Fig. 1. (A) Time-lapse sequence of a Lilium longiflorum (lily) pollen tube growing facing an extracellular NO point-source (SNAP on agarose; left on the image). Pollen tube slows as it moves into the NO-gradient, but direction proceeds unchanged for 12 minutes. A new growth axis then starts to be defined, forming a sharp right angle from the original axis (97.7±3.6°, n=28). The pollen tube then regains normal growth rate (16-20 minutes). Scale bar: 30 µm. (See Movie 1 at http://dev.biologists.org/supplemental) (B) Lily pollen tube showing three consecutive re-orientation responses induced by moving the same source to the locations marked with arrows. The growth axis changed reproducibly by right angles after each challenge by the NO source in front of the pollen tube tip. (C) Artificial NO source measurements using a vibrating self-referenced polarographic probe to NO. The graph shows a typical exponential NO gradient decay from the point source at different step distances. Although variations between sources were detected, these measurements show that within the effective distance (see A) the NO concentration is in the range of 5-10 nmol l^–1, and the NO flux is in the range of 0.1-0.2 pmol cm^–2 s^–1 (values well within the physiological range accepted for NO action). (D) Time-lapse sequence of a pollen tube being challenged with a diluted NO artificial source in the presence of sildenafil citrate (Viagra^TM) (numbers in the top right-hand corner represent minutes after detection of the response). Using these diluted sources, most pollen tubes do not show any response, often growing into the pipette. For this experiment, pollen tubes were first incubated on standard medium and challenged with the diluted NO source. If a pollen tube showed no response, i.e. if it was demonstrated to be insensitive to such low amounts of NO, the medium was perfused with sildenafil citrate and the same pollen tube is challenged with the same NO source. Despite the lower amount of NO, reverse re-orientation angles were observed in the presence of sildenafil citrate (109.8± 9.8°, n=9) showing a sensitization effect, from unresponsive to the peak response (see movie 1 at http://dev.biologists.org/supplemental).