetrical stomata. (d) Stomatal cluster in a
flp-1 mus-1 double mutant containing one stoma (left) displaying normal bilateral
symmetry, and another stoma (right) with disrupted symmetry. (e) A tumour like
cell cluster in a fama-1 single mutant (which entirely lacks guard cells). (f) A
tumour-like cluster in a fama-1 mus-1 double mutant (that displays no obvious
stomatal skewing) (g) Stomatal cluster in tmm-1 single mutant containing
bilaterally symmetrical stomata. (h) Cluster in a tmm-1 mus-1 double mutant
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containing two bilaterally symmetrical stomata (left), and two with disrupted
symmetry (right). (i) Single Guard Cell (SGC) in 35S:CDKB1;1 N161
overexpression mutant line. (j) 35S:CDKB1;1 N161 mus-1 double mutants contain
SGCs (left) as well as stomata with disrupted bilateral symmetry (right). All scale
bars = 5 µm.
SUPPORTING FIGURE S10. Comparison of conserved amino acid motifs
indicates that MUS likely encodes an atypical kinase (modified from Castells
and Casacuberta, 2007).
Comparison of conserved amino acid motifs (red box) in Kinase VII domains of
typical (DFG) and atypical receptor-like kinases (adapted from Castells &
Cascuberta, 2007). All proteins shown are in Arabidopsis except for MARK.
MUS: MUSTACHES; MARK: MAIZE ATYPICAL RECEPTOR KINASE; TMKL1:
TRANSMEMBRANE KINASE LIKE1; SUB: STRUBBELIG; CRR1: CRINKLY4RELATED1; CRR2: CRINKLY4-RELATED2 BRI1:BRASSINOSTEROID
INSENSITIVE1. Note that in the wild-type MUS protein, a glutamic acid (E) is
substituted for aspartic acid (D). The presence of this substitution suggests that
MUS might be incapable of phosphorylation.
SUPPORTING TABLE S1. Nature and location of mutations carried in three
mus alleles.
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SUPPORTING MOVIE S1. 35Spro:EB1:EB1: GFP movement in a WT stoma.
SUPPORTING MOVIE S2. 35Spro:EB1:EB1: GFP movement in a skewed mus-1
stoma.
SUPPORTING MOVIE S3. 35Spro:EB1:EB1: GFP movement in an abnormally
expanded mus-1 stoma.
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