Progress 10/01/15 to 09/30/18
Outputs
Target Audience:The goal of this project is to enhance our understanding of the fundamental molecular processes by which the phytohormone, GA, regulates plant growth and development. In the short term, the research community is the primary target audience. Our research findings were presented at Midwest Plant Cell Dynamics Meetings (June 22-24th 2016), (May 30th-June 2nd2017) and (May29th- June 1st, 2018) as well as at the Pan American Membrane Biology Workshop (June 24th-June 27th, 2018). In addition, two manuscriptsare currently in preparation for submission to peer reviewed journals. In the long term, this project will potentially lead to the development of new strategies for the generation of crops with enhanced yield and quality for sustainable food, feed-stock and bio-fuel production. Therefore, the audience for the long-term applications of this project include growers, food processors, bioenergy producers and the general public. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?The project has provided research training in molecular genetics, biochemistry and imaging for young scientists at the undergraduate and graduate levels. The P.I.'s primary goal for the graduate students is for them to become independent research scientists. Toward this goal, they are encouraged to design well-controlled experiments, to critically interpret their own results and to organize this data into manuscripts that they write.Graduate students are also encouraged to attend local and national meetings and to establish professional contacts in their fields of interest and to present their findings through poster and oral presentations.The graduate students engaged in this project presented their findings at national meetings and are currently writing up their results for publication. Equally important for the training of graduate students is for them to develop mentoring skills. Five UW-Madison undergraduates, J. Grenier, M. Kachina Kimmel, C. Rollo. T. Sargent, H. Lier were engaged in independent research directly related to the project aims. These students have been being directly mentored by the graduate students involved in the project along with the PI. In addition to gaining "hands-on" research experience, the undergraduate research students receive guidance to develop communication and critical thinking skills through lab presentations and the reading and discussion of primary research papers related to their projects. How have the results been disseminated to communities of interest?Our research findings were presented at Midwest Plant Cell Dynamics Meetings (June 22-24th 2016), (May 30th-June 2nd2017) and (May29th- June 1st, 2018) as well as at the Pan American Membrane Biology Workshop (June 24th-June 27th, 2018). In addition, two manuscriptsare currently in preparation for submission to peer reviewed journals. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
Aim 1) Our in vitro and in planta biochemicalstudies have demonstrated that PUX1, a negative regulator of the chaperone AtCDC48, which is required for plant cell division and growth, directly binds to the Arabidopsis Gibberellin (GA) receptors, GID1a, b, and c (GA-INSENSITIVE DWARF1) through a region of PUX1 containing the UBX domain and that the N-terminus domain of GID1. In addition, we have demonstrated that the PUX1 colocalizes in the nucleus with GID1 and CDC48. These results are consistent with our hypothesis that GA-signaling via GID1 and PUX1 may directly regulate plant growth and development by controlling the levels and/or localization of CDC48. Aim 2) Previously we have shown that PUX1 promotes the disassembly and inactivation of the CDC48 complex. To further understand how PUX1 regulates CDC48-dependent plant growth and development we have investigated whether the activity of PUX1 is regulated by post-translational processing and the mechanism by which PUX1 mediates CDC48 disassembly. Various experiments aimed at defining the nature of PUX1 post-translational modification(s) have been inconclusive due to technical issues; alternative approaches are currently in progress to determine the cleavage sites within PUX1 protein and whether this process is regulated by GA-signaling. Binding of PUX1 to the N-terminal domain of CDC48 is required for disassembly of the hexameric complex. Our recent studies have identified mutations in several CDC48 N-terminal domain amino acids disrupt PUX1-dependent CDC48 disassembly but not binding. Further analysis of how these mutations affect PUX1-dependent regulation of CDC48 in vitro including purification of wild-type and mutant PUX1/CDC48 complexes for X-ray crystallographic analysis as well as the characterization of how the N-terminal CDC48 mutations that uncouple PUX1 binding and disassembly affect GA-dependent plant growth are in progress. Aim 3) Plants that are defective for GA biosynthesis and/or signaling are dwarfed. In contrast, plants with elevated levels of GA or GA signaling show enhanced growth due increased levels of cell division and elongation. Consistent with our hypothesis that GA/GID1 signaling regulates the levels of active hexameric CDC48, our recent analysis has demonstrated that of pux1 mutants show similar phenotypes as plants with enhanced GA responses. Compared to wild-type plants, pux1 mutants flower significantly earlier, have increased root growth length, and are more resistant to Paclobutrazol, a GA biosynthesis inhibitor. In addition, our studies have suggested that GA inhibits PUX1-mediated CDC48 disassembly. Ongoing experiments will determine whether plants lacking PUX1 have elevated levels of GA and/or GA signaling during various stages of plant growth and development.
