The pollen tube is a cellular protuberance formed by the pollen grain, or male gametophyte, in flowering plants. Its principal metabolic activity is the synthesis and assembly of cell wall material, which must be precisely coordinated to sustain the characteristic rapid growth rate and to ensure geometrically correct and efficient cellular morphogenesis. Unlike other model species, the cell wall of the Arabidopsis (Arabidopsis thaliana) pollen tube has not been described in detail. We used immunohistochemistry and quantitative image analysis to provide a detailed profile of the spatial distribution of the major cell wall polymers composing the Arabidopsis pollen tube cell wall. Comparison with predictions made by a mechanical model for pollen tube growth revealed the importance of pectin deesterification in determining the cell diameter. Scanning electron microscopy demonstrated that cellulose microfibrils are oriented in near longitudinal orientation in the Arabidopsis pollen tube cell wall, consistent with a linear arrangement of cellulose synthase CESA6 in the plasma membrane. The cellulose label was also found inside cytoplasmic vesicles and might originate from an early activation of cellulose synthases prior to their insertion into the plasma membrane or from recycling of short cellulose polymers by endocytosis. A series of strategic enzymatic treatments also suggests that pectins, cellulose, and callose are highly cross linked to each other.

The delivery of cell wall material and membrane to growing plant cell surfaces requires the spatial and temporal coordination of secretory vesicle trafficking. Given the small size of vesicles, their dynamics is difficult to quantify. To quantitatively analyze vesicle dynamics in growing pollen tubes labeled with the styryl dye FM1-43, we applied spatiotemporal correlation spectroscopy on time-lapse series obtained with high-speed confocal laser scanning microscopy recordings. The resulting vector maps revealed that vesicles migrate toward the apex in the cell cortex and that they accumulate in an annulus-shaped region adjacent to the extreme tip and then turn back to flow rearward in the center of the tube. Fluorescence recovery after photobleaching confirmed vesicle accumulation in the shoulder of the apex, and it revealed that the extreme apex never recovers full fluorescence intensity. This is consistent with endocytotic activity occurring in this region. Fluorescence recovery after photobleaching analysis also allowed us to measure the turnover rate of the apical vesicle population, which was significantly more rapid than the theoretical rate computed based on requirements for new cell wall material. This may indicate that a significant portion of the vesicles delivered to the apex does not succeed in contacting the plasma membrane for delivery of their contents. Therefore, we propose that more than one passage into the apex may be needed for many vesicles before they fuse to the plasma membrane and deliver their contents.

The cell wall is one of the structural key players regulating pollen tube growth, since plant cell expansion depends on an interplay between intracellular driving forces and the controlled yielding of the cell wall. Pectin is the main cell wall component at the growing pollen tube apex. We therefore assessed its role in pollen tube growth and cytomechanics using the enzymes pectinase and pectin methyl esterase (PME). Pectinase activity was able to stimulate pollen germination and tube growth at moderate concentrations whereas higher concentrations caused apical swelling or bursting in Solanum chacoense Bitt. pollen tubes. This is consistent with a modification of the physical properties of the cell wall affecting its extensibility and thus the growth rate, as well as its capacity to withstand turgor. To prove that the enzyme-induced effects were due to the altered cell wall mechanics, we subjected pollen tubes to micro-indentation experiments. We observed that cellular stiffness was reduced and visco-elasticity increased in the presence of pectinase. These are the first mechanical data that confirm the influence of the amount of pectins in the pollen tube cell wall on the physical parameters characterizing overall cellular architecture. Cytomechanical data were also obtained to analyze the role of the degree of pectin methyl-esterification, which is known to exhibit a gradient along the pollen tube axis. This feature has frequently been suggested to result in a gradient of the physical properties characterizing the cell wall and our data provide, for the first time, mechanical support for this concept. The gradient in cell wall composition from apical esterified to distal de-esterified pectins seems to be correlated with an increase in the degree of cell wall rigidity and a decrease of visco-elasticity. Our mechanical approach provides new insights concerning the mechanics of pollen tube growth and the architecture of living plant cells.