Volume 1, Issue 4, Pages 703-714 (July 2008) Targeting of Pollen Tubes to Ovules Is Dependent on Nitric Oxide (NO) Signaling  Prado Ana Margarida , Colaço Renato , Moreno Nuno , Silva Ana Catarina , Feijó José A.   Molecular Plant  Volume 1, Issue 4, Pages 703-714 (July 2008) DOI: 10.1093/mp/ssn034 Copyright © 2008 The Authors. All rights reserved. Terms and Conditions

Figure 1 Detection of Abnormal Pollen Tube Guidance in Self- and Cross-Pollinations between Atnos1 and Wt Plants. Fluorescence microscopy images of Wt and Atnos1 pistils in self and cross-pollinations after 6 h. Staining of callose walls with aniline-blue. (A) Wt self-pollinated pistil, arrow pointing to Wt pollen tube targeting to the micropyle. Scale bar: 40 μm. (B) Cross-pollination between ♀ Wt X ♂Atnos1 arrow pointing to Atnos1 pollen tubes at the micropyle entrance. Scale bar: 80 μm. (C, D) Atnos1 self-pollinated pistil. (C) Arrow pointing to abnormal pollen tube tip with comb-like shape; (D) arrow pointing to pollen tube that shows a swollen tip near the micropyle (mp) entrance (white dashed line). (E) Cross-pollination between ♀ Atnos1 X ♂Wt arrow pointing to swollen tip of a pollen tube growing on top of an ovule wall. Scale bars: 24 μm. Molecular Plant 2008 1, 703-714DOI: (10.1093/mp/ssn034) Copyright © 2008 The Authors. All rights reserved. Terms and Conditions

Figure 2 Identification of Putative NO Production Spots in Restricted Areas of the Micropyle of Arabidopsis Floral Mutants. (A, B) Fluorescence microscopy images of Arabidopsis floral mutant kannadi. (A) and (B) show exposed ovules labeled with DAF-2DA at the micropyle entrance area. The DAF-2DA signal probes for NO presence. Labeling is observed in both images, being restricted to a cell layer in the immediate vicinity of the micropyle entrance area. Dashed white line shows contour of ovules. (C) Graph shows the variation of levels of expression of Atnos1 (Arabidopsis thaliana nitric oxide synthase 1), nr1 and nr2 (nitrate reductase 1 and 2) transcripts in different tissues namely the pistil at different hours after pollination (HAP). Molecular Plant 2008 1, 703-714DOI: (10.1093/mp/ssn034) Copyright © 2008 The Authors. All rights reserved. Terms and Conditions

Figure 3 Lilium Pollen Tube Targeting to the Ovules. (A–C) Lilium longiflorum pollen tubes targeting the micropyle of Lilium isolated ovules in semi-vivo preparations. (B) and (C) pollen tube cell wall stained with aniline-blue. (B) The pollen tube curls near the micropyle region. (C) Pollen tube making a curve along the base of the micropyle region. Scale bars: 75 μm. (D, E) Targeting abrogation and pollen tube growth, respectively. (D) and (E) show the superior and inferior halves of the semi-vivo preparation where isolated ovules and pollen tubes are under the presence of CPTIO, a NO scavenger, in the medium. (D) Abrogation of targeting by pollen tube showing a balloon tip in the proximity of the micropyle region. (E) Pollen tubes growing in the inferior half of the semi-vivo preparation, which is deprived of ovules. (F) Schematic diagram of the semi-vivo preparation to illustrate that the line of pollen divides the preparation into two halves (frontier between halves is represented by a green line). The top half presents isolated ovules (represented in green) that are aligned with the micropyle facing the row of pollen. Growing pollen tubes are represented by black lines and dots. Black dots represent the balloon pollen tube tips. (G) Graph showing pollen tube tip morphological differences and targeting events (% ± SE) in semi-vivo preparations treated with CPTIO, D-Ser and CPTIO + D-Ser. In all treatments with D-Ser, a decrease in the exploded and balloon tips as well as the promotion of targeting were observed. Molecular Plant 2008 1, 703-714DOI: (10.1093/mp/ssn034) Copyright © 2008 The Authors. All rights reserved. Terms and Conditions

Figure 4 Cytosolic Free Calcium (c[Ca2+]) Dynamics during NO Challenging. Lilium pollen tubes were microinjected with Oregon-Green BAPTA-Dextran (10 KDa) and imaged in a confocal microscope to visualize the typical tip-focused gradient (A). Tubes growing straight with rates > 8 μm min−1 (B) were challenged with a SNP-filled pipette source as described in Prado et al. (2004) until the typical re-orientation response takes place (C). The plot in (D) shows the alteration in the normalized fluorescence level of the first 15 μm from the apex of a pollen tube during a typical experiment (dots  =  experimental values; line  =  moving average, n  =  2). As the tube enters the diffusion gradient from the pipette, the levels of c[Ca2+] start to raise (1.5 min onwards) until reaching a first peak. This peak coincides with a slowdown, sometimes full halt, of growth. The levels of c[Ca2+] then somewhat decrease, and start to raise again to a peak that corresponds to the turning point, and growth restart (4–6 min). After that, the pollen tubes resume growth at normal rate in the new growth axis (>7 min), and the levels of c[Ca2+] return to basal levels (here normalized to ∼100–110 arbitrary units of fluorescence on a 8-bit scale). Molecular Plant 2008 1, 703-714DOI: (10.1093/mp/ssn034) Copyright © 2008 The Authors. All rights reserved. Terms and Conditions