Publications
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Progress 10/01/16 to 09/30/17
Outputs
Target Audience:This project seeks to enhance our understanding of the fundamental molecular processes by which the phytohormone, GA, regulates plant growth and development. In the short term, the research community is the primary target audience. During 2017 our research findings were presented at the Midwest Plant Cell Dynamics Meeting (Madison, Wisconsin: May 30th-June 2nd2017). In addition, our findings are currently being prepared for submission as a peer reviewed scientific research publication. In the long term, this project will potentially lead to the development of new strategies for the generation of crops with enhanced yield and quality for sustainable food, feed-stock and bio-fuel production. Therefore, the audience for the long term applications of this project include growers, food processors, bioenergy producers and the general public. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?The project provided research training in molecular genetics, biochemistry and imaging for young scientists at the undergraduate and graduate levels. The P.I.'s primary goal for the graduate students is for them to become independent research scientists. Toward this goal, they are encouraged to design well-controlled experiments, to critically interpret their own results and to organize this data into manuscripts that they write.Graduate students are also encouraged to attend local and national meetings, to establish professional contacts in their fields of interest, and to present their findings through poster and oral presentations.The graduate students engaged in this project presented their findings at the Midwest Plant Cell Dynamics Meetings held in St. Louis, Missouri (June 22-24th 2016) and in Madison, Wisconsin (May 30th-June 2nd2017). In addition, we are currently preparing a manuscript describing their findings for submission during 2018. Equally important for the training of graduate students is for them to develop mentoring skills. Three UW-Madison undergraduates, J. Grenier, M. Kachina Kimmel, and C. Rollo have been engaged in independent research directly related to the project aims. These students have been being directly mentored by the graduate students involved in the project along with the PI. In addition to gaining "hands-on" research experience, the undergraduate research students receive guidance to develop communication and critical thinking skills through lab presentations and the reading and discussion of primary research papers related to their projects. How have the results been disseminated to communities of interest?Our research findings were presented at the Midwest Plant Cell Dynamics Meeting (Madison, Wisconsin: May 30th-June 2nd2017). In addition, our findings are currently being prepared for submission as a peer reviewed scientific research publication. What do you plan to do during the next reporting period to accomplish the goals?Having demonstrated that the PUX1 UBX domain is required for the interaction of PUX1 and GID1 we will next determine the regions of GID1 required for their association in vitro. For these studies, we are currently analyzing the binding of a series of GID1 truncation proteins including the N-terminus of the GID1 GA receptor which contains the conserved DELLA protein interaction motifs, LExLE and VHYNP as well as a Small Ubiquitin-like Modifier Protein (SUMO) Interacting Motif (SIM), which facilitate the GA-independent association of GID1 and SUMO-conjugated DELLA. For the structural analysis studies, we will validate and optimize the use of co-expression to generate PUX1/CDC48 and PUX1/GID1 complexes in sufficient quantities for X-ray crystallography and/or high-resolution cryo-EM tomography. In addition, we will determine using mass spec analysis of purified GS-PUX1 protein, whether PUX1-38 undergoes proteolytic to generate the 34kD PUX1 isoform. Lastly, we will generate CFP-tagged CDC48 constructs with enhanced solubility and activity, and determine the utility of using FRET to monitor the process of PUX1-mediated CDC48 disassembly in vitro and in vivo.
Impacts
What was accomplished under these goals?
