Progress 10/01/13 to 09/30/16
Outputs
Target Audience:The target audience for this research is the plant biology community within Vermont and the US. We described our research results through an invited minisymposium talk at the 2014 meeting of the American Society of Plant Biology, an invited symposium talk in 2015 at the PanAmerican Plant Membrane Meeting in San Pedro de Atacama, Chile, an invited short talk in 2015 at the Plant Cell Wall Gordon Conference and through a poster session in 2016 at the International Workshop on Plant Membrane Biology in Anapolis, MD. These meetings are attended by plant biology faculty, graduate students and post-doctoral researchers interested in the function of plant cell walls. Publication of results from this work reached an international target audience of plant biologists involved in plant cell biology and cell wall biology research. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?Two female PhD students (Emily Larson and Suryatapa Jha) have been trained as part of this research project. Both students have had the opportunity to present their work at national and international meetings, both as poster presentations and short invited oral talks. These students have also participated in the annual university research forum and had presented a poster of their research. Emily Larson has finished her PhD and was mentored in the writing of two published manuscripts, both as a first author. Suryatapa Jha is currently writing a manuscript describing her work on the VPS26C retromer complex in Arabidopsis and will submit this by the end of Spring, 2017. Both Sury and Emily have also had the opportunity to mentor undergraduate students performing research in the lab. How have the results been disseminated to communities of interest?Results from this research have been disseminated to communities of interest through oral presentation at national meetings and in the form of peer-reviewed publications. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
One focus of this research was to identify and characterize proteins within an endosomal trafficking pathway critical for polarized growth of root hairs and cell wall organization in Arabidopsis. We initiated this by characterization of VTI13, a member of the VTI family of SNAREs involved in vesicle fusion events. These studies led to two publications. In the first [17], we show that VTI13 localizes to the vacuole membrane and an early endosomal compartment in roots. Genetic analysis was used to demonstrate that VTI13 is also required for polarized growth of root hairs and cell wall organization in roots and root hairs of Arabidopsis seedlings. VTI13 was also found to be required for the localization of SYP41, a SNARE that resides within the trans-Golgi network and functions in secretory and vacuolar transport pathways. In the second publication [21] we describe a method for labeling the surface of cell walls of live seedlings, eliminating the need for fixation or sectioning of plant material. This antibody labeling approach allows one to rapidly investigate the organization of wall components in the epidermis of various mutants to define the function of gene products involved in secretion or endosomal trafficking of wall components. Our second major focus has been to characterize a novel VPS26 subunit of the large retromer complex required for polarized growth in root hairs and cell wall organization in roots of Arabidopsis. Mutant alleles of VPS26C are defective in xyloglucan organization in roots and exhibit short root hairs when grown in the presence of mannitol or salt and this phenotype can be complemented by the introduction of a GFP-VPS26C fusion under the transcriptional control of its endogenous promoter. VPS26C shares only 28% amino acid sequence identity with VPS26A and VPS26B (the other VPS26 family members in Arabidopsis), but shares greater than 50% sequence identity with a subset of VPS26 amino acid sequences found in both animal and plants. Phylogenetic analysis showed that VPS26C is part of an ancient clade [22], suggesting that it may have evolved a novel retromer function. Consistent with this, we have shown that vps26a and vps26b mutants exhibit normal root hair growth. Based on work described above for a shared endosomal trafficking pathway including both VTI11 and the large retromer [2], we investigated whether VTI13 and VPS26C might share a common endosomal trafficking pathway as well. Analysis of a vti13 vps26c double mutant demonstrated that vps26c is a suppressor of the vti13 root hair phenotype, implicating VTI13 and VPS26C function in a common endosomal pathway required for cell wall organization and root hair growth. Based on these studies, we propose a model for VTI113 and VPS26C function in trafficking cargo to the vacuole and recycling proteins from the late endosome to the Golgi or trans-Golgi network. To confirm that VPS6C interacts with other large retromer complex proteins required for root hair growth, we examined null mutants in Arabidopsis for all of the other know large retromer complex proteins. This included null mutants for vps26a, b and c, vps29 and vps35a, b and c. We examined root hair growth for each of these mutants and compared it with that of wild type on normal MS media and MS media containing mannitol. Only vps35a and vps29 mutants exhibited shorter root hairs than wild type seedlings when grown in the presence of mannitol (similar to what was observed for vps26c; Fig. 2), predicting that VPS26C may form a complex with VPS35A and VPS29 to form a retromer subunit. To test this model, we used an approach called Bimolecular Fluorescence Complementation (BiFC). It has been shown that VPS26 and VPS35 proteins physically interact with each other in the large retromer subunit [7]. Therefore we fused the gene for VPS26C with the N-terminal half of YFP (yellow fluorescent protein) and each of the genes for VPS35 (A, B and C) with the C-terminal half of YFP. In addition, we constructed a mCherry-VPS29 fusion protein, as it has been shown that all three proteins within the large retromer subunit must be overexpressed together in these assays [7]. Agrobacterium strains containing the genes for these three fluorescent protein fusions were used to transfect tobacco leaves, and confocal analysis was used to determine 1) if VPS26C and one of the VPS35 proteins physically interacted (thus reconstructing a functional YFP signal) and 2) if the YFP signal colocalized with the signal from mCherry-VPS29. Only the VPS35A-cYFP fusion exhibited a physical interaction with VPS26C-nYFP and this signal did colocalize with mCherry-VPS29 (Fig. 3). These studies establish that VPS26C, VPS29 and VPS35A form a large retromer subunit in plants that is required for cell wall organization and root hair growth. We are preparing a manuscript describing this work and plan to submit it by the end of spring, 2017.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2014
Citation:
Larson ER, Domozych DS, Tierney ML. 2014. 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. Ann Bot. 114(6):1147-59.
