Progress 07/01/09 to 06/30/13
Outputs
OUTPUTS: For additional information, please contact John Cushman at 775-784-1918 or jcushman@unr.edu PARTICIPANTS: Nothing significant to report during this reporting period. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
For additional information, please contact John Cushman at 775-784-1918 or jcushman@unr.edu
Publications
- No publications reported this period
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Progress 01/01/11 to 12/31/11
Outputs
OUTPUTS: Dr. Cushman presented five invited lectures on various aspects of the project for lay and scientific audiences including: 1) UNR Foundation Board of Directors at the University of Nevada, Reno, NV on June 23, 2011; 2) 2011 International Botanical Congress in Melbourne, AU on July 25, 2011; 3) Metabolon, Inc. in Research Triangle Park, NC on September 23, 2011; 4) NC-1-168 Regional Meeting at Michigan State University in East Lansing, MI on November 12, 2011; 5) Oak Ridge National Laboratory in Oak Ridge, TN on December 15, 2011. A scientific poster presentation was presented at the American Society of Plant Biologists (ASPB) annual meeting in Minneapolis, MN on August 6-11, 2011. A lay brochure featuring metabolomic profiling aspects of the ice plant project was also developed in collaboration with Metabolon, Inc. Manuscripts on the microarray-based transcriptome profiling and one on unbiased metabolic profiling are in preparation. A "Plants Have Rhythm" educational display and teaching module for K-12 and public outreach was completed and presented at the 2011 ASPB annual meeting in Minneapolis, MN on August 6-11, 2011. The display won the 2011 ASPB Educational Booth competition and was featured in the ASPB Education Booth for the duration of the meeting. The display was also presented at the Nevada Agricultural Experiment Station Ag Field Day on September 10, 2011. Reno, NV. PARTICIPANTS: Dr. Karen Schlauch (Department of Biochemistry and Molecular Biology, UNR) serves as a co-PI on this project and is responsible for various bioinformatic and statistical data analyses. Dr. Mike Covington (Rice University) has agreed to assist with the analysis of circadian mRNA expression patterns. To date, two graduate students (Katia Silvera and Bahay Gulle) have been trained as future independent scientists and are receiving advising and mentoring from the project director about their various research activities and their career development. One lab manager (Rebecca Albion, Staff Research Associate II) has been trained on the project in a wide range of preparative and analytical techniques related to the specific requirement of the project including sample preparation and analysis for metabolomic profiling. Two postdoctoral researchers (Richard Tillett and Matt Wheatley have been trained on the project to conduct microarray and Illumina RNA-Seq data analysis and proteomic analysis, respectively. Two visiting scientists from Vietnam (Hoang Thi Kim Hong and Truong Thi Bich Phuong) were trained in proteomics and tissue culture aspects of the project during the 2011 summer months. Lastly, two undergraduates (Jeremiah Smith and J. Evan Villaluz) have been trained on the project. TARGET AUDIENCES: The project results and outcomes are being targeted to the scientific community at national and international scientific meetings, specifically to research scientists and undergraduate and graduate students and post-doctoral researchers conducting research on CAM functional genomics, circadian clock biology, as well as on molecular mechanisms of CAM induction and regulation. Outreach projects have been targeted through poster presentations and oral presentations at national and international scientific meetings and poster and display presentations to the lay public. A special "Plants Have Rhythm" educational display was completed and presented to both scientific audiences and the lay public. The planned teaching module targeted K-12 (high-school) students and served as a general public outreach activity. PROJECT MODIFICATIONS: For research aim 1, we switched from microarray-based mRNA profiling to a digital RNA-Seq expression profiling using Illumina (Solexa) sequencing-by-synthesis to assess relative transcript abundance changes within objective 1.
