Progress 09/01/07 to 08/31/10
Outputs
OUTPUTS: Activities: We performed experiments to determine the role of phosphatidic acid (PA)-protein phosphatase 2A (PP2A) interactions in regulating hormonal responses to abiotic stress conditions in rice and Arabidopsis. Our data support the hypothesis that PA negatively regulates PP2A by binding to the regulatory/scaffolding subunit of the heterotrimeric holoenzyme complex. We developed rice as a model monocot system for PP2A studies, investigated the role of PA-PP2A interactions in regulating root growth during salt stress, and pursued experiments to isolate the PA binding domains of PP2A A-subunits in rice and Arabidopsis. Outputs: The PI has developed the project and attained a tenure-track faculty position at the Ohio State Agricultural Research and Development Center (OARDC), the Ohio State University, focused on the biochemistry of plant responses to abiotic stress conditions. We provided mentoring and research opportunities to Honors students from Brown University and a student from an under-represented minority group through the Leadership Alliance (LA). One of our Brown undergraduates from an under-represented minority (Latino) earned an authorship on our Plant Physiology MS, and is pursuing a research career in the University of California system. Our LA student's research was presented at the Brown summer research symposium and the LA's national conference (East Brunswick, N.J.), and has earned the student authorship on our M.S. This student is currently enrolling in graduate school in molecular/cellular biology. Events: We have presented results obtained at several conferences including: -Shoemaker, E., Blakeslee, J., DeLong, A. (2010). Does Phosphatidic Acid Modulate the Effect of Protein Phosphatase 2A in Salt Stress Signaling Leadership Alliance National Symposium (LANS), 7/30/10-8/1/10, East Brunswick, NJ (talk). -Shoemaker, E., Blakeslee, J., DeLong, A. (2010). Phosphatidic Acid Modulation of Protein Phosphatase 2A. Brown Summer Research Symposium, 7/10 (poster). - Blakeslee, J.J., DeLong, A. (2010). Phosphatidic acid is a stress-induced regulator of protein phosphatase 2A. April 2010 New England Arabidopsis Meeting (NEAM), 4/7/10, Providence, RI (talk). - Blakeslee, J.J., DeLong, A. (2010). Phosphatidic acid inhibits protein phosphatase 2A activity in Arabidopsis and rice. 3rd Pan-American Membrane Biology Workshop: Jan13th- 16th, Puebla, Mexico (talk and poster). Research was selected for an early career talk and awarded a travel grant. - Blakeslee, J.J., Heath, J.T., Skottke, K.R., DeLong, A. (2008). Analysis of RCN1- Specific Regulation of Protein Phosphatase 2A. Gordon Research Conference: Plant Molecular Biology, July 13th-18th, Holderness, NH (poster). Products: -A semi-hydroponic rice growing system was developed and constructed at Brown University. This system will be adapted and installed at the OARDC. -amiRNAi lines designed to suppress expression of the rice PP2AA-subunit gene RPA1 were generated through a collaboration with the laboratory of Prof. Pamela Ronald, U.C. Davis, and are being characterized for gene expression and phenotype(s). Dissemination: See above, Events. PARTICIPANTS: Individuals: Joshua Blakeslee, Brown University, PI: Formulated hypotheses, designed and conducted experiments, mentored undergraduate honors students, mentored Leadership Alliance student. Alison DeLong, Brown University, research mentor: formulated hypotheses, helped design experiments, mentored undergraduate honors students, mentored Leadership Alliance student. Collaborators: Pamela Ronald, University of California, Davis, collaborator: constructed rice amiRNAi lines to suppress expression of rice PP2A A-subunit gene RPA1. Training/Professional Development: Professional development: The PI has developed the research performed through the funding period into an independent project and attained a tenure-track faculty position at the Ohio State Agricultural Research and Development Center, Ohio State University focused on the biochemistry of plant responses to abiotic stress conditions. Trainig of Honors students, Brown University: Throughout the project, we provided mentoring and research opportunities to Honors students from Brown University. Students were trained in confocal & standard microscopy and image analysis techniques, molecular cloning, Agrobacterial plant transformation and transformant screening, protein expression and purification, and basic plant physiology. As a result of these research opportunities, one of our undergraduates from an under-represented