Figure S1. cobl10 has larger seeds than wild type due to increased embryonic cell
size. (a) Naturally dehydrated mature seeds of wild type (left) and cobl10 (right). (b)
Representative images of fully expanded seedlings of wild type (left) and cobl10
(right). (c) DIC image of cotyledon cells of wild-type embryos collected from
rehydrated mature seeds after removing seed coats. (d) DIC image of cotyledon cells
of cobl10 embryos collected from rehydrated mature seeds after removing seed coats.
(e) Quantitative analysis of seed weight. Data were collected from three independent
experiments using 100 naturally mature and dehydrated seeds in each experiment.
Asterisk indicates significant difference (Student’s t-test, P<0.01). (f) Quantitative
analysis of cotyledon sizes of wild type and cobl10 mutants. Asterisk indicates
substantial difference (Student’s t-test, P<0.01). Data were collected from 30
seedlings. (g) Quantitative analysis of embryonic cell sizes of wild type and cobl10
mutants. Asterisk indicates substantial difference (Student’s t-test, P<0.01). Data were
collected from 200 cells from 10 embryonic cotyledons. Bars for (a,b), 1 mm, for
(c,d), 20 µm.
Figure S2. cobl10 has normal pollen development, hydration and germination. (a)
Representative Alexander staining of a wild-type anther. Inset shows DAPI staining of
pollen. (b) Representative Alexander staining of a cobl10 anther. Inset shows DAPI
staining of pollen. (c) Scanning electron micrograph (SEM) of a representative
wild-type pollen grain. (d) SEM of a representative cobl10 pollen grain. (e) SEM of
wild-type pollen hydrated on a wild-type stigma. (f) SEM of cobl10 pollen hydrated
1
on a wild-type stigma. Bars for (a,b), 100 µm; for (a,b) insets, 10 µm. Bars for (c,d), 5
µm; for (e,f), 10 µm.
Figure S3. cobl10 pollen showed normal germination and growth in vitro. (a) qrt
pollen germinated in vitro for 4 hrs. (b) cobl10;qrt pollen germinated in vitro for 4 hrs.
(c) In vitro pollen germination percentage. In total, 468 or 524 pollen were measured
for wild type and cobl10, respectively, from three independent experiments. (d) In
vitro pollen growth speed. Data were collected from three independent experiments in
which 24 to 32 pollen tubes were measured in each experiment for wild type or
cobl10. All measured pollen tubes were 150 µm to 300 µm long. Wild type and
cobl10 pollen were not significantly different in germination percentage or tube
growth rate (Student’s t-test, P>0.05). Bars, 100 µm.
Figure S4. Pollen tubes of cobl10 did not differ from those of wild type in
carbohydrate deposition in vitro. (a) A wild type pollen tube stained with calcofluor
white. (b) A cobl10 pollen tube stained with calcofluor white. (c) Two wild type
pollen tubes stained with ruthenium red. (d) Two cobl10 pollen tubes stained with
ruthenium red. (e) A wild type pollen tube stained with aniline blue. (f) A cobl10
pollen tube stained with aniline blue. Images shown are representative of 36
individual tubes from 3 experiments for each stain. Bright field image is shown above
fluorescent image for (a,b). Bars for (a-d), 10 µm, for (e,f), 20 µm.
2
Figure S5. COBL10ΔC-citrine relocalized to the endoplasmic reticulum. A
representative pollen tube expressing COBL10ΔC-citrine (green) and Calnexin-RFP
(red) was shown. Note that except for ER localization, COBL10ΔC-citrine can still be
detected in a few punctate vesicles. BF represents bright field image. Bar, 10 µm.
Figure S6. The apical plasma membrane localization of COBL10 relies on an intact
PIG complex. (a) Schematic illustration of the seth1-4 and seth2 mutant alleles used
in this study. Arrows indicate binding sites of the primers used for charactering these
mutants. (b) Representative bright field images of in vitro growing pollen tubes from
heterozygous +/seth2 plants expressing COBL10-Citrine, showing a short pollen tube
and a long pollen tube. Inset is a representative short pollen tube from heterozygous
+/seth1-4 plants. (c) Fluorescent image of tubes shown in (b). The tips of pollen tubes
are pointed at by an arrowhead (presence of COBL10-Citrine signal at the apical
plasma membrane) or by arrows (absence of COBL10-Citrine signal at the apical
plasma membrane). Bars, 10 µm. (d) Quantitative analysis of the COBL10-Citrine
signal at the apical plasma membrane of pollen tubes from heterozygous +/seth2
plants. The similar result was obtained from heterozygous+/seth2 plants. N.D. = not
determined. Data were collected from three independent in vitro germination assays.
Figure
S7.
COBL10
function
is
partly
redundant
with
COBL11.
(a)
ProCOBL11:SP-Citrine-COBL11 partially rescued the sterility defect of cobl10 mutants
by silique analysis. Percentage of unfertilized ovules in siliques of cobl10 transformed
3
with COBL11. Data were collected from 3 independent experiments, each of which
used 5 sequential siliques from 10 independent plants. Samples with the different
letters (a, b, and c) are significantly different from each other by Fisher’s least
significant difference (LSD) method. (b) COBL11 localized at the apical plasma
membrane and in punctate vesicles, similar to that of COBL10. Top: Bright field
image; bottom: fluorescent image. Bar, 10 µm. (c) A representative wild-type silique.
(d) A representative cobl10 silique. (e) A representative silique from a homozygous
ProCOBL10:SP-Citrine-COBL11;cobl10 plant.
Table S1. Primers used in this study.
Movie S1. Pollen tubes expressing SP-Citrine-COBL10 during in vitro growth. Note
that the apical plasma membrane localization of COBL10 is dynamically maintained.
Bar, 10 µm.
4