Vacuolar Protein Sorting 26C encodes an evolutionarily conserved large retromer subunit in eukaryotes that is important for root hair growth in Arabidopsis thaliana.

The large retromer complex participates in diverse endosomal trafficking pathways and is essential for plant developmental programs, including cell polarity, programmed cell death and shoot gravitropism in Arabidopsis. Here we demonstrate that an evolutionarily conserved VPS26 protein (VPS26C; At1G48550) functions in a complex with VPS35A and VPS29 necessary for root hair growth in Arabidopsis. Bimolecular fluorescence complementation showed that VPS26C forms a complex with VPS35A in the presence of VPS29, and this is supported by genetic studies showing that vps29 and vps35a mutants exhibit altered root hair growth. Genetic analysis also demonstrated an interaction between a VPS26C trafficking pathway and one involving the SNARE VTI13. Phylogenetic analysis indicates that VPS26C, with the notable exception of grasses, has been maintained in the genomes of most major plant clades since its evolution at the base of eukaryotes. To test the model that VPS26C orthologs in animal and plant species share a conserved function, we generated transgenic lines expressing GFP fused with the VPS26C human ortholog (HsDSCR3) in a vps26c background. These studies illustrate that GFP-HsDSCR3 is able to complement the vps26c root hair phenotype in Arabidopsis, indicating a deep conservation of cellular function for this large retromer subunit across plant and animal kingdoms.

SNARE VTI13 plays a unique role in endosomal trafficking pathways associated with the vacuole and is essential for cell wall organization and root hair growth in arabidopsis.

BACKGROUND AND AIMS: Root hairs are responsible for water and nutrient uptake from the soil and their growth is responsive to biotic and abiotic changes in their environment. Root hair expansion is a polarized process requiring secretory and endosomal pathways that deliver and recycle plasma membrane and cell wall material to the growing root hair tip. In this paper, the role of VTI13 (AT3G29100), a member of the VTI vesicular soluble NSF attachment receptor (SNARE) gene family in Arabidopsis thaliana, in root hair growth is described. METHODS: Genetic analysis and complementation of the vti13 root hair phenotypes of Arabidopsis thaliana were first used to assess the role of VTI13 in root hair growth. Transgenic lines expressing a green fluorescent protein (GFP)-VTI13 construct were used to characterize the intracellular localization of VTI13 in root hairs using confocal microscopy and immunotransmission electron microscopy. KEY RESULTS: VTI13 was characterized and genetic analysis used to show that its function is required for root hair growth. Expression of a GFP-VTI13 fusion in the vti13 mutant background was shown to complement the vti13 root hair phenotype. GFP-VTI13 localized to both the vacuole membrane and a mobile endosomal compartment. The function of VTI13 was also required for the localization of SYP41 to the trans-Golgi network. Immunohistochemical analysis indicated that cell wall organization is altered in vti13 root hairs and root epidermal cells. CONCLUSIONS: These results show that VTI13 plays a unique role in endosomal trafficking pathways associated with the vacuole within root hairs and is essential for the maintenance of cell wall organization and root hair growth in arabidopsis.

CCDC22 and CCDC93, two potential retriever-interacting proteins, are required for root and root hair growth in Arabidopsis.

Endomembrane trafficking is essential for plant growth and often depends on a balance between secretory and endocytic pathways. VPS26C is a component of the retriever complex which has been shown to function in the recycling of integral plasma membrane proteins in human cell culture and is part of a core retriever complex in Arabidopsis that is required for root hair growth. In this work, we report a characterization of the Arabidopsis homologues of CCDC22 and CCDC93, two additional proteins required for retriever function in humans. Phylogenetic analysis indicates that CCDC22 (AT1G55830) and CCDC93 (AT4G32560) are single copy genes in plants that are present across the angiosperms, but like VPS26C, are absent from the grasses. Both CCDC22 and CCDC93 are required for root and root hair growth in Arabidopsis and localize primarily to the cytoplasm in root epidermal cells. Previous work has demonstrated a genetic interaction between VPS26C function and a VTI13-dependent trafficking pathway to the vacuole. To further test this model, we characterized the vti13 ccdc93 double mutant and show that like vps26c, ccdc93 is a suppressor of the vti13 root hair phenotype. Together this work identifies two new proteins essential for root and root hair growth in plants and demonstrate that the endosomal pathway(s) in which CCDC93 functions is genetically linked to a VTI13-dependent trafficking pathway to the vacuole.

Rapid Assessment of Cell Wall Polymer Distribution and Surface Topology of Arabidopsis Seedlings.

The deposition and modulation of constituent polymers of plant cell walls are profoundly important events during plant development. Identification of specific polymers within assembled walls during morphogenesis and in response to stress conditions represents a major goal of plant cell biologists. Arabidopsis thaliana is a model organism that has become central to research focused on fundamental plant processes including those related to plant wall dynamics. Its fast life cycle and easy access to a variety of mutants and ecotypes of Arabidopsis have stimulated the need for rapid assessment tools to probe its wall organization at the cellular and subcellular levels. We describe two rapid assessment techniques that allow for elucidation of the cell wall polymers of root hairs and high-resolution analysis of surface features of various vegetative organs. Live organism immunolabeling of cell wall polymers employing light microscopy and confocal laser scanning microscopy can be effectively performed using a large microplate-based screening strategy (see Figs. 1 and 2). Rapid cryofixation and imaging of variable pressure scanning electron microscopy also allows for imaging of surface features of all portions of the plant as clearly seen in Fig. 3.

Using monoclonal antibodies to label living root hairs: a novel tool for studying cell wall microarchitecture and dynamics in Arabidopsis.

BACKGROUND: The Arabidopsis root hair represents a valuable cell model for elucidating polar expansion mechanisms in plant cells and the overall biology of roots. The deposition and development of the cell wall is central to the root hair expansion apparatus. During this process, incorporation of specific wall polymers into the growing wall architecture constitutes a critical spatio-temporal event that controls hair size and growth rate and one that is closely coordinated with the cell's endomembrane, cytoskeletal and signal transduction apparatuses. RESULTS: In this study, the protocol for live cell labeling of roots with monoclonal antibodies that bind to specific wall polymers is presented. This method allows for rapid assessment of root hair cell wall composition during development and assists in describing changes to cell wall composition in transgenic mutant lines. Enzymatic "unmasking" of specific polymers prior to labeling allows for refined interpretation of cell wall chemistry. Live cell immunofluorescence data may also be correlated with transmission electron microscopy-based immunogold labeling. CONCLUSIONS: Live Arabidopsis root hairs may be labeled with cell wall polymer-specific antibodies. This methodology allows for direct visualization of cell wall dynamics throughout development in stable transgenic plant lines. It also provides an important new tool in the elucidation of the specific interactions occurring between membrane trafficking networks, cytoskeleton and the cell wall deposition/remodeling mechanism.