minority group (Latino) earned an authorship on our plant physiology MS, and is currently pursuing other research opportunities in the University of California system. Training of under-represented students through the Leadership Alliance: In the final year of the project, we provided mentoring and a summer research opportunity to a student from an under-represented minority group through the Leadership Alliance. The Leadership Alliance is a consortium of universities gathered together to provide research opportunities and training to traditionally under-represented groups, with the goal of increasing the presence of these groups in academic research, business, and the public sector (http://www.theleadershipalliance.org/). Our LA student designed and conducted plant physiology experiments designed to investigate the role of phosphatidic acid-PP2A interaction in controlling root growth under salt stress conditions. The results of the student's summer research have been presented at both the Brown University summer research symposium and the Leadership Alliance's national conference in East Brunswick, N.J., and have earned the student an authorship on our forthcoming M.S. (see below). Finally, as a result of the summer research experience, the student is currently enrolling in graduate school and pursuing a career in molecular/cellular biology. TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Our work has yielded significant insights into the role of the membrane signaling molecule phosphatidic acid (PA) in regulating protein phosphatase 2A (PP2A) activity. Initially, we outlined two mechanisms by which PA might regulate PP2A activity. In the first mechanism, PA binding to the A subunit of the PP2A heterotrimeric enzyme complex relocalizes the PP2A holoenzyme to a cellular membrane compartment and alters activity by sequestering PP2A within a specific substrate milieu. In the second mechanism, PA alters PP2A activity by binding to the A subunit and allosterically altering enzyme activity. Our data have provided support for the hypothesis that PA allosterically regulates both rice and Arabidopsis PP2A activity via A-subunit binding. When PP2A enzymatic assays were supplemented with a vesicular suspension of phosphatidic acid, PA substantially inhibited PP2A activity. Both Arabidopsis and rice PP2A complexes were highly sensitive to PA, as PP2A activity in whole-plant extracts was reduced by approximately 50% at 10 uM PA in both species; indicating that PA responsiveness is conserved across PP2A complexes in monocots and dicots. We hypothesized that PA acts on PP2A by binding to the regulatory/scaffolding A-subunit of the PP2A complex. To investigate this hypothesis, we measured PP2A activity in the Arabidopsis rcn1 mutant, which lacks the PP2AA1 subunit (but retains functional PP2AA2 and PP2AA3 subunits). Interestingly, PP2A activity in rcn1 is as strongly inhibited by PA as in the wild-type control. PA inhibition of PP2A activity is also conserved in pp2aa2 pp2aa3 mutants, which lack both the PP2AA2 and PP2AA3 subunits. These data suggest that PA binding and responsiveness is conserved across all A subunits in Arabidopsis and rice. To provide further support for our phosphatidic acid-A subunit interaction model, we developed a method to co-immunoprecipitate active, functional PP2A holoenzyme complexes from Arabidopsis YFP-tagged A subunit lines. We assayed PP2A activity from immunoprecipitated complexes, and confirmed the ability of PA to inhibit phosphatase activity. Inhibition of PP2A activity shows specificity for phosphatidic acid, as the membrane phospholipid phosphatidylglycerol was significantly less inhibitory than PA on PP2A activity in both immunoprecipitated PP2A complexes and whole plant extracts. We also investigated the specificity of PP2A-PA interactions using a 'Far-Western' blotting approach, in which protein lipid-interactions are assayed using lipids immobilized on a nitrocellulose matrix. Using this system, we demonstrated PA binding by the A subunits both rice and maize, supporting the idea that the A-subunit of the PP2A complex is responsible for PA binding, with only a minor contribution possibly made by the C-subunit. We are using this system to assay lipid-binding specificities of full-length YFP- and HA-tagged RPA1/RCN1 constructs, as well as RPA1/RCN1 deletion constructs, which will be used to determine PA binding regions. These experiments will be continued as part of the research program of a new laboratory in the Department of Horticulture and Crop Science at the Ohio State University.