Aim 1) Structure/function studies will examine the binding of the GA receptor GID1, PUX1 and CDC48/p97 in vitro, and determine what protein domains are involved in this interaction: Previously we have shown using purified proteins that PUX1 physically interacts with the three Arabidopsis Gibberellin (GA) receptors, GID1a, b, and c (GA-INSENSITIVE DWARF1) and together with GID1 forms a ternary complex with CDC48, in vitro. To confirm the interaction of GID1 and PUX1, we generated Arabidopsis cell lines expressing N-terminal protein G/Streptavidin-binding peptide (GS)-tagged-PUX1 and -GID1 fusion proteins. PUX1 was found to co-purify with tandem affinity purified (TAP) GS-GID1 by immunoblot and LC/MS/MS indicating that PUX1 and GID1 physically interact in vivo. To determine the domains and binding sites required for PUX1 and GID1 interaction, we generated GST-tagged PUX1 domain and truncation mutant proteins including PUX1 N-terminal (aa 1-100), middle UBX domain (aa 88-211) and UBX-C terminus (aa 101-252). In this reporting period, we have assessed their binding to purified GID1a, b, and c. These binding studies have revealed that PUX1 association with GID1 is mediated through the UBX domain which is also required for its interaction and disassembly of hexameric CDC48. Taken together, these experiments have indicated that PUX1 binds to both the GA receptor, GID1, and CDC48 through its UBX domain. Expression, purification and in vitro binding studies of GID1 truncation mutant constructs is in progress to define the region(s) of GID1 necessary for its interaction with the PUX1 UBX domain. We have also initiated studies to characterize mechanism by which PUX1 promotes CDC48 subunit dissociation. Size exclusion chromatography coupled to multi-anglelight scattering studies are in progress to determine the stoichiometry of purified PUX1, hexameric CDC48 and the PUX1/CDC48 complex. In addition, preliminary work indicates that we can generate through co-expression of PUX1 with either GID1 or CDC48 in E. coli, PUX1/GID1 and PUX1/CDC48 complexes. To generate sufficient quantities of PUX1/CDC48 and PUX1/GID1 for X-ray crystallography, we are currently optimizing the co-expression conditions to enhance the yield of these proteins complexes. Aim 2) Determine whether the full length 38kDa form of PUX1 undergoes GA- and GID1-dependent post-translational processing and whether this processing event regulates PUX1-dependent CDC48/p97 inactivation: Coimmunoprecipiation studies have shown that the Gibberellin receptor, GID1 interacts with two forms of PUX1, a 38kD full-length and a 34 kD putative truncated form in plant total protein extracts. To determine 1) whether the 34kD form is the result of post-translational proteolytic processing of the 38kD form and 2) if GA modulates the relative abundance of PUX1-38 versus PUX1-34 in vivo, we have generated transgenic Arabidopsis plants and cell lines that express GFP-tagged and N-terminal protein G/Streptavidin-binding peptide (GS)-tagged PUX1 fusion proteins. Similar to endogenous PUX1, we have detected by immunoblot analysis full-length and truncated forms of GS-tagged PUX1. Full-length and truncated forms of GS-tagged PUX1 have been submitted for mass spectrometry analysis to determine the nature of the cleavage site. Determination of the site of PUX1-38 cleavage will be used to inform experiments outlined in aim 2B of the proposed studies. Aim 3) Test whether GA and GID1 signaling inhibits PUX1-mediated CDC48/p97 inactivation thereby promoting plant growth: In vitro studies have demonstrated that PUX1 promotes the formation of an inactive heteromeric complex comprised of PUX1 and a CDC48 protomer. To address whether GA modulates the levels of active vs inactive CDC48 in plant cells, we have quantitated the levels of hexameric CDC48 vs disassembled PUX1/CDC48 complexes in cytosolic extracts of suspension cultured Arabidopsis cells treated in the presence or absence of GA, by velocity sedimentation and immunoblot analysis using anti-PUX1 and -CDC48 antibodies. Consistent with our hypothesis that GA regulates the levels of active hexameric CDC48, we observed elevated levels of the ~8S PUX1/CDC48 in untreated cells, relative to GA-treated cells. To determine if GID1 is required for the observed GA-dependent inhibition of CDC48 disassembly we are currently testing whether gid1 mutants, defective for individual or all three GID1 isoform (i.e. gid1a, b,c), show increased levels of the inactive ~8S PUX1/CDC48 complex. In addition, development of a complementary