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= Pollen Tube =

Overview
Pollen tube elongation is an integral stage in the plant life cycle. Once a pollen grain has implanted on a compatible stigma via self-incompatibility mechanisms, the germination process is initiated. During this process, the pollen grain undergoes a conformational change whereupon a given section begins to protrude outwards to form a tube-like structure, known as the pollen tube. This structure rapidly descends down the length of the style via tip-directed growth, reaching rates of 1cm/h, whilst carrying two non-motile sperm cells. Upon reaching the ovule the pollen tube ruptures, thereby delivering the sperm cells to the female gametophyte, ultimately resulting in a double fertilization event. The first fertilization event produces a diploid zygote and the second fertilization event produces a triploid endosperm.

The actin cytoskeleton has proven to be critical in assisting pollen tube growth. In terms of spatial distribution, actin filaments are arranged into three different structures within the pollen tube. Each unique arrangement, or pattern, contributes to the maintenance of polarized cell growth characteristic of the pollen tube. In the apical region - the site of tip-directed growth- actin filaments are less abundant, however they are highly dynamic. Furthermore, small vesicles accumulate in the apex, indicating that this region is the site of critical vesicle targeting and fusing events. Such events are essential for regulating the velocity and direction of pollen tube growth. In the subapical region, actin filaments are arranged into a collar-like structure. Reverse-fountain cytoplasmic streaming occurs at the subapex; the direction of cytoplasmic streaming is reversed and continues along the axial actin cables comprising the shank. The shank region comprises the central part of the pollen tube. In this region, actin filaments are arranged into axial bundles of uniform polarity, thereby enabling the transport of various organelles and vesicles from the base of the pollen tube to the tip, propelling overall tube growth.

Study of Actin Filament Dynamics in The Pollen Tube via Actin Markers
Both the spatial distribution and dynamics of the actin cytoskeleton are regulated by actin-binding proteins (ABPs). In order to experimentally observe distributional changes that take place in the actin cytoskeleton during pollen tube growth, green fluorescent proteins (GFPs) have been put to use. GFPs were mainly selected for the purposes of dynamic visualization due to the fact that they provided an efficient means for the non-invasive imaging of actin filaments in plants. Amongst the various GFPs employed during experimentation were GFP-mTalin, LIM-GFP and GFP-fimbrin/ABD2-GFP. However, each of these markers either disrupted the natural structure of the actin filaments or unfavorably labeled such filaments. For example, GFP-mTalin resulted in excessive filament bundling and GFP-fimbrin/ABD2-GFP did not label actin filaments located in the apical or subapical regions of the pollen tube. In light of these drawbacks, Lifeact-mEGFP has been designated as the prominent marker of choice for actin filaments in the pollen tube; Lifeact-mEGFP is able to detect all three arrangements of actin filaments, and it has minimal effects on the natural structure of actin filaments. Lifeact-mEGFP has been used as a marker to study the dynamics of actin filaments in the growing pollen tubes of tobacco, lilies and Arabidopsis.

Through studies conducted with GFP, it has been confirmed that the dynamic state of actin filaments located in the apical region are essential for pollen tube growth. Experimentation of actin filaments stained with GFP-mTalin have yielded results confirming that tip-localized actin filaments are highly dynamic. Such experimentation has made a connection between the dynamics of tip-localized actin filaments and their role in the formation of actin structures in the subapical region. Furthermore, experimentation of actin filaments located in the apical dome of Arabidopsis indicates that actin filaments are continuously produced from the apical membrane of the pollen tube; the production of these actin filaments are mediated by formins. These findings have provided evidence supporting the theory that actin filaments located in the apical region are highly dynamic and are the site of vesicle targeting and fusing events. Experimentation of etiolated hypocotyl cells as well as BY-2 suspension cells show that highly dynamic actin filaments produced from the apical membrane can either be turned over by filament severing and depolarizing events, or they can move from the apex to the apical flank, resulting in decreased accumulation of actin filaments in the apical region of the pollen tube.

Experimentation of actin filament dynamics in the shank region were also conducted with the use of GFP. Findings indicated that maximum filament length in this region significantly increased, and the severing frequency significantly decreased. Such findings indicate that actin filaments located in the shank region are relatively stable compared to actin filaments located in the apical and subapical regions.

ABPs and Their Role in the Regulation of Actin Dynamics in the Pollen Tube
ABPs regulate the organization and dynamics of the actin cytoskeleton. As stated previously, actin filaments are continuously synthesized from the apical membrane. This indicates the presence of membrane-anchored actin nucleation factors.Through experimentation, it has been theorized that formins are representative of such actin nucleation factors. For example, formin AtFH5 has been identified as a major regulator of actin filament nucleation, specifically for actin filaments synthesized from the apical membrane of the pollen tube. Genetic knockouts of AtFH5 resulted in a decreased abundance of actin filaments in both apical and subapical regions of the pollen tube, thereby providing more evidence to support the theory that AtFH5 nucleates actin filament assembly in apical and subapical regions of the pollen tube.

Class I formin AtFH3 is another actin nucleation factor. AtFH3 nucleates actin filament assembly of the longitudinal actin cables located in the shank region of the pollen tube. More specifically, AtFH3 uses the actin/profilin complex in order to interact with the end of actin filaments, thereby initiating actin filament nucleation. Furthermore, actin filament binding proteins organize actin filaments into bundles and maintain longitudinal arrangements of actin bundles located in the shank region of pollen tubes. In vitro experimentation of Arabidopsis show that FIMBRIN5 is an actin bundling factor that stabilizes actin filaments. Dysfunctional FIMBRIN5 causes disorganization of actin filaments in the pollen tube and alters the arrangement of longitudinal actin cables. As a result, cytoplasmic streaming patterns are altered, thereby decreasing the velocity and direction of pollen tube elongation. Overall, non-functioning FIMBRIN5 impedes pollen germination as well as polarized tube growth.