Publications
- Blakeslee, J.J., Skottke, K.R., Shoemaker, E., DeLong, A. (2010). Phosphatidic acid inhibits protein phosphatase 2A activity via interactions with the regulatory/scaffolding A-subunit, in preparation.
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Progress 09/01/08 to 08/31/09
Outputs
OUTPUTS: Activities: Throughout the reporting period, we have focused on pursuing experiments described in the experimental approach section of the proposal (pages: 8-12). Specifically, we have focused on experiments designed to characterize the role of PP2A in salt stress responses in rice, as well as assaying the role of phosphatidic acid in allosterical regulation of PP2A activity in rice and Arabidopsis. Additionally, we have pursued experiments designed to isolate the phosphatidic acid binding domains of PP2A A-subunits in both rice and Arabidopsis. Finally, we have mentored undergraduate students involved in independent research projects at Brown University. One undergraduate student assisted in performing experiments designed to assay the role of PP2A in mediating salt stress responses in rice roots. The student learned how to grow rice hydroponically, prepare samples, and analyze and present data. We are currently mentoring a second undergraduate student who is involved in constructing truncated versions of A-subunit proteins to isolate phosphatidic acid binding domains. This student is being trained in standard molecular biology techniques, as well as protein expression, purification, and analysis. Events: Results obtained in our experimental studies (described in the outcomes/impacts section below) will be presented at the third Pan-American Plant Membrane Biology Workshop, in Pueblo, Mexico, which has been re-scheduled for Jan. 14-21, due to the recent swine flu outbreak. The research funded by the award has been selected for an early career talk at the Membrane Biology Workshop and has been awarded a travel grant to cover meeting travel and expenses. Products: As mentioned in a previous update, we have developed and constructed a rice culture/growing system for use in the Brown University Plant Environmental Center greenhouse (prior to these experiments, rice had not been grown in the University facilities). Using this system, we have been able to maximize our seed harvest, and minimize generation time to roughly 2-2.5 months for all cultivars used. This system has allowed us to become completely self-sufficient regarding our experimental rice seed needs. Additionally, we are in the final stages of our construction of amiRNAi lines to suppress expression of the single-copy rice PP2AA-subunit gene RPA1. We have completed construction of amiRNAi plasmids, which are currently being transformed into rice plants through a collaboration with the laboratory of Prof. Pamela Ronald, at U.C. Davis, and should be completed by approximately 2/10. Dissemination: Results and data obtained were presented in a research seminar at the University of Nebraska-Lincoln on Nov. 12, 2009. Additional experimental data will be presented at the Third Pan-American Membrane Biology Workshop, in Pueblo, Mexico, in Jan. 2010. PARTICIPANTS: Joshua Blakeslee, PI, Post-doctoral research associate, MCB Dept., Brown University. Designed and conducted experiments, mentored undergraduate research students. Alison DeLong, PI, Associate professor, MCB Dept., Brown Univeristy. Mentored Joshua Blakeslee and undegraduate students. Pamela Ronald, collaborator, Professor, Plant Pathology; Vice President of Feedstocks, Joint Bioenergy Institute; University of California, Davis. Transformation of rice RPA1 RNAi lines. TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
Our experimental work to date has provided increasing support for the hypothesis that PA allosterically regulates both rice and Arabidopsis PP2A activity via A-subunit binding. When PP2A enzymatic assays were supplemented with a vesicular suspension of phosphatidic acid, PA was able to substantially inhibit PP2A activity. Arabidopsis PP2A appears to be highly sensitive to PA, as PP2A activity in whole-plant extracts is reduced by approximately 50% at a PA concentration of 10 uM. This concentration is much lower than has been reported for the PA-sensitive phosphatase ABI1; 50% inhibition of ABI1 occurs at approximately 100 uM PA (Zhang et al., 2004). PP2A activity in the Arabidopsis rcn1 mutant, which lacks the PP2AA1 subunit, is as strongly inhibited by PA as the wild-type control. PA inhibition of PP2A activity is also conserved in pp2aa2 pp2aa3 mutants, which lack both the PP2AA2 and PP2AA3 subunits. In support of