Impacts
Research Aim 1. Additional 454 transcriptome GS-FLX TITANIUM pyrosequencing from ice plant flower, seed pod, leaf and root tissue has now resulted in 822 Mb of sequence data from 4.0 M reads with mean unigene read length of 1,586 base pairs and mean read depth of 67x. An estimated 79-103% of the ice plant transcriptome is represented, which will provide a useful reference for upcoming Illumina sequencing. Data analysis of the custom NimbleGen oligonucleotide microarray experiment using leaves sampled over a 48 h time course has been completed and several cohorts of circadianly regulated genes representing about 18% of transcripts have been identified with 2 to 200-fold changes in transcript abundance being observed. Measurements of titratable acidity, starch, and soluble sugar content have been completed with the CAM-deficient mutant showing expected patterns of starch deficiency and hyperaccumulation of soluble sugars. Research Aim 2. Two-dimensional difference in gel electrophoresis has been completed for assessing total protein changes for both diel and circadian changes in protein abundance and phosphorylation events from both well watered (C3 photosynthesis performing) and water deficit stressed plants (CAM performing). Between 125-136 proteins exhibited significant increases or deceases in abundance during the shift from C3 photosynthesis to CAM in wildtype plants. Wildtype plants performing C3 photosynthesis showed only 32 proteins with significant temporal changes in abundance, whereas plants performing CAM showed 153 proteins, indicating much greater protein abundance changes in CAM. More than 100 phospho-proteins have been identified. Research Aim 3. Unbiased metabolic profiling has been completed for both diel and circadian time course sampling every 8 h over a 72 h period from wild type and CAM-deficient mutant plants performing C3 photosynthesis and CAM induced by drought stress. Titratable acidity, starch, and soluble sugar analyses have also been completed. A total of 199 named and 304 unnamed (total 503) metabolites were identified by combined LC- and GC-MS analysis. Research Aim 4. A new non-antibiotic, non-herbicide ice plant selectable marker gene MPR1, which acetylates a toxic analogue of proline (azetidine-2-carboxylic acid, A2C), was designed and synthesized as an ice plant codon optimized gene. The gene was placed under the control of the CaMV 35S promoter cloned into the pCAMBIA1302 T-DNA vector, transferred to a binary Agrobacterium host, and transformed into embyogenic ice plant callus from hypocotyl explants. Initial results indicate that stably transformed ice plants have been recovered and these are currently being scored for alternative ER-localized green fluorescence protein reporter gene expression. Broader Impacts. One undergraduate student was recruited and has been working in the lab during 2011 as part of the "hands-on" training opportunity. The "Plants Have Rhythm" educational display and teaching module for K-12 and public outreach project was completed and presented at the American Society of Plant Biologists (ASPB) annual meeting as one of two 2011 ASPB Education Booth competition winners.
Publications
- Haider MS, Barnes JD, Cushman JC, Borland AM. (2012) A CAM and starch-deficient mutant of the facultative CAM species, Mesembryanthemum crystallinum reconciles sink demands by repartitioning carbon during acclimation to salinity stress. J. Exp. Bot. In press.
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Progress 01/01/10 to 12/31/10
Outputs
OUTPUTS: Dr. Cushman presented an invited lecture on the project entitled "Circadian clock transcriptome regulation in C3 photosynthesis versus Crassulacean acid metabolism in the common ice plant" at the CAM 2010 Workshop, at the Smithsonian Tropical Research Institute, Panama City, Panama on March 23, 2010. One manuscript on the microarray-based transciptome profiling experiments is in preparation. PARTICIPANTS: Dr. Karen Schlauch (Department of Biochemistry and Molecular Biology, UNR) serves as a co-PI on this project and is responsible for various bioinformatic and statistical data analyses. Dr. Mike Covington (Rice University) will also assist with the analysis of circadian mRNA expression patterns. To date, two graduate students (Katia Silvera and Bahay Gulle) were trained as future independent scientists and are receiving advising and mentoring from the project director about their various research activities and their career development. Two undergraduates (Jeremiah Smith and J. Evan Villaluz) have been trained on the project. One lab manager (Rebecca Albion, Staff Research Associate II) has been trained on the project in a wide range of preparative and analytical techniques related to the specific requirement of the project including sample preparation and analysis for metabolomic profiling. Lastly, two postdoctoral researchers (Richard Tillett and Matt Wheatley) are being trained on the project to conduct microarray and Illumina RNA-Seq data analysis and proteomic analysis, respectively. TARGET AUDIENCES: The project results and outcomes are being targeted to the scientific community at national and international scientific meetings, specifically to research scientists and undergraduate and graduate students and post-doctoral researchers conducting research on CAM functional genomics, circadian clock biology, as well as on molecular mechanisms of CAM induction and regulation. Outreach projects are also being targeted through poster presentations and oral presentations at national and international scientific meetings and poster and display presentations to the lay public. A special "Plants Have Rhythm" educational display will target both the scientific and lay public. The planned teaching module will target K-12 (high-school) students and serve as general public outreach. