Supplemental Figure 1. 2+ - Plant Cell · Supplemental Data. Zhang et al. (2010). Plant Cell...

Supplemental Data. Zhang et al. (2010). Plant Cell 10.1105/tpc.110.076257

Supplemental Figure 1. VLN5 retains conserved residues at both type 1 and type 2 Ca2+-binding

sites in the G1 domain.

Multiple sequence alignment was performed with DNAMAN6.0.40. Secondary structural

elements of human villin were predicted by Predict Protein Server

(http://cubic.bioc.columbia.edu/pp/). The six gelsolin-homology domains (G1 to G6) and the villin

headpiece (VHP) domain are marked with lines above the sequence. Alpha-helices and β-strands

are represented with revolving lines and broad arrows below the sequence, respectively. Amino

acids with 100% conservation were marked with black blocks, whereas amino acids with greater

than 50% identity were marked with gray blocks. The protein accession numbers are as follows:

human villin (HV; NP_009058), lily 135-ABP (AAD54660), Arabidopsis thaliana VLN1

(NP_029567), VLN2 (NP_565958) and VLN5 (NP_200542). Type 1 and type 2 Ca2+ ion

coordinating residues are highlighted with green and yellow, respectively. Residues for site 1 and

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site 2 calcium-regulation sites within the G1 domain are indicated with asterisks and closed circles,

respectively.

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Supplemental Figure 2. VLN5 is expressed preferentially in mature pollen and at higher levels

than other Arabidopsis villins.

The expression data for Arabidopsis villins was extracted from a currently available database

(http://www.bar.utoronto.ca/efp/development/; Schmid et al., 2005). The expression data was

normalized with the GCOS (Gene Chip Operating Software) method, Target intensity (TGT) value

of 100, which was expressed as GCOS expression signal and was plotted against Arabidopsis

tissues. Error bars represent SD (n = 3-5). (A) VLN1; (B) VLN2; (C) VLN3; (D) VLN4 and (E)

VLN5. Arabidopsis tissues are as follows: (1) Dry seed; (2) Imbibed seed, 24 h; (3) 1st node; (4)

Flower stage 12, stamens; (5) Cauline leaf; (6) Cotyledon; (7) Root; (8) Entire rosette after

transition to flowering; (9) Flower stage 9; (10) Flower stage 10/11; (11) Flower stage 12; (12)

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Flower stage 15; (13) Flower stage 12, carpels; (14) Flower stage 12, petals; (15) Flower stage 12,

sepals; (16) Flower stage 15, carpels; (17) Flower stage 15, petals; (18) Flower stage 15, sepals;

(19) Flower stage 15, stamen; (20) Flowers stage 15, pedicels; (21) Leaf 1 + 2; (22) Leaf 7, petiole;

(23) Leaf 7, distal half; (24) Leaf 7, proximal half; (25) Hypocotyl; (26) Root; (27) Rosette leaf 2;

(28) Rosette leaf 4; (29) Rosette leaf 6; (30) Rosette leaf 8; (31) Rosette leaf 10; (32) Rosette leaf

12; (33) Senescing leaf; (34) Shoot apex, inflorescence; (35) Shoot apex, transition; (36) Shoot

apex, vegetative; (37) Stem, 2nd internode; (38) Mature pollen; (39) Seeds stage 3 w/ siliques; (40)

Seeds stage 4 w/ siliques; (41) Seeds stage 5 w/ siliques; (42) Seeds stage 6 w/o siliques; (43)

Seeds stage 7 w/o siliques; (44) Seeds stage 8 w/o siliques; (45) Seeds stage 9 w/o siliques; (46)

Seeds stage 10 w/o siliques; (47) Vegetative rosette.

(F) The expression signal of Arabidopsis villins in mature pollen was plotted. The results show

that the expression of VLN5 is higher than that of other Arabidopsis villins in mature pollen. Error

bars represent SD (n = 3-5).

