Supplemental Data. Wu et al. (2010). Plant Cell /tpc

Transcription

1 Supplemental Figure 1. FIM5 is preferentially expressed in stamen and mature pollen. The expression data of FIM5 was extracted from Arabidopsis efp browser ( which was represented as GCOS (Gene Chip Operating Software) expression signal, and plotted against different Arabidopsis tissues. Tissues labeled by numbers 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) 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. Column represented for expression in mature pollen is highlighted in red.

2 Supplemental Figure 2. Alignment of the FIM5 protein sequence with sequences of other fimbrin family members. Protein sequences were aligned using DNAMAN. α-helices in each CH domain are indicated by bars above the alignment. The color of the bar indicates the CH domain: CH1, orange; CH2, yellow; CH3, green; and CH4, blue. Linker regions between CH domains are colored light blue. Residues that constitute putative actin binding sites are underlined in pink. The actin binding sites included ABS1 (residues in ABD1; in ABD2), ABS2 (residues in ABD1; in ABD2), and ABS3 (residues in ABD1; in ABD2). The numbers shown

3 above the alignment correspond to the FIM1 sequence. Identical amino acids (100% identity) and similar amino acids (greater than 75% identity) are colored olive and orange, respectively. Note that both FIM1 and FIM5 have an extended C-terminus. Names and database accession numbers for the sequences are as follows: FIM1 (Arabidopsis thaliana fimbrin1, NP_194400), FIM5 (NP_198420), Sac6p (Saccharomyces cerevisiae, NP_010414) and L-plastin (NP_002289).

4 Supplemental Figure 3. Pollen tube growth defects in fim5 mutants were rescued by expression of FIM5-GFP driven by the FIM5 native promoter. (A) RT-PCR analysis showed that FIM5 expression was restored in fim5 mutants after transformation with the complementation construct (C-fim5-1 and C-fim5-2 refer to transformants carrying the complementation construct in the fim5-1 and fim5-2 backgrounds, respectively). eif4a was amplified as an internal control. Forty PCR cycles were performed for both transcripts, and three replicates were conducted. (B) The defect in pollen tube growth of fim5 mutants was rescued by complementation. Pollen tube lengths were measured after 3 h of in vitro germination at 25ºC. A minimum of 100 pollen tubes were measured in each experiment, and three replicates were conducted. Data represent the mean ± SE. An Anova test revealed that no significant difference was present between C-fim5 lines and WT (p = 0.828).

5 Supplemental Figure 4. FIM5-GFP, driven by the FIM5 promoter, decorates actin filaments in both pollen grains and pollen tubes. FIM5-GFP decorates actin filaments in pollen grains (A-B) and in pollen tubes (C-I). In some pollen tubes, FIM5-GFP accumulates behind the tip, corresponding to the fringe or collar region. The step size of optical section was 0.5 µm for pollen grains and 0.4 µm for pollen tubes. Maximal projection of confocal optical sections throughout the entire pollen grain or pollen tube was performed. Scale bars = 10 µm in (A-I).

6 Supplemental Figure 5. Filamentous structures decorated by FIM5-GFP are dispersed by LatB treatment. To determine whether filamentous structures decorated by FIM5-GFP were actin filaments, fim5 pollen tubes carrying FIM5-GFP constructs were subjected to LatB treatment. (A) Pollen tubes expressing FIM5-GFP constructs contain filamentous structures. (B) Pollen tubes subjected to treatment with the vehicle (DMSO) exhibit similar structures after 30 min of treatment. (C) Filamentous structures dispersed after 30 min of 100 nm LatB treatment. (D) Filamentous structures returned after LatB wash out. Scale bar = 20 µm.

7 Supplemental Figure 6. FIM5-GFP co-localizes with actin filaments in pollen grains and pollen tubes. Pollen grains from fim5-1 lines complemented with FIM5-GFP driven by FIM5 promoter were dipped onto the pollen germination medium and germinated for 2 h at 25ºC. Pollen grains and pollen tubes were then processed for fixation with the addition of 300 µm MBS followed by staining with Alexa-568 phalloidin (see the details in the method section of F-actin staining with fluorescent phalloidin). (A-B) Representative Images showing FIM5 co-localizes with all F-actin structures in pollen grains; (C-I) Representative Images showing FIM5 co-localizes with all F-actin structures in pollen tubes of differing lengths. The red panels in (a) show actin filaments labeled with Alexa-568 phalloidin; the green panels in (b) show distribution of FIM5-GFP; the panels in (c) represent merged signals of corresponding (a) and (b) panels. Scale bars = 5 µm in (A-B) or 10 µm in (C-I).

8 Supplemental Figure 7. Actin filaments become redistributed, form thick bundles and display diverse patterns in fim5 pollen grains. Besides the patterns of actin filaments distribution shown in Figure 5B-D, actin filaments also display other patterns in fim5 pollen grains and form thick bundles. (A-D) Actin distribution in fim5-1 pollen grains; (E-H) Actin distribution in fim5-2 pollen grains. Scale bar = 5 µm.

