Pollen tubes are one of the fastest growing eukaryotic cells. Rapid anisotropic growth is supported by highly active exocytosis and endocytosis at the plasma membrane, but the subcellular localization of these sites is unknown. To understand molecular processes involved in pollen tube growth, it is crucial to identify the sites of vesicle localization and trafficking. This report presents novel strategies to identify exocytic and endocytic vesicles and to visualize vesicle trafficking dynamics, using pulse-chase labelling with styryl FM dyes and refraction-free high-resolution time-lapse differential interference contrast microscopy. These experiments reveal that the apex is the site of endocytosis and membrane retrieval, while exocytosis occurs in the zone adjacent to the apical dome. Larger vesicles are internalized along the distal pollen tube. Discretely sized vesicles that differentially incorporate FM dyes accumulate in the apical, subapical, and distal regions. Previous work established that pollen tube growth is strongly correlated with hydrodynamic flux and cell volume status. In this report, it is shown that hydrodynamic flux can selectively increase exocytosis or endocytosis. Hypotonic treatment and cell swelling stimulated exocytosis and attenuated endocytosis, while hypertonic treatment and cell shrinking stimulated endocytosis and inhibited exocytosis. Manipulation of pollen tube apical volume and membrane remodelling enabled fine-mapping of plasma membrane dynamics and defined the boundary of the growth zone, which results from the orchestrated action of endocytosis at the apex and along the distal tube and exocytosis in the subapical region. This report provides crucial spatial and temporal details of vesicle trafficking and anisotropic growth.

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In Arabidopsis thaliana, urease transcript levels increased sharply between 2 and 4 d after germination (DAC) and were maintained at maximal levels until at least 8 DAC. Seed urease specific activity declined upon germination but began to increase in seedlings 2 DAC, reaching approximately 75% of seed activity by 8 DAC. Urea levels showed a small transient increase 1 DAC and then approximately paralleled urease activity, reaching maximal levels at approximately 9 DAC. Urease inhibition with phenylphosphorodiamidate resulted in a 2-to 4-fold increase in urea levels throughout seedling development. Arginine pools (0-8 DAC) changed approximately in parallel with the urea pool. Consistent with arginine being a major source of urea, arginase activity increased 10-fold in the interval O to 6 DAC. Allopurinol, a xanthine dehydrogenase inhibitor, had no effect on urea levels up to 3 DAC but reduced the urea pool by 30 to 40% during the interval 5 to 8 DAC, suggesting that purine degradation contributed to the urea pool well after germination, if at all. In aged Arabidopsis seeds, there was a correlation between phenylphosphorodiamidate inactivation of urease and germination inhibition, the latter overcome by NH,NO, or amino acids. Since urease activity, urea precursor, and urea increase in young seedlings, and since urease inactivation results in a nitrogen-reversible inhibition of germination, we propose that urease recycles urea-nitrogen in the seedling.

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