Förster resonance energy transfer (FRET)-based assay to directly measure GA-dependent modulation of hexameric CDC48 containing fluorescent fusion protein YFP- and CFP-tagged CDC48 subunits activity in plant cells is in progress. We have successfully demonstrated that YFP-CDC48 proteins are ATPase active, undergo PUX1-mediated hexamer disassembly in vitro and rescue Atcdc48 loss-of-function mutants. However, our first-generation CFP-tagged CDC48 construct displays aberrant aggregation, likely due to oligomerization of the CFP tag. Work is in progress to generate functional CFP-tagged CDC48 constructs with reduced aggregation properties that will be subsequently analyzed in vitro and transformed into plants. Transgenic plants expressing functional YFP- and CFP-tagged CDC48 will be analyzed using standard confocal and FRET microscopy to directly monitor the effects of GA on the subcellular distribution of CDC48 and its oligomeric structure in wild-type, pux1 and gid1 mutant plant cells.
Publications
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Progress 10/01/15 to 09/30/16
Outputs
Target Audience:The goal of this project is to enhance our understanding of the fundamental molecular processes by which the phytohormone, GA, regulates plant growth and development. In the short term, the research community is the primary target audience. During 2016 our research findings were presented at the Midwest Plant Cell Dynamics Meeting (St. Louis, Missouri: June 22-24th 2016)and the American Society of Plant Biologist annual meeting (Austin, Texas; July 9-13th 2016). In addition, our findings are currently being prepared for submission as apeer reviewed scientific research publication. In the long term, this project will potentially lead to the development of new strategies for the generation of crops with enhanced yield and quality for sustainable food, feed-stock and bio-fuel production. Therefore, the audience for the long term applications of this project include growers, food processors, bioenergy producers and the general public. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?The P.I.'s primary goal for the graduate students engaged in this project is for them to become independent research scientists. Toward this goal they are encouraged to design well-controlled experiments, to critically interpret their own results and to organize this data into manuscripts that they write.Graduate students are also encourage to attend local and national meetings and to establish professional contacts in their fields of interest and to present their findings through poster and oral presentations.The graduate students engaged in this project presented their findings at the Midwest Plant Cell Dynamics Meeting (St. Louis, Missouri: June 22-24th 2016) and are currently preparing a manuscript describing their findings for submission during 2017. Equally important for the training of graduates students is for them to develop mentoring skills.Currently one UW-Madison undergraduate, Curtis Rollo is conducting an independent research projects directly related to the project aims. This student is being directly mentored by the graduate students involved in the project along with the PI. In addition to gaining "hands-on" research experience this students receives guidance to develop communication and critical thinking skills through the reading and discussion of primary research papers related to his project. How have the results been disseminated to communities of interest?The main research findings generated during the previous reporting period (and described in research accomplishment section) were presented at the Midwest Plant Cell Dynamics Meeting (St. Louis, Missouri: June 22-24th 2016) and the American Society of Plant Biologists' annual meeting (Austin, Texas; July 9-13th 2016). In addition, our findings are currently being prepared for submission as a peer reviewed scientific research publication. What do you plan to do during the next reporting period to accomplish the goals?To further characterize the mechanism by which the Gibberellin (GA) hormone and the Gibberellin receptor, GID1, regulate active versus inactive levels of CDC48/p97 our primary goals in the next reporting period will be to (1) determine whether PUX1-34 is generated through proteolytic processing of full-length PUX1-38, 2) define the domains of GID1 involved in the GID1/PUX1/CDC48 interaction, and (3) assess whether GA affects the subcellular localization of PUX1 and CDC48 in vivo.
Impacts
What was accomplished under these goals?