the hypothesis put forth in our proposal, rice PP2A activity responds to PA in a manner identical to that observed in Arabidopsis, with 50% inhibition at a PA concentration of 10 uM. These data suggest that PA binding and responsiveness is conserved across all A subunits in Arabidopsis and rice, and that all PP2A complexes containing a functional A subunit contribute to PA responses. Therefore, while relocalization of PP2A complexes following PA binding remains a formal possibility, our data suggest that it is much more likely that PA exerts its effect on PP2A function via allosteric regulation. To investigate the PP2A-PA interaction further, we have developed a method to isolate active, functional PP2A holoenzyme complexes from our Arabidopsis YFP-tagged A subunit lines using co-immunoprecipitation. We have assayed PP2A activity from immunoprecipitated complexes, and have confirmed the ability of phosphatidic acid to inhibit phosphatase activity. Inhibition of PP2A activity shows specificity for phosphatidic acid, as addition of the membrane phospholipid phosphatidylglycerol was siginificantly less inhibitory than phosphatidic acid on PP2A activity in both immunoprecipitated PP2A complexes and whole plant extracts. We are currently investigating the specificity of PP2A-PA interactions using a "Far-Western" blotting approach, in which lipids are immobilized on a nitrocellulose matrix and assayed for their ability to bind proteins from either whole plant or bacterial expression systems. Using this system, we have been able to demonstrate PA binding by the A subunits of the PP2A complexes of the monocot crop species rice and maize. Binding studies support the idea that the A-subunit of the PP2A complex is primarily responsible for PA binding, with only a minor contribution possibly made by the C-subunit. We are currently using this system to assay full-length YFP- and HA-tagged RPA1/RCN1 constructs expressed in bacteria and Arabidopsis suspsension cell cultures for lipid binding specificity. We have also begun the construction of RPA1/RCN1 deletion constructs for use in our Far-Western blotting assays in order to determine the specific PA binding regions of these proteins.
Publications
- Blakeslee, J.J., DeLong, A. (2010). Phosphatidic Acid Inhibits Protein Phosphatase 2A Activity in Arabidopsis and Rice. Early Career Talk, 3rd Pan-American Membrane Biology Workshop, Jan. 13-16th 2010, Puebla, Mexico.
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Progress 09/01/07 to 08/31/08
Outputs
OUTPUTS: Activities: Throughout the reporting period, we have focused on pursuing experiments described in the experimental approach section of the proposal (pages: 8-12). Specifically, we have focused on experiments designed to both develop rice as a model monocot crop species for PP2A studies and assay for the role of phosphatidic acid in regulating PP2A activity via either relocalization or allosteric inhibition in rice and Arabidopsis. Additionally, we have mentored undergraduate students involved in research projects at Brown University. One undergraduate student assisted in generating confocal microscopy images of the localization of protein phosphatase 2A subunits in Arabidopsis roots. The student learned how to prepare samples and use the confocal microscope (i.e. setting exposure time, laser intensities, focal planes, etc.), and earned authorship on our Plant Physiology paper. Additionally, we are currently mentoring a second undergrad student, and have trained him in basic molecular biology techniques, as well as Agrobacterial plant transformation. Events: We have presented results obtained in our experimental studies (described in the outcomes/impacts section below) at the Gordon Research Conference for Plant Molecular Biology in Holderness, NH (July 13-18th 2008). Products: We have developed and constructed a rice culture/growing system for use in the Brown University Plant Environmental Center greenhouse (prior to these experiments, rice had not been grown in the University facilities). In our system, the plants are grown in a 1:1 mix of Cornell rice growing media and Turface. Seeds are germinated in a growth chamber and then transferred to a hydroponic growing tray with extensive aeration. Plants are grown for several weeks under a cultivar-specific fertilization and iron supplementation regime, as we have found that different cultivars exhibit profound differences in sensitivity to fertilizer concentration which can result in drastically reduced seed set. Using this system, we have been able to maximize our seed harvest, and minimize generation time to