Progress 05/01/09 to 08/31/12
Outputs
OUTPUTS: The goal of this project, which utilized the crop Pisum sativum (garden pea) and the plant model system Arabidopsis thaliana, has been to investigate the molecular link between sterol metabolism and cell differentiation in plant development. The first objective was a metabolic analysis of sterol composition of developing seeds as well as other aerial tissues of the garden pea, and this work was completed and published in Phytochemistry, an in international journal. The second objective of this project was the functional characterization of a candidate sterol-binding domain, the steroidogenic acute regulatory (StAR)-related lipid transfer (START) domain from homeodomain leucine-zipper transcription factors. The Arabidopsis GLABRA2 (GL2) transcription factor, which acts in a complex regulatory circuit controlling epidermal patterning, was investigated. Although the putative ligand for the GL2 START domain is not yet known, this project was set up to test a model in which the transcription factor is regulated by a positive feedback loop that requires a functional START domain. Projects relating to this work have been published in research journals, but the main body of work will be submitted at the end of this calendar year. The long-term aim of both objectives of this project is to define sterol signaling networks that act during embryogenesis and seed development of flowering plants. Outcomes from this project were presented in seminars that were held both nationally and internationally. The work was presented in seminars to other universities, and also at a USDA/CSREES/AFRI Project Director's Meeting in Bethesda, MD in May 2009. Other invited seminars included as Keynote Speaker at the Washington University Plant Biology Annual Retreat at Shaw Nature Reserve outside of St. Louis, MO, in September 2009. Over the funding period there have been a total of fourteen seminars given to other universities ranging from the University of Calgary in Canada, to the Pennsylvania State University in University Park, PA. The interdisciplinary nature of the project led to collaborations with researchers in the areas of chemistry, statistics, as well as biology. Experimental approaches were further enhanced in including the broader scientific community. In summary, the most significant outputs and dissemination activities, aside from publications have been in interactions, discussions, and presentations to other scientists who work in related areas of research. There has also been a substantial output in training scientists to work in the area of molecular biology. During the project, five undergraduates, one graduate student, one postdoctoral fellow, and four technical personnel were trained in the field of plant molecular biology and biotechnology as they contributed to the project output. PARTICIPANTS: The individuals who worked on the project included one postdoctoral fellow, Bala Krishna P. Venkata (Keck Graduate Institute, Claremont, CA)(until 1-2009), who performed site-directed mutagenesis, Agrobacterium-mediated transformation, and analysis of mutant phenotypes. This work was critical in determining the function of the START domain within the homeodomain transcription factor. One graduate student, Aashima Khosla, Ph.D. candidate (Kansas State University) (9-2010 to 8-2012) worked on purification of START domain proteins for structural analysis. Three technical personnel contributed to the project. Cindy C. Cordova, research technician (Keck Graduate Institute, Claremont, CA)(until 3-2009) worked on the growth and harvesting of Pisum sativum plant embryos and other organs for sterol analysis, and also helped in mutant characterization of the Arabidopsis transgenic lines. Hanh T. Nguyen, research technician (Kansas State University)(6-2009 to 8-2009) continued the project on characterization of transgenic Arabidopsis lines, while Achira Mukhopadhyay, research technician (Kansas State University)(1-2010 to 8-2010) helped with mutagenesis and suppressor screens as well as lipid analysis work. More recently Paige N. Cox (5-2011 to 8-2012) worked on construction of additional mutant lines as well as a yeast two hybrid screen for interactors of the homeodomain transcription factor GL2. In addition, six undergraduate students were trained in the field of plant biotechnology. Meghan Herde, undergraduate (Kansas State University)(10-2009 to 8-2012) and Shawna Cikanek, undergraduate (Kansas State University)(10-2009 to 9-2010) worked on mutant genotyping and characterization. Preston Stephens, undergraduate (Kansas State University)(10-2009 to 7-2012) worked on yeast two hybrid interactors of GL2, as well as homo- and heterodimerization between family members. Allison Boehler, undergraduate (Kansas State University)(10-2010 to 8-2012), worked on the EMS mutagenesis screen and the identification of suppressors of a semi-dominant allele of GL2. James Arpin, undergraduate (Kansas State University)(1-2011 to 8-2012) initiated a project on steryl glucosides in plants, lipid analysis. Key collaborations on the first part of the project were with Shozo Fujioka (RIKEN, Japan) for sterol analysis of plant tissues from Pisum sativum, and Leigh Murray (Department of Statistics, Kansas State University), for statistical analysis. Martin Hulskamp (Department of Botany, Cologne, Germany) provided the original GL2:EYFP construct that we used for the second part of the project. We collaborated with Sean Cutler (University of California, Riverside) in experiments to decipher the lipid/sterol ligands of plant-derived START domains. Seth DeBolt (Department of Horticulture, University of Kentucky) has been our collaborator in studying steryl glucosides, and Ruth Welti (Division of Biology, Kansas State University) has collaborated with us in the area of lipidomics, specifically in detection of steryl glucosides using mass spectrometry TARGET AUDIENCES: This project is in the category of fundamental research, and if it were to be renewed, it would be under the umbrella of the foundational program at the USDA. In this project we obtained information for the crop, Pisum sativum. The garden pea is one of the top four legume crops worldwide. Research on this crop may be of interest to plant biologists, breeders, seed companies and ultimately, growers. The pea, because it is propagated by seed, is likely to benefit from increased knowledge on the underlying molecular basis of embryogenesis, ie. seed development. The major focus of this work has been on sterols, which are small fat soluble molecules with both structural and signaling functions in cell biology of the eukaryotes. Any information learned is therefore valuable for researchers in the areas of chemistry, biochemistry and biology. Since the subject pertains to plant sterols, plant biologists are a particularly appropriate target audience for the work in this project. In addition, we address the function of START domains, which are found broadly in both animals and plants, as well as in some species from other taxa. Therefore, scientists that work with proteins containing START domains, regardless of experimental organism, may benefit from knowledge gained with respect to the function of START domains in the plant model system, Arabidopsis. It is the hope that the knