Functional Analysis of NET2A at the Actin-Membrane Interface of Plants

Abstract

Recently, NET2A was characterised as a novel, plant-specific actin-binding protein
that binds actin at the plasma membrane of growing pollen tubes (Deeks et al. 2012;
Dixon. 2013). However, a function for NET2A has not yet been identified.
Throughout the course of this investigation, several strategies were employed to
elucidate the roles of NET2A in Arabidopsis, including reverse-genetic analysis, in
situ localisation studies of NET2A in developing pollen grains and growing pollen
tubes, as well as several protein-protein interaction screens.
During this study, multiple NET2 subfamily members were shown to interact with
plasma membrane integral proteins, demonstrating novel mechanisms by which the
actin cytoskeleton is linked to the plasma membrane in plants. Through these
interactions, NET2 proteins are implicated in the regulation of pollen tube growth in
vivo during fertilisation, and regulation of the cytoskeleton in response to
extracellular signals. A role for NET2A in pollen grain development was also studied
during this investigation, in which NET2A was observed to associate with the actin
cytoskeleton of developing pollen grains, and undergo dynamic reorganisations in
subcellular localisation timed to specific developmental events in male
gametogenesis.
Reverse-genetic analysis of individual NET2 subfamily members has identified no
phenotypic growth defects in pollen grain development or fertilisation. Several lines
of evidence suggest functional redundancy and cooperation between individual
NET2 proteins, including their involvement with common interacting partners at the
plasma membrane, and interactions with one another in NET2 hetero-oligomers.
Additionally, this project describes the discovery of a novel microtubule-associated
protein in Arabidopsis. This protein, named herein as MAP7A, was demonstrated to
associate with microtubules directly, and localise to the pollen tube plasma
membrane and generative cell nucleus.
Described in this thesis are the potential roles for NET2A and MAP7A in the
regulation of the plant cytoskeleton during anisotropic cell growth.