WT glc/GLC - The Plant · PDF fileWT glc/GLC % of ovules reached by . ... pistils two types of unvisited ovules were observed : the pollen tube reached the funiculus but not the micropyle

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0 8 14

WT

glc/GLC

glc/GLC:450.2

% o

f o

vu

les

re

ac

he

d

by

po

lle

n t

ub

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Hours after pollination

A * **

WT B glc/GLC

11.7% 88.3%

PT

PT

PT

PT

PT

B

B B

B

Supplemental Figure 1 - Pollen tube guidance and burst at the embryo-sac micropylar end of wt, glc/GLC and glc/GLC;450.3 ovules. A) Flowers from both genotypes were emasculated and pollinated 24 hours later with pollen of

LAT52::GUS pollen specific marker. Pistils were sampled at the indicated time points after pollination and scored for GUS

presence at the micropylar end of the embryo-sac. In each time point, 160-240 ovules per genotype were examined. Asterisks on

column of the 8HAP time point indicate statistical significance from glc/GLC (Fisher exact test p values : * 3.9e-9, **1.2e-7), the

differences in all other comparisons were not statistically significant (p>0.05). At 8HAP 230 glc/GLC ovules were sampled, out of

which 148 were GUS positives. We can expect 115 of these ovules to be GLC. Since the arrival rate of pollen tubes at the

micropyle of WT ovules at 8 HAP was 91.1%, we estimate that 105 of the GUS positives were due to GLC ovules, and the

remaining 43 GUS positives were from the glc ovules. Thus, the pollen tube arrival was estimated to be 37.4% for the glc ovules

(43/115, 37.4%), and that an estimated 62.6% of the glc mutant ovules did not have pollen tube arrival at this time point.

B) Representative images from the 8 hours after pollination time point. In glc/GLC pistils two types of unvisited ovules were

observed : the pollen tube reached the funiculus but not the micropyle (top ovule, right side image), the pollen tube did not reach

the funiculus (top ovule, middle image). The percentage of occurrences of the two types is indicated inside black boxes (scale bars

50 μm). Black arrows point to pollen tube (PT) or indicate pollen tube burst (B).

Supplemental Data. Leshem et al. Plant Cell. (2012). 10.1105/tpc.112.096420

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WT (Ler)

# of ovules containing Total # of % of ovules containing p value

sperm cells ovules sperm cells (Fisher exact test)

glc/GLC

Supplemental Table 1 . Presence of sperm nuclei in the embryo-sac 24 hours

after pollination

Emasculated flowers were pollinated with pollen of the HTR10 sperm-specific RFP marker. Sperm presence

in the embryo-sac was detected in ovules 24 hours later by fluorescence microscope using the red

fluorescence filter as in material and methods. Observations were made on 10 glc/GLC pistils and 7 for wt.

a – Of which 71 ovules (53.7%) contained single sperm as shown in Supplemental Figure 2.

132a 451 29.3

15 308 4.9

1.22e -17

Supplemental Figure 2 - Sperm localization in glc ovules 24 hours after pollination. Representative images from Supplemental Table 1. Emasculated glc/GLC flowers were

pollinated with pollen of the HTR10 sperm-specific RFP marker. Sperm presence in the

embryo-sac was detected by fluorescence microscope using the red fluorescence filter as in

material and methods. Right side image is a close up of the smaller ovule in the left image.

White arrows indicate sperm nucleus. Scale bar , left image 100 μm, right image 50 μm.


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Z

CCN

SH

SH

Supplemental Figure 3 - Complete Z stack cross section confocal images of HTR10:RFP in glc ovule expressing DD65 central cell marker. Fluorescent micrographs were captured

by MRC-1024 confocal microscope

(Bio-Rad, Hercules, CA) 24 hours after

pollination and presented here at 2 μm

increments. The green (DD65) and red

(HTR10) fluorescent channels were

merged. Solid white arrow indicates the

probable position of the zygote (Z).

Dashed white arrow indicate position of

the central cell nucleus (CCN). Blue

arrows point to a central-cell

cytoplasmic protrusion at the

micropylar end of the embryo-sac called

the synergid hook (SH). Scale bars 40

μm.


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Gene model

cDNAs

Supplemental Figure 4 - The known population of cDNAs of At1g65450 and its variants. Adapted from TAIR. Blue colored bars indicates full length products.


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Supplemental Figure 5 -

Central cell-specific GUS activity in wt ovule driven by DD65 promoter fusion. White dotted line indicates the embryo sac micropylar end, where the EC and the two

SC are located. Scale bar 50μm.


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Supplemental Figure 6 - Putative Promoter Analysis.

