2012 Annual Report
1a.Objectives (from AD-416):
Small GTPases termed ROPs (for Rho of plants) have been implicated in regulation of diverse signaling processes, including cell form, pattern formation, hormonal, biotic and abiotic stress responses. Pollen tube growth is an excellent system with which to study polar cell growth and dissect the roles of ROPs and their interacting proteins, the RhoGEFS (for Rho Guanine exchange factors) and the RhoGDIs (for Rho guanine dissociation inhibitor). ROPs have conserved cysteine residues that are substrates for S-acylation, which is performed by a family of proteins called S-palmitoyl transferases, or PATs. Mutations of cysteine residues in a type I ROP had profound effects on cell polarity, suggesting that S-acylation of these cysteine residues is also an important way to regulate ROP function. We therefore want to understand the functions of S-acylation in the regulation of ROP small GTPases. We will determine whether transient S-acylation occurs on all type-I ROPs, and if it occurs on type-II ROPS. We will determine if acylation influences whether and how ROPs interact with RhoGDIs. We will determine how mutations that compromise RhoGEF function in pollen affect the membrane distribution of ROPs. We will characterize the tissue specificity and individual roles for pollen-expressed PATs. We will determine the effects of silencing pollen-expressed S-palmitoyl transferases and determine the distribution of ROPs in such mutant lines. We will determine the association between ROP activation and S-acylation and the catalytic mechanisms involved in S-acylation. The conservation of these G-domain cysteines in the Rho family suggest that transient S-acylation may not be limited to plant small GTPases and thus the results of our proposed experiments might have implications for the roles of GTPases in other organisms. The research is expected to show whether transient S-acylation is general to all ROPs and whether S-acylation is required for ROP function in signaling cascades.
1b.Approach (from AD-416):
We will obtain T-DNA insertion lines, or, when necessary, generate silenced lines for the ROPs, RhoGDIs, RhoGEFS and PATs, and characterize their phenotypes.
We will biochemically fractionate membranes and use pulldown assays to determine the location of ROP, Rho-GEFs, RhoGDIs and PATs, in wild type and in mutant lines for each of these components.
We will use fluorescent protein fusions for each of these components in order to image their location in growing pollen tubes.
We will assay for in vivo interactions of these components, using Bimolecular fluorescence complementation assays.
This project involves analysis of the functions of signaling pathways during pollen tube growth; therefore, it relates to objective 1 of the parent project. This invloves elucidating the molecular interactions that occur during pollen development,pollen-pistil interactions, and sperm-egg interactions. PGEC scientists characterized the phenotypes of T-DNA insertions into several of the pollen-expressed Arabidopsis S-Palmitoyl Transferases (PATs) by carrying out pollen germination assays and scoring for reduction of seed set. No phenotypes were found in single mutants, suggesting that gene redundancy might exist. They also showed that some of the fluorescent protein fusion constructs of the Arabidopsis PATs introduced into stable transgenic plants exhibited different localization patterns than when the same constructs were transiently delivered into tobacco pollen. This finding shows that gene expression studies are best conducted in the native plant species.