Larson ER, Tierney ML, Tinaz B, Domozych DS. 2014. Using monoclonal antibodies to label living root hairs: a novel tool for studying cell wall microarchitecture and dynamics in Arabidopsis. Plant Methods. 10(1):30.
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Progress 10/01/13 to 09/30/14
Outputs
Target Audience: During this reporting period my graduate student, Suryatapa Jha, presented an oral presentation of her data as an invited minisymposium speaker at the annual meeting of the American Society of Plant Biologists. We have also published a manuscript from this work, describing a method to probe the organization of root and root hair cell walls with monoclonal antibodies using live seedlings. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided? My graduate student, Suryatapa Jha, has presented her research at the UVM student research day and as an invited minisymposium speaker at the annual meeting of the American Society of Plant Biologists in 2014. How have the results been disseminated to communities of interest? We have published one manuscript describing a method for labeling cell walls of live seedings with monoclonal antibodies raised against specific cell wall epitopes. Sury has also presented her work in poster and oral form at a national meeting. What do you plan to do during the next reporting period to accomplish the goals? We are currently in the process of generating a set of suppressor mutants of the vps26c phenotypes that should provide us with the identity of other proteins in the shared VPS26C/VTI13 endsomal trafficking pathway required to maintain root hair growth and root cell wall organization. We are also using a genetic approach,making double mutants with VTI13 and other retromer subunit mutants to define the other components of the large subunit of the retromer containing VPS26C. These studies will be complemented by using immunoprecipitation with GFP antibodies to identify retromer subunits that interact with VPS26C and other SNARE proteins that interact with VTI13.
Impacts
What was accomplished under these goals?
In this past year, we have focused on the function of a novel VPS26 gene family member, VPS26C (At1g48550). VPS26C is predicted to function in a protein conplex (the retromer) to recycle membrane proteins from the late endosomes and vacuole to the ER and Golgi. Null mutants of VPS26C are defective in root hair growth when seedlings are grown in the presence of mannitol (an osmotic stress) or in the presence of high salt and these phenotypes can be complemented with a GFP-VPS26C gene fusion. This suggests that VPS26C functions in an endosomal pathway required for growth in plants exposed to osmotic or salt stress. Preliminary studies, using these complemented seedlings, indicate that VPS26C is localized to the cytoplasm. We have also generated genetic data supporting a role for both VPS26C and the SNARE VTI13 in a common endosomal pathway important in cell wall organization and assembly. Immunohistochemistry of seedling roots with LM15, a monoclonal antibody recognizing xyloglucan in the cell wall, showed that while root epidermal and hair cells are strongly labeled in wild type seedlings, little to know labeling can be observed in either the vti13 or vps26c mutants. However, the vti13, vps26c double mutant has restored root hair growth and cell wall organization simlar to wild type seedlings. A phylogeny of VPS26C genes has been established in plants and shows that while VPS26C is represented in many angiosperms and several non-plant eukaryotes, it is absent from monocots.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2014
Citation:
Larson ER, Tierney ML, Tinaz B, Domozych DS. 20145. Using monoclonal antibodies to label living root hairs: a novel tool for studying cell wall microarchitecture and dynamics in Arabidopsis. Plant Methods. 10(1):30
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