Wild-type plants were transformed with the putative promoter sequences of 450.2 and 450.3

fused to the GUS reporter gene. GUS activity was detected in mature unfertilized wild-type

ovules of “450.2 promoter” A) and “450.3 promoter” B-C). Scale bar 50μm. N= 10

transgenic plants in A, 3 in B and 6 in C.

C B

A


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450.1 450.2 450.3

Central Cell Degenerating Synergid Cell

? Molecule X

Sperm cell Fusion / Communication

Signaling Molecule X

Cytoplasmic

Protein Complex Y

Complex Y (Signal Z)

?

Supplemental Figure 7 - Alternative modes of action of At1g65450 isoforms in central cell – sperm cell interaction. 1- At1g65450 isoforms might be involved in enzymatic reaction leading to formation of signaling molecule X

which is needed for communication with the sperm cell. Molecule X might act in the CC cytoplasm, CC

plasmalemma or be translocated to the synergid cell.

2- At1g65450 isoforms might inhibit protein complex Y which negatively regulates the communication with

the sperm cell. Complex Y site of action might be in the CC cytoplasm, CC plasmalemma or be translocated.

Complex Y might effect the communication of the sperm cell with the central through mediator signal Z .


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WT (Ler)

# of ovules containing Total # of % of ovules containing p value

sperm cells ovules sperm cells (Fisher exact test)

glc/GLC

Supplemental Table 1 . Presence of sperm nuclei in the embryo-sac 24 hours

after pollination

Emasculated flowers were pollinated with pollen of the HTR10 sperm-specific RFP marker. Sperm presence

in the embryo-sac was detected in ovules 24 hours later by fluorescence microscope using the red

fluorescence filter as in material and methods. Observations were made on 10 glc/GLC pistils and 7 for wt.

a – Of which 71 ovules (53.7%) contained single sperm as shown in Supplemental Figure 2.

132a 451 29.3

15 308 4.9

1.22e -17

Supplemental Figure 2 - Sperm localization in glc ovules 24 hours after pollination. Representative images from Supplemental Table 1. Emasculated glc/GLC flowers were

pollinated with pollen of the HTR10 sperm-specific RFP marker. Sperm presence in the

embryo-sac was detected by fluorescence microscope using the red fluorescence filter as in

material and methods. Right side image is a close up of the smaller ovule in the left image.

White arrows indicate sperm nucleus. Scale bar , left image 100 μm, right image 50 μm.


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Supplemental Table 2. List of T-DNA knock out lines screened in this study.

GENE Knockout line T-DNA location glc phenotype

At1g65340 SAIL_439_D01 exon no

At1g65352 SALK_091605 300 UTR 3' no

SALK_094384 300 UTR 3' no

SAIL_131_A05 300UTR 3' no

At1g65365 SALK_088234 exon no

SAIL_1307_D03 exon no


SALK_070386 exon no

At1g65410 GABI_284C11 intron no


CSHL_GT2255 exon no


SALK_069579 exon no

SALK_064308 exon no

SALK_058846 intron no


SAIL_59_G06 intron no

SAIL_59_G06 exon no

At1g65445 SAIL_703_E05 300 UTR 5' no

SAIL_258_A02 300 UTR 5' no

SM_3_1166 exon no

At1g65450 SAIL_342_B04 exon no

SM_3_25133 300 UTR 5 no

SM_3_25281 300 UTR 5 no

At1g65470 GABI_095A01 exon no


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SALK_063910 300 UTR3 no


SALK_135487 promotr no



SAIL_100_C05 300 UTR 5' no


SALK_051134 exon no

SALK_122065 intron no


SALK_137811 300 UTR 5 no

SAIL_786_B04 300 UTR 3 no

At1g65550 SALK_099492 intron no

SAIL_714_A11 intron no


SAIL_136_E01 exon no

At1g65560 SAIL_785_B08 intron no

SAIL_267_B07 exon no

At1g65570 SM_3_24141 intron no

SM_3_24121 intron no

At1g65580 SALK_037438 300UTR5' no

SAIL_253_E02 300UTR5' no

At1g65585 SAIL_752_E06 exon no

SALK_014148 exon no


SALK_022428 exon no

At1g65640 SAIL_893_E09 exon no

At1g65650 SALK_112037 300UTR3' no

SALK_111941 300UTR3' no


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SALK_018538 exon no

SAIL_1244_B12 exon no


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WT glc/GLC - The Plant · PDF fileWT glc/GLC % of ovules reached by . ... pistils two types of unvisited ovules were observed : the pollen tube reached the funiculus but not the micropyle