roughly 2-2.5 months for all cultivars used. To date we have been able to generate over 400 grams of viable seed, which we have been able to use in our rice PP2A enzyme and salt dose response assays. Dissemination: Results and data obtained were presented at the Brown University annual Molecular Biology, Cellular Biology, and Biochemistry Graduate Program Retreat. Additionally, we presented our data in a talk at the New England Arabidopsis Meeting (a bi-monthly meeting of plant biology laboratories in the New England area) at the Whitehead Institute (Cambridge, MA) in February 2008. PARTICIPANTS: Joshua Blakeslee, PI, Post-doctoral research associate, MCB Dept., Brown University. Designed and conducted experiments, mentored undergraduate research students. Alison DeLong, PI, Associate professor, MCB Dept., Brown Univeristy. Mentored Joshua Blakeslee and undegraduate students. TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
Our experiments to date have yielded promising preliminary data on the ability of phosphatidic acid (PA) to regulate PP2A activity. In our original proposal, we outlined two mechanisms by which phosphatidic acid might regulate PP2A activity. In the first mechanism, PA binding to the A subunit of the PP2A heterotrimeric enzyme complex promotes re-localization of the PP2A holoenzyme complex to a specific cellular membrane compartment, and alters activity by sequestering PP2A within a specific substrate milieu. In the second mechanism, PA alters PP2A activity by binding to the A subunit and allosterically altering enzyme activity. We have made good progress in our initial characterization of PP2A function and stress responses in rice. Previous studies have identified PP2A subunits in rice, but assay conditions for PP2A enzyme complexes have not been established. We have optimized the assay conditions for PP2A enzymatic assays using rice tissue extracts, and are currently performing PA dosage curves in this optimized system. We have performed salt dosage-response curves to establish a range of NaCl concentrations suitable for future stress experiments. Based on these data, we have selected a NaCl concentration of 150 mM for future experiments, much higher than the 80 mM NaCl commonly used in Arabidopsis salt-stress experiments. We have also completed preliminary dose-response experiments assaying the effects of increasing concentrations of the PP2A inhibitor cantharidin on rice growth and root development. Concurrent with these studies in rice, we have continued the analysis of PA-regulation of PP2A function in Arabidopsis. Thus far, studies in Arabidopsis using YFP-tagged A subunit constructs have not supported the hypothesis that stress-induced PA production leads to a re-localization of the PP2A enzyme complex. It is possible, however, that PA binding may lead to a re-localization of PP2A complexes between different membrane microdomains or at a level below our detection limit. We also assayed the ability of PA to allosterically regulate PP2A activity via A-subunit binding. We were able to supplement our PP2A enzymatic assays with a vesicular suspension of phosphatidic acid, and found that PA was able to substantially inhibit PP2A activity. PP2A activity appears to be highly sensitive to PA, as activity is reduced by approximately 50% at a PA concentration of 10 uM. This concentration is much lower than has been reported for other PA-sensitive phosphatases such as ABI1, in which 50% inhibition occurs at approximately 100 uM PA (Zhang et al., 2004). Interestingly, the rcn1 mutant, which lacks the PP2AA1 subunit, but which retains functional PP2AA2 and PP2AA3 subunits, responds to PA in a manner identical to the wild-type control. These data suggest that PA binding and responsiveness is conserved across all A subunits, and that PP2A complexes containing any A subunit contribute to PA responses. These data are encouraging, as the common putative PA binding regions conserved across the Arabidopsis A subunit family are also conserved in the rice single-copy PP2A A-subunit, RPA1.
Publications
- Blakeslee, J.J., Zhou, H.W., Heath, J.T., Skottke, K.R., Barrios, J.A., Liu, S.Y., DeLong, A. (2008). Specificity of RCN1-mediated protein phosphatase 2A regulation in meristem organization and stress response in roots. Plant Physiology, 146 (2): 539-53
- Blakeslee, J.J., Heath, J.T., Skottke, K.R., DeLong, A. (2008). Analysis of RCN1-Specific Regulation of Protein Phosphatase 2A. Meeting abstract & poster, Gordon Research Conference: Plant Molecular Biology, Holderness, NH, July 13th-18th, 2008.
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