9 Supplemental Figure 8. Thicker actin bundles are present in fim5-1 pollen grains. The appearance of thicker actin bundles in fim5-1 pollen grains was confirmed by analyzing the fluorescence pixel intensity of actin filaments along the long axis of pollen grains using Image J. (A) WT Col-0 pollen grain. A total of 94 fluorescence peaks were identified. (B) fim5-1 pollen grain. A total of 72 fluorescence peaks were identified. (C) Plot of average peak number. Black column represents the number of fluorescence peak of WT col-0 pollen; white column represents the number of fluorescence peak of fim5-1. Five pollens for each genotype were selected randomly to analyze the number of fluorescence peak, and error bars represent mean ± SE. *P < 0.05, by a students test. (D) Black column represents the width of fluorescence peak of WT col-0 pollen; white column represents the width of fluorescence peak of fim5-1. Five pollens for each genotype were selected randomly to analyze the width of fluorescence peak, and at least 25 fluorescence peaks were measured for each pollen. Peak width was determined at half height of the fluorescence peak. Error bars represent mean ± SE. *P < 0.05, by nested ANOVA test.

10 Supplemental Figure 9. Actin filaments are reorganized and form thicker bundles in fim5-1 pollen grains revealed by fluorescent phalloidin staining fixed with paraformaldehyde. Pollen grains were dipped onto the surface of pollen germination medium, rinsed for five minutes in PEM (0.1 M PIPES, ph 7.0, 1 mm MgCl 2, 5 mm EGTA) containing 18% sucrose and fixed for 1h with 4% paraformaldehyde in the same buffer. The following procedure was similar to that of F-actin staining with MBS fixation described in the method section. The occurrence of thick actin bundles are confirmed by analyzing the fluorescence peak number and average fluorescence peak width by Image J as described in Supplemental Figure 8. (A) Pollen grains stained with Alexa-488 phalloidin. (a-b) WT Col-0 pollen grains; (c-h) fim5-1 pollen grains. Scale bars = 5 µm in (a-h). (B) Plot of peak number. Five pollen grains for each genotype were selected randomly to identify the peak number, and error bars represent mean ±

11 SE. *P <0.05 (Student s t-test). (C) Plot of average fluorescence peak width. Five pollen grains were selected randomly and at least 40 fluorescence peaks were measured for each pollen grain. Peak width was determined at half height. Error bars represent mean ± SE. **P <0.01 (Nested ANOVA test).

12 Supplemental Figure 10. Uniform normal distribution of actin filaments was restored in complemented fim5-1 pollen grains carrying the FIM5-GFP construct. (A) In WT Col-0 pollen, actin filaments were uniformly distributed. (B) In fim5-1 pollen, actin filaments were disorganized, as shown in Figure 5. (C D) In fim5-1 pollen grains carrying pcambia1301fim5 pro :Fim5-EGFP-NOS, actin filaments displayed a uniform, normal organization indistinguishable from WT Col-0 pollen grains. Scale bar = 5 µm.

13 Supplemental Table 1. Primer information used in this study Primer name Primer sequence fim5-1 LP 5 -TTTAGGACGGTGAGGCATATG-3 fim5-1 RP 5 -GCGAGTGTGATCTCAAGTTCC-3 fim5-2 LP 5 -GTGGGACTGGACTCGGAAGC-3 fim5-2 RP 5 -GACATAGTGGACGGCTCAG-3 LB 5 -AACGTCCGCAATGTGTTATTAAGTTGTC-3 F5f1 5 -GCGGCCGCATGTCTAGTTACGTTG-3 F5r1 5 -TCTAGACCGCCTGCATCTTTATTATTC-3 F5f2 5 -GACATAGTGGACGGCTCAG-3 F5r2 5 -GTGGGACTGGACTCGGAAGC-3 F5f3 5 -GAGCCTCCAACAACAATCAGACA-3 F5r3 5 -GCATCTTTATTATTCTCACCATC-3 eif4afor 5 -GGGTATCTATGCTTACGGTTTCG-3 eif4arev 5 -CAGAGAACACTCCAA CCTGAATC-3 FIM5For 5 -GGATCCATGTCTAGTTACGTTGGTGTT-3 (with the BamH I site underlined) FIM5Rev 5 -CCCGGGTGCATCTTTATTATTCTCA-3 (with the Sma I site underlined) FIM5proFor 5 -GTCGACCTAACATTAACATCTACAA-3 (with the Sal I site underlined) FIM5proRev (5 -GGATCCTTTTTCTAATCGATTTCT-3 ) (with the BamH I site underlined) EGFPFor 5 -CCCGGGATGGTGAGCAAGGGCGAGGAG-3 (with the Sma I site underlined) EGFPRev 5 -GTGAATTCTTACTTGTACAGCTCGTCCATG-3 (with the EcoR I site underlined) FIM5KGFor 5 -GAATTCTAATGTCTAGTTACGTTGGTGT-3 (underlined EcoRⅠ site) FIM5KGRev 5 -GAATTCTCATGCATCTTTATTATTCTC-3 (underlined EcoRⅠ site)