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Supplemental Figure 3. The level of VLN1 and VLN2 transcripts was not reduced in VLN5 RNAi

flowers.

Flowers from WT Col-0 and three VLN5 RNAi lines (Line 1–3) were subjected to RT treatment.

Tubulin 2 was used as an internal loading control, whereas VLN1 and VLN2 primer pairs were

used to detect whether VLN5 was silenced specifically. The number of PCR cycles was 25 for

Tubulin 2 and 35 for VLN1 and VLN2, respectively.

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Supplemental Figure 4. VLN5 loss-of-function does not affect pollen germination rate.

Pollen grains from WT Col-0 and homozygous vln5 plants were germinated on germination

medium. The germination rate was plotted versus time. White columns, gray columns and black

columns represent WT Col-0, vln5-1 and vln5-2, respectively. Error bars represent ± SE, n = 500.

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Supplemental Figure 5. Pollen tube growth rate was reduced in VLN5 RNAi lines.

WT Col-0, black bar; Line 1, gray bar; Line 2, white bar, Line 3, crosshatched bar. Error bars

represent mean values ± SE, n ≥ 97. Pollen tube growth rate of VLN5 RNAi pollen tubes was

significantly different from that of WT Col-0 pollen tubes as determined by ANOVA followed by

Dunnett post hoc multiple comparisons, **P < 0.01.

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Supplemental Figure 6. Root hair length of vln5 homozygous mutant plants is not significantly

different from that of WT Col-0.

(A) Micrographs of root hairs after germination for four days. (a) WT Col-0; (b) vln5-1; (c) vln5-2.

Bar = 300 µm in (a) for (a-c).

(B) The length of root hairs was plotted against each genotype. The average length of root hairs is

235.4 ± 30.7 (n = 144), 246.4 ± 18.9 (n = 155) and 247.5 ± 18.9 (n = 179) for WT Col-0, vln5-1

and vln5-2, respectively. Error bars represent ± SE. There is no significant difference of the length

of root hairs between vln5 mutants and WT Col-0 (P = 0.62 for vln5-1 and P = 0.56 for vln5-2).

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Supplemental Figure 7. Actin distribution in WT Col-0 and VLN5 loss-of-function pollen tubes.

(A) Actin distribution in WT Col-0 pollen tubes. (a-f) showing different actin staining patterns in

WT Col-0 pollen tubes.

(B) Actin distribution in vln5-1 pollen tubes. (a-f) showing different actin staining patterns in

vln5-1 pollen tubes.

(C) Actin distribution in vln5-2 pollen tubes. (a-f) showing different actin staining patterns in

vln5-2 pollen tubes.

(D) Actin distribution in VLN5 RNAi line 1 pollen tubes. (a-f) showing different actin staining

patterns in VLN5 RNAi line 1 pollen tubes.

(E) Actin distribution in VLN5 RNAi line 2 pollen tubes. (a-f) showing different actin staining

patterns in VLN5 RNAi line 2 pollen tubes. Bar = 10 µm.

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Supplemental Figure 8. VLN5 loss-of-function does not alter the level of actin polymer in pollen

tubes.

Quantification of actin polymer level in WT Col-0 and vln5 mutant pollen tubes. The relative

amount of F-actin was determined by measuring the fluorescence pixel intensity of phalloidin

staining. WT Col-0, black bar; vln5-1, white bar; vln5-2, gray bar. Error bars represent ± SE (n >

39), (P = 0.13 for vln5-1 and P = 0.87 for vln5-2 by a student’s t-test).

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Supplemental Figure 9. Pollen tube growth of VLN5 RNAi plants is hypersensitive to LatB

treatment.

To determine the effect of LatB on pollen tube growth rates of VLN5 RNAi plants, 3 nM LatB

was added to the germination medium. The growth rate of pollen tubes from WT Col-0, and VLN5

RNAi plants in standard germination medium was normalized to 100%. WT Col-0 grew

significantly better than did VLN5 RNAi pollen in the presence of LatB. WT Col-0, black bar;

Line 1, gray bar; Line 2, white bar, Line 3, crosshatched bar. Error bars represent mean ± SE (n ≥

65), **P < 0.01 (Student’s t-test).

