Rates of exocytosis and endocytosis in Arabidopsis root hairs and pollen tubes.

Exocytosis and endocytosis are pivotal in many biological processes, but remain difficult to quantify. Here we combine a new algorithm for estimating vesicle size with a detailed morphological analysis of tip-growing cells, in which exocytosis is highly localized and therefore more readily quantified. Cell preservation was rendered as life-like as possible by rapid freezing. This allowed us to produce the first estimates of exocytosis rates in the root hairs and pollen tubes of the model plant Arabidopsis. To quantify exocytosis and endocytosis rates during cell growth, we measured the diameter of vesicles located in the tips of Arabidopsis root hairs and pollen tubes and the widths of cell walls and the cell lumen in longitudinal thin transmission electron microscopic sections. In addition, we measured growth velocities of Arabidopsis root hairs and pollen tubes, using video microscopy. The number of exocytotic vesicles required for cell wall expansion, and the amount of excess membrane inserted into the plasma membrane to be internalized, were estimated from the values that were obtained. The amount of excess membrane that is inserted into the plasma membrane during cell growth was estimated as 86.7% in root hairs and 79% in pollen tubes. This membrane has to be recycled by endocytosis. From counting of the total number of vesicles that is present in thin EM sections through the pollen tube tip, we estimated the average number of vesicles that is present in the tip of pollen tubes. By calculating the total amount of membrane and cell wall material that is required for continued cell growth, assuming that all vesicles are exocytotic, we estimated that pollen tubes continue to grow for 33 s when delivery of vesicles to the tip is inhibited. We arrested vesicle delivery to the tip by application of cytochalasin D. After cytochalasin D application, pollen tubes continued to grow for 30-40 s, which is in the same range as the estimated value of 33 s and shows that in this time frame, the availability of exocytotic vesicles is not a limiting factor.