Aim 1) Structure/function studies will be examine the binding of the GA receptor GID1, PUX1 and CDC48/p97 in vitro, and determine what protein domains are involved in this interaction:The Gibberellin (GA) receptor, GID1 (GA-INSENSITIVE DWARF1), interacts in a GA independent manner with the ubiquitin regulatory X (UBX)-containing protein, PUX1, which negatively regulates CDC48/p97 by promoting the disassembly of the active homohexameric complex through direct protein binding. These findings suggest a previously undefined molecular mechanism by which GA controls plant growth and development. In this reporting period we have utilized in vitro binding studies with purified proteins to show that GID1, PUX1 and CDC48 form a ternary complex. These findings have been validated through the analysis of CDC48 and PUX1 complexes partially purified from Arabidopsis cell suspension cytosolic protein lysates. To determine the domains and binding sites required for PUX1 and GID1 interaction (ie.. structure/function studies), we generated GST-tagged PUX1 domain and truncation mutant proteins including PUX1 N-terminal (aa 1-100), middle UBX domain (aa 88-211) and UBX-C terminus (aa 101-252) and assessed their binding to purified GID1a, b, and c. These binding studies have revealed that PUX1 association with GID1 is mediated through the UBX domain which is also required for its interaction and disassembly of hexameric CDC48.Taken together, these experiments have indicated that PUX1 binds to both the GA receptor, GID1, and CDC48 through its UBX domain. Aim 2) Determine whether the full length 38kDa form of PUX1 undergoes GA- and GID1-dependent post-translational processing and whether this processing event regulates PUX1-dependent CDC48/p97 inactivation:Coimmunoprecipiation studies have shown that the Gibberellin receptor, GID1 interacts with two forms of PUX1, a 38kD full-length and a 34 kD putative truncated form in plant total protein extracts. To determine 1) whether the 34kD form is the result of post-translational proteolytic processing of the 38kD form and 2) if GA modulates the relative abundance of PUX1-38 versus PUX1-34 in vivo, we have generated transgenic Arabidopsis plants and cell lines that express GFP-tagged and N-terminal protein G/Streptavidin-binding peptide (GS)-tagged PUX1 fusion proteins.Transgenic GFP- and GS-tagged PUX1 plants will be analyzed by immunoblot analysis to determine if the GFP- and GS-tagged PUX1 fusion proteins, similar to the native PUX1, undergo proteolytic processing in a GA dependent manner. If so, the processed form of GS-tagged PUX1 will be purified and subjected to mass spectrometry analysis to determine the nature of the cleavage site. Determination ofthe site of PUX1-38cleavagewill be used to inform experiments outlined in aim 2B of the proposed studies. Aim 3) Test whether GA and GID1 signaling inhibits PUX1-mediated CDC48/p97 inactivation thereby promoting plant growth:In vitro studies have demonstrated that PUX1 promotes the formation of an inactive heteromeric complex comprised of PUX1 and a CDC48 protomer. To address whether GA modulates the levels of active vs inactive CDC48 in plant cells we have quantitated the levels of hexameric CDC48 vs disassembled PUX1/CDC48 complexes in cytosolic extracts of suspension cultured Arabidopsis cells treated in the presence or absence of GA, by velocity sedimentation and immunoblot analysis using anti-PUX1 and -CDC48 antibodies. Consistent with our hypothesis that GA regulates the levels of active hexameric CDC48, we observed elevated levels of the ~8S PUX1/CDC48 in untreated cells, relative to GA-treated cells. To determine if GID1 is required for the observed GA-dependent inhibition of CDC48 disassembly we are currently testing whether gid1 mutants show increased levels of the inactive ~8S PUX1/CDC48 complex. In addition, we have initiated the development of a complementary assay to directly measure GA-dependent modulation of CDC48 activity in plant cells. For these studies we have generated and demonstrated that fluorescent fusion protein YFP- and CFP-tagged CDC48 proteins are ATPase active and undergo PUX1-mediated hexamer disassembly in vitro. Transgenic plants expressing functional YFP- and CFP-tagged CDC48 will be generated in the next reporting period to directly monitor GA and subcellular site of CDC48 disassembly in wild-type, pux1 and gid1 mutant plant cells.
Publications
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