Volume 6, Issue 4, Pages 1074-1090 (July 2013) Acquisition of LURE-Binding Activity at the Pollen Tube Tip of Torenia fournieri  Satohiro Okuda, Takamasa Suzuki, Masahiro M. Kanaoka, Hitoshi Mori, Narie Sasaki, Tetsuya Higashiyama  Molecular Plant  Volume 6, Issue 4, Pages 1074-1090 (July 2013) DOI: 10.1093/mp/sst050 Copyright © 2013 The Authors. All rights reserved. Terms and Conditions

Figure 1 Pollen Tube Attraction at Different Developmental Stages. (A) Schematic drawing indicating pollen tubes grown under each condition (i–iv). HBE, hours before the pollen tubes emerged from the cut end of the style; HOM, hours when the pollen tubes were growing on the media after emerging from the style; HAP, hours after pollen grains were dusted on the medium in in vitro condition (i) or hours after pollination in semi-in vitro condition (ii–iv). (B–I) Pollen tube attraction assay was conducted under each condition. The pollen tubes grown for 12 HAP without a style were not attracted to LURE2 (B, C). The pollen tubes elongated for 12 HAP through the 5-mm (D, E) or 10-mm (F, G) cut style were not attracted to LURE2, but those elongated for 12 HAP with the 15-mm cut style (H, I) were attracted to LURE2. Scale bar = 20 μm. (J) Pollen tube attraction assay at different developmental stages. Data are the means ± SD. The total numbers of pollen tubes examined are indicated above each bar (n) for more than three replicates. Statistical significance of differences was determined by one-way ANOVA (analysis of variance) with Turkey–Kramer post-hoc test (** P < 0.01, compared with others). Molecular Plant 2013 6, 1074-1090DOI: (10.1093/mp/sst050) Copyright © 2013 The Authors. All rights reserved. Terms and Conditions

Figure 2 Visualization of LURE2 Binding at the Pollen Tube Tip. (A) Schematic diagram of LURE2 binding assay. A pistil was pollinated, cut, and incubated with ovules in liquid medium for elongation of the pollen tubes. The pollen tubes were dipped into LURE solution to allow interaction with LURE. After interaction with LURE, the pollen tubes were transferred into cross-linker solution for cross-linkage of pollen tube plasma membrane proteins and LURE peptides for 5 min, followed by fixation with PFA. The treated pollen tubes were immunostained with anti-LURE2 antibodies or an anti-AtLURE1 antibody and observed by phase-contrast microscopy. (B–I) Immunostaining of pollen tubes. Fluorescence was detected on the tip region of the pollen tubes provided with LURE2 (B, C). Negative controls did not show signals at the pollen tube tip with pre-immune serum (D, E). No signal was observed on pollen tubes grown in vitro (F, G). Pollen tubes provided with AtLURE1 were stained with an anti-AtLURE1 antibody. Fluorescence was not detected on the tip region of the pollen tubes provided with AtLURE1 (H, I). (B, D, F, H) Phase-contrast bright field images. (C, E, G, I) Fluorescent images. Arrowheads indicate several pollen tube tips. Scale bar = 100 μm. Inset, magnified view. Scale bar = 10 μm. (J–O) Confocal laser scanning microscope imaging of immunostained pollen tubes (15 mm_12 HAP pollen tubes). (K) The signal was detected on the peripheral of the pollen tube tip. Dashed lines indicate positions of vertical sections; the left (proximal) line for (L) and the right (apical) line for (M). (L, M) Vertical sections of a three-dimensional image constructed from confocal images, one of which is shown in (K). The signal was mainly localized on the peripheral of the pollen tube and modestly as a dot (an arrowhead) in the cytoplasm. (N, O) The plasmolyzed pollen tube showed a signal concentrated on the peripheral of the cytoplasm in the tip region. An arrowhead and an arrow show the peripheral of the cytoplasm and cell wall in the tip region, respectively. (J, N) Bright field images. (K–M, O) Fluorescent images. Scale bar = 5 μm. Molecular Plant 2013 6, 1074-1090DOI: (10.1093/mp/sst050) Copyright © 2013 The Authors. All rights reserved. Terms and Conditions

Figure 3 Pollen Tube Binding LURE2 during Different Developmental Stages. (A–H) Immunostaining analysis of the pollen tubes at different stages. The pollen tubes grown for 12 HAP without a style showed no association with LURE2 (A, B). The pollen tubes elongated for 12 HAP through the 5-mm (C, D), 10-mm (E, F), and 15-mm (G, H) cut styles showed binding of LURE2 on the tip. (A, C, E, G) Phase-contrast bright field images. (B, D, F, H) Fluorescent images. Scale bar = 10 μm. (I) Pollen tube LURE2 binding activity. Data are the means ± SD. Total numbers of observed pollen tubes are indicated above each bar (n) for more than three replicates. Statistical significance of differences was determined by Welch’s t-test (* P < 0.05; ** P < 0.01). Molecular Plant 2013 6, 1074-1090DOI: (10.1093/mp/sst050) Copyright © 2013 The Authors. All rights reserved. Terms and Conditions

Figure 4 Comparison of Gene Expression Profiles. (A) Hierarchical clustering of the 24 RNA-Seqs representing nine conditions was performed using pvclust, an R package for hierarchical clustering with P-values (Suzuki and Shimodaira, 2006). Gray values are approximately unbiased P-values (%). (B–L) M–A plot for pairwise comparison, after scaling for reads per million (RPM) mapped reads in each sample. Each dot represents a gene. Red dots indicate genes estimated as differentially expressed genes with false discovery rates (FDR) <0.01 using TbT normalization (Kadota et al., 2012). The horizontal axis indicates the average expression level of a gene across two groups, and the vertical axis indicates the log ratio (Sample b relative to Sample a). Molecular Plant 2013 6, 1074-1090DOI: (10.1093/mp/sst050) Copyright © 2013 The Authors. All rights reserved. Terms and Conditions

Figure 5 Differential Gene Expression in Pollen Tubes at Several Developmental Stages. Contigs were considered expressed using an RPM threshold >1. (A) A three-way comparison among 15 mm_12 HAP, 15 mm_6 HAP, and the set of other conditions. (B) A three-way comparison among 15 mm_12 HAP, 5/10 mm_12 HAP including contigs expressed in 5 mm_12 HAP, or 10 mm_12 HAP and the set of other conditions. (C–E) Number of contigs annotated with putative GO terms. The total number of annotated contigs with RPM >1 under each condition is indicated beside the color legend. (C) Cellular component. (D) Molecular function. (E) Biological process. Molecular Plant 2013 6, 1074-1090DOI: (10.1093/mp/sst050) Copyright © 2013 The Authors. All rights reserved. Terms and Conditions