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Supplemental Figure 10. VLN5 loss-of-function renders the growth of pollen tubes resistant to

cytochalasin D (CD).

To determine the effect of CD on pollen tube growth rates, 200 nM and 500 nM CD were added to

the germination medium. The growth rate of pollen tubes from WT Col-0 and vln5 homozygous

mutant plants in standard germination medium was normalized to 100%. vln5 pollen tubes grew

significantly better than did WT Col-0 plant in the presence of CD. WT Col-0, black bar; vln5-1,

gray bar; vln5-2, crosshatched bar. Error bars represent mean ± SE, n ≥ 121, *P < 0.05 and **P <

0.01 (student’s t-test). Experiments were repeated three times for each treatment.

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Supplemental Figure 11. The amount of VLN5 sedimented was decreased in the presence of

Ca2+/calmodulin (CaM).

A high-speed cosedimentation assay was employed to determine whether the binding of VLN5 to

actin filaments was regulated by Ca2+/CaM. Three micromolar polymerized actin was incubated

with 500 nM VLN5 with or without 50 µM CaM in the presence of 1 mM free Ca2+. The mixtures

were centrifuged at 200,000g for 1 h to separate bound versus unbound VLN5.

(A) SDS-PAGE separation showing that the amount of VLN5 bound to actin filaments was

decreased in the presence of Ca2+/CaM. Lanes 1 and 2 represent samples of supernatant and pellet

for actin alone; lanes 3 and 4 represent samples of supernatant and pellet for actin plus 500 nM

VLN5; lanes 5 and 6 represent samples of supernatant and pellet for actin plus 500 nM VLN5 in

the presence of 50 µM CaM, respectively.

(B) Plot of the percentage of VLN5 in the pellet. Error bars represent mean ± SE (n = 3), *P <

0.05 (student’s t-test).

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Supplemental Figure 12. Ca2+/CaM inhibits the bundling activity of VLN5.

A low-speed cosedimentation assay was employed to determine whether the bundling activity of

VLN5 was regulated by Ca2+/CaM. Three micromolar polymerized actin was incubated with 500

nM VLN5 with or without 50 µM CaM in the presence of 1 mM free Ca2+. The mixtures were

centrifuged at 13,600g for 30 min.

(A) SDS-PAGE separation showing that the amount of sedimented actin filaments was decreased

in the presence of CaM. Lanes 1 and 2 represent samples of supernatant (S) and pellet (P) for actin

alone; lanes 3 and 4 represent samples of supernatant and pellet for actin plus 500 nM VLN5;

lanes 5 and 6 represent samples of supernatant and pellet for actin plus 500 nM VLN5 in the

presence of 50 µM CaM, respectively.

(B) Plot of the percentage of VLN5 and actin in the pellet. Error bars represent mean ± SD (n = 3),

(*P < 0.05 by a student’s t-test).

(C-E) Micrographs of actin filaments stained with rhodamine-phalloidin.

(C) Individual actin filaments in the absence of villin. The image was captured at a 500-ms

exposure time.

(D) Actin filament bundles formed in the presence of 0.5 µM VLN5. The image was captured at

150-ms exposure time.

(E) Actin bundles formed in the presence of 0.5 µM VLN5 with the addition of CaM. The image

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was captured at a 150-ms exposure time. Bar in (D) = 10 µm.

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Supplemental Figure 13. Direct visualization of VLN severing activity by time-lapse TIRFM.

The detail method is described in the method section and Figure 11A legend.

(A) Time-lapse images of actin filaments severing in the presence of 0.5 nM human villin at 1 µM

free Ca2+. See Supplemental Movie 4 for the entire time series.

(B) Time-lapse images of actin filaments severing in the presence of 5 nM VLN5 at 100 nM free

Ca2+. See Supplemental Movie 5 for the entire time series.