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Research Aim 1. The custom NimbleGen oligonucleotide microarray was hybridized with cDNA from leaves sampled every 4 h over the course of a 48 h time course. Parts of the data set were determined to be of low quality and were repeated. The data from the 12 repeated array hybridizations is now being analyzed. Tissue collection for a new cDNA library containing mature flowers, seed pods and seeds has been completed. RNA isolation from these tissues has been isolated for cDNA library construction and 454 sequencing and submission will occur in February 2011. Sample processing has been completed and RNA isolations initiated for a second 72 h experiment for wildtype and CAM-deficient mutant plants grown under well watered conditions and dehydration stress conditions to examine both diel and circadian changes in transcript abundance using Illumina RNA-Seq technology. Subsampling for titratatrable acidity, polysaccharide and starch measurements, and RNA isolations has been completed. Research Aim 2. Sampling for both diel and circadian changes in protein abundance and phosphorylation events from both well watered (C3 photosynthesis performing) and water deficit stressed plants (CAM performing) was completed for the 72 h diel/circadian timecourse for wild type plants and CAM-deficient mutants. Subsampling for protein isolation is ongoing. Research Aim 3. Diurnal and circadian time course samples have been collected. Sampling for both diel and circadian changes in relative metabolite abundance was completed for wild type plants and CAM-deficient mutants for plants performing C3 photosynthesis and CAM. Tissues were ground, subsampled and lyophilized for metabolite analysis. Sample submission will occur in February 2011. Research Aim 4. A candidate bZip transcription factor (C32923), with strong induction under salinity stress and strong, persistant circadian expression patterns under constant light and temperature conditions was selected for detailed analysis. A new non-antibiotic, non-herbicide ice plant selectable marker gene MPR1, which acetylates a toxic analogue of proline, has now been obtained for stable ice plant transformation trials. We designed the codon optimized de novo resynthesized expression cassette, which should result in higher translation of the selectable marker gene, and will be synthesized in February 2011. Broader Impact Aim 1. This "hands-on" training opportunity will be offered starting in May 2010.Two undergraduate students have been recruited and have been working in the lab during 2010. High-school students will be recruited from the Upward Bound program for Summer 2011 research experience. Aim 2. The "Plants Have Rhythm" educational display and teaching module for K-12 and public outreach project will be developed during years 1 and 2 and will debut during year 2 at the American Society of Plant Biologists (ASPB) annual meeting. A graphic designer and digital media artist, Lori Kunder, has been retained to work on design and layout of the display and video content from colleagues has been requested, with the goal of completing the display by mid 2011.
Publications
- Sunagawa H, Cushman JC, Agarie S. (2010) Crassulacean acid metabolism alleviates reactive oxygen species in the facultative CAM plant, the common ice plant, Mesembryanthemum crystallinum. Plant Production Science. 13: 246-260.
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Progress 01/01/09 to 12/31/09
Outputs
OUTPUTS: Crassulacean acid metabolism (CAM), a photosynthetic pathway found in approximately 7% of all vascular plant species that improves water use efficiency up to 10-fold relative to C3 species, provides an exquisite example of circadian and environmentally regulated photosynthetic adaptation. CAM plants display distinctive circadian clock outputs and inverse stomatal rhythms, which are not found in C3 or C4 plants. The long-term goals of the proposed research are to identify the regulatory and signaling pathways essential for the circadian control of CAM and inverse stomatal behavior, respectively. The common or crystalline ice plant will be used as a model to determine the environmentally induced, circadian controlled changes in mRNA and protein abundance, and reversible protein phosphorylation events during the transition from C3 photosynthesis to CAM by conducting mRNA expression profiling. Metabolite changes will also be monitored to determine if they participate in possible feedback control circuits of upstream circadian clock gene expression outputs. Key regulatory and signaling factors that function in the CAM circadian clock will also be analyzed. Increased understanding of the molecular mechanisms responsible for controlling the expression and regulation of CAM will provide new knowledge about the molecular basis of this important water-saving photosynthetic adaptation that will provide novel strategies for improving crop productivity in the context of global climate change. This project will provide unique "hands-on" training opportunities in plant research for undergraduate, graduate