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First published online 5 May 2004
doi: 10.1242/dev.01153

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Nitric oxide is involved in growth regulation and re-orientation of pollen tubes

¹ Instituto Gulbenkian de Ciência, PT-2780-156 Oeiras, Portugal
² University of Missouri-Rolla, Department of Biological Sciences, 105 Schrenk Hall, 1870 Miner Circle, Rolla, MO 65409, USA
³ Centro de Biotecnologia Vegetal, Faculdade de Ciências, Universidad de Lisboa, Campo Grande, Ed.C2. PT-1749-016 Lisboa, Portugal

View larger version (146K): [in a new window]   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 (ViagraTM) (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).

View larger version (42K): [in a new window]   Fig. 2. Detection of intracellular NO in a growing pollen tube of lily using the NO-specific fluophore DAF2-DA (1'). Fluorescence is seen in the cytosol, with less intensity in the apical domain, and is very bright on round cytoplasmic organelles. The spatiotemporal dynamics of intracellular NO is shown in the form of kymographs in which we averaged an active representative region inside each pollen tube at each time-point as a color-coded line (see top wedge), and plotted these lines as a function of time (y-axis) and pollen tube length (x-axis). For the sake of clarity, the pollen tube tip was aligned with the right side, and therefore the slope on the left side of the kymograph reflects the growth rate. The chronological order of each time point is read from top to bottom as illustrated by the arrow on the y-axis. In a non-challenged pollen tube (control), no significant variation along time is seen. Apical depletion and subapical accumulation of NO are clearly visible. Incubation with the NO-scavenger CPTIO (44') almost suppressed the signal from the cytosol, but the round organelles are still distinguishable. Kymograph analysis shows the overall decrease after CPTIO addition, but the apical/subapical pattern, polarity and dimensions are maintained.

View larger version (107K): [in a new window]   Fig. 3. The DAF-2DA-positive organelles are peroxisomes. (A-D) Confocal images of growing lily pollen tubes incubated in DAF-2DA (green) and in organelle-specific dyes (red). No co-localization was found in the endomembrane system (A, BodipyTR), mitochondria (B, Rhodamine 123) or acidic organelles (C, LysotrackerRed). (D) Peroxisomes were then tagged by transient transformation of pollen grains with a construct containing the pollen-specific LAT52 promoter driving an ECFP-peroxisome targeting signal (PxTS) fusion. Pollen tubes were observed 10 hours after germination. The NO-producing organelles (DAF-2, green) show an almost complete co-localization with the ECP signal (PxTS-CFP, blue), as shown in the merged image. Scale bars: 15 µm.

View larger version (94K): [in a new window]   Fig. 4. An increase in intracellular NO precedes re-orientation. The time-lapse sequence (A-C) shows the changes in intracellular NO after challenge with an extracellular point source, as reported by DAF-2DA fluorescence. In A, a DAF-2DA loaded pollen tube was followed for 4 minutes. The inserted kymograph shows the typical NO pattern, and no significant variation with time. (B) Challenging with an external source produces a rise in fluorescence within 1 minute. After 10 minutes, the low NO concentration domain disappears (arrow) simultaneously with growth arrest (slope on the left side of the kymograph) and soon after the NO concentration peaks. (C) As the concentration stabilizes, the negative NO tip gradient starts to be defined and re-orientation occurs. Scale bars: 15 µm. (D) The average pixel intensity variations of the DAF-2DA signal plotted as a function of time at the tip of a growing pollen tube before (yellow) and after extracellular NO challenge (white). Accumulation of intracellular NO is obvious soon after the pollen tube moves into the gradient, but builds up strongly from a threshold point. When the peak point is reached, growth is arrested. As soon as growth is regained in the new axis, the level of NO drops to a stable value, which is about twice that seen before challenge. Addition of the NO-scavenger CPTIO totally inhibited the re-direction response as illustrated in E-G. A DAF-2DA stained tube (E) was challenged with an extracellular NO point source in the presence of CPTIO. While the signal decreased as in Fig. 2, the growth of the tube slowed but slowly regained normal growth without any change of direction (F). Evolution of intracellular NO shows that after some initial increase, this reaction is immediately followed by a decrease to levels below the initial level (G and inserted kymograph). Scale bar: 16 µm.