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.