(C) Time-lapse images of actin filaments severing in the presence of 5 nM VLN5 at 10 µM free


(D) Time-lapse images of actin filaments severing in the presence of 5 nM VLN5 at 100 µM free


(E) Time-lapse images of actin filaments severing in the presence of 5 nM VLN5 at 1 mM free

Ca2+. See Supplemental Movie 8 for the entire time series. Scale bar = 20 µm.

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Supplemental Table 1. List of primers used in genotyping, plasmid construction and RT-PCR

Name Primer sequence(5’-3’) DescriptionLP5-1 CCAAGAATCAGAGGTTCCACC For

genotyping of

VLN5 T-DNA

insertion lines

RP5-1 AAAATTCAGGTCTGGCGAATC

LP5-2 AAAGATCCTTCTCGAAGCAGC

RP5-2 GAGGATCACTCTCTCCATCCC

LP2-1 CCAAGAATCAGAGGTTCCACC

RP2-1 AAAATTCAGGTCTGGCGAATC

LP2-2 AAAGATCCTTCTCGAAGCAGC

RP2-2 GAGGATCACTCTCTCCATCCC

LBGABI ATATTGACCATCATACTCATTGC

LBSAIL GCCTTTTCAGAAATGGATAAATAGCCTTGCTTCC

v5pF GGGGACAAGTTTGTACAAAAAAGCAGGCTACTCGTTAGTCCGTTTTGTT For VLN5

promoter

GUS fusion

construction

v5pR GGGGACCACTTTGTACAAGAAAGCTGGGTCTCTGGTTTTTGCAAATCTTT

v5IF TCTAGACCATGGCTAAATATAAGAAACCAATC For VLN5

RNAi

construction

v5IR GGATCCATTTAAATCCTCTGAGTCGGTTTTAAGG

pLat52F GAATTCTGTCGACATACTCGACTCAG

pLat52R CTCGAGTTTAAATTGGAATTTTTTTT

v5F1 GCCCATGGCGTTTTCCATGAGAGATTTA For VLN5

full-length

CDS cloning

and VLN5

insertion lines

RT-PCR

analysis

v5R1 CGGCGGCCGCTTAGAAGAGATTGACAGACAT

v5F2 CTCGGTAAAGATTCCAGCCA

v5R2 CAATGTATGGCTTCGGTTCG

v5F3 ACAAGTTGACCCAAAGAAGA

v5R3 TTAGAAGAGATTGACAGACA

v1TF TCTTACTCTTGGTCTGAAAT

v1TR TTAGAAAAGATGAAGAGATA

v2TF CATCGTTGTTATTTGGCACT

v2TR CTAGAACAAGTCGAACTTCT

eIF4AF GGGTATCTATGCTTACGGTTTCG

eIF4AR CAGAGAACACTCCAACCTGAATC

v5F1 GCCCATGGCGTTTTCCATGAGAGATTTA For RT-PCR

(VLN5 tissue

distribution)

V5bR CTTAACCAGGCCTTGAACGTTAACTCCTTG

Tubulin2F GGTATCCAGGTCGGAAATGC

Tubulin2R TCCCGTAGTCAACAGAAAGT

qVLN5F GTTTCGGGTTCAAGGTTCTG For Real-time

PCR (vln5

RNAi lines)

qVLN5R GAGGAAGTAAGATTGCCACACC

qeIF4AF TGACCAGAGGCTGAATGAAGT

qeIF4AR CGTAAGCATAGATACCCCTAAGAA

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Supplemental References: Schmid, M., Davison, T.S., Henz, S.R., Pape, U.J., Demar, M., Vingron, M., Scholkopf, B., Weigel,

D., and Lohmann, J.U. (2005). A gene expression map of Arabidopsis thaliana development. Nat Genet 37, 501-506.

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Supplemental Figure 1. 2+ - Plant Cell · Supplemental Data. Zhang et al. (2010). Plant Cell...

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