Abstract
Pollen tubes are an excellent system for studying the cellular dynamics and complex signaling pathways that coordinate polarized tip growth. Although several signaling mechanisms acting in the tip-growing pollen tube have been described, our knowledge on the subcellular and molecular events during pollen germination and growth site selection at the pollen plasma membrane is rather scarce. To simultaneously track germinating pollen from up to 12 genetically different plants we developed an inexpensive and easy mounting technique, suitable for every standard microscope setup. We performed high magnification live-cell imaging during Arabidopsis pollen activation, germination, and the establishment of pollen tube tip growth by using fluorescent marker lines labeling either the pollen cytoplasm, vesicles, the actin cytoskeleton or the sperm cell nuclei and membranes. Our studies revealed distinctive vesicle and F-actin polarization during pollen activation and characteristic growth kinetics during pollen germination and pollen tube formation. Initially, the germinating Arabidopsis pollen tube grows slowly and forms a uniform roundish bulge, followed by a transition phase with vesicles heavily accumulating at the growth site before switching to rapid tip growth. Furthermore, we found the two sperm cells to be transported into the pollen tube after the phase of rapid tip growth has been initiated. The method presented here is suitable to quantitatively study subcellular events during Arabidopsis pollen germination and growth, and for the detailed analysis of pollen mutants with respect to pollen polarization, bulging, or growth site selection at the pollen plasma membrane.
Highlights
The pollen tube (PT) of flowering plants is formed by the pollen grain vegetative cell and represents a cell of enormous specialization, responsible for the transport of the two male gametes through the female tissues of the pistil to the ovule
We focused on early cell polarization events during pollen activation and germination by studying the spatiotemporal localization of green fluorescent protein (GFP)-labeled Armadillo Repeat Only 1 (ARO1), which is known to be essential for polar PT growth (Gebert et al, 2008)
Pollen germination and PT growth of up to 12 genetically different plants can be simultaneously observed over many hours when using the 12-well micro-germination setup
Summary
The pollen tube (PT) of flowering plants is formed by the pollen grain vegetative cell and represents a cell of enormous specialization, responsible for the transport of the two male gametes through the female tissues of the pistil to the ovule. Pollen can be germinated in vitro to study the cellular dynamics and complex signaling pathways that coordinate polar tip growth (Qin and Yang, 2011) From these studies we know that intensive exo- and endocytosis at the tip supported by regulated vesicle trafficking and cytoskeleton dynamics, as well as coordinated changes in cell wall properties are essential cellular activities of the growing PT (for review see Geitmann, 2010; Guan et al, 2013). Great advances have been made during the past years in identifying key signaling molecules for the proper elongation of the PT tip, such as Rho GTPases, calcium ions, and phosphoinositides (for review see Cheung and Wu, 2008; Qin and Yang, 2011; Steinhorst and Kudla, 2013) These key regulators are components of distinct signaling pathways forming a complex network that controls the cellular activities of tip-growing PTs (Guan et al, 2013). There are still significant gaps in our knowledge of PT growth regulation, especially with regard to the question when and how symmetry breaking in the apparently unpolar pollen vegetative cell occurs, and what the molecular mechanism for selecting the growth site is
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