and post-doctoral students. Undergraduate students will be selected from established outreach programs at the University of Nevada (UNR) that target students from disadvantaged backgrounds to participate in developing a "Plants Have Rhythm" educational display and K-12 teaching module to inform and excite high school, college students, and the general public about CAM and the existence of circadian rhythms in plants and their adaptive significance. PARTICIPANTS: Dr. Karen Schlauch (Department of Biochemistry and Molecular Biology, UNR) serves as a co-PI on this project and is responsible for various bioinformatic and statistical data analyses. To date, two graduate students (Katia Silvera and Bahay Gulle, Matt Wheatley, and Richard Tillett) were trained as future independent scientists and are receiving advising and mentoring from the project director about their various research activities and their career development. Two undergraduates (Jeremiah Smith and J. Evan Villaluz) have been trained on the project. One lab manager has been trained on the project in a wide range of preparative and analytical techniques related to the specific requirement of the projects: Rebecca Albion, Staff Research Associate II. Finally, one postdoctoral researcher (TBD) will also be trained. TARGET AUDIENCES: The project results and outcomes are being targeted to the scientific community at national and international scientific meetings, specifically to research scientists and undergraduate and graduate students and post-doctoral researchers conducting research on CAM functional genomics, circadian clock biology, as well as on molecular mechanisms of CAM induction and regulation. Outreach projects are also being targeted through poster presentations and oral presentations at national and international scientific meetings and poster and display presentations to the lay public and local stakeholders at an annual Agriculture Field Day held by the Nevada Agricultural Experiment Station. A special "Plants Have Rhythm" educational display will target both the scientific and lay public. The planned teaching module will target K-12 (high-school) students and serve as general public outreach. PROJECT MODIFICATIONS: For research aim 1, we are switching from microarray-based mRNA profiling to a digital RNA-Seq expression profiling using Illumina (Solexa) sequencing-by-synthesis to assess relative transcript abundance changes.
Impacts
Research Aim 1. To determine the environmentally induced, circadian controlled changes in mRNA abundance during the transition from C3 photosynthesis to CAM by conducting mRNA expression profiling using cDNA sequence data sets derived from massively parallel DNA sequencing. The 454 transcriptome pyrosequencing has been completed. Results from GS-FLX TITANIUM sequencing from a normalized cDNA library from C3 and CAM performing leaves, roots, and flowers generated 2,380,901 reads resulting is 765 MB of sequence. These data, along with included 27,347 ESTs derived from Sanger sequencing, were combined to create 41,374 contigs and 96,490 singletons. The contigs were then used to design a custom, probe optimized, NimbleGen oligonucleotide microarray containing 133,848 probes (4 per gene). The custom array was then hybridized with cDNA from leaves sampled every 4 h over the course of a 48 h time course. Data has been received and is currently being analyzed. We have also completed a second round of tissue collection for wildtype plants grown under well watered conditions and dehydration stress conditions (in progress) in order to perform the integrated mRNA digital expression profiling, proteomic, and metabolic analyses outlined in aims 1-3. Research Aim 2. To determine if environmentally induced, circadian controlled changes in mRNA abundance are accompanied by protein abundance changes and reversible protein phosphorylation events that occur during the transition from C3 photosynthesis to CAM. Diurnal and circadian time course samples have been collected for plants performing C3 photosynthesis and are in progress for CAM performing plants. Research Aim 3. To analyze the interaction and possible feedback control circuits between downstream metabolic outputs and upstream circadian clock gene expression outputs by conducting integrated metabolite profiling in wild type and CAM-deficient mutant ice plant leaves during the transition from C3 photosynthesis to CAM. This research has not yet been initiated. Diurnal and circadian time course samples have been collected for plants performing C3 photosynthesis and are in progress for CAM performing plants. Research Aim 4. To identify regulatory and signaling factors that function in the circadian clock output of CAM. This research has not yet been initiated. Broader Impact Aim 1. Creation of "Hands-on" training opportunities in Integrative CAM Functional Genomics. This training opportunity will be offered starting in May 2010. Aim 2. Development of a "Plants Have Rhythm" educational display and teaching module for K-12 and public outreach. This outreach project will be developed during years 1 and 2 and will debut during year 2 at the American Society of Plant Biologists (ASPB) annual meeting.
Publications
- Sunagawa H, Agarie S, Cushman JC. (2010) Crassulacean acid metabolism alleviates reactive oxygen species in the facultative CAM plant, the common ice plant, Mesembryanthemum crystallinum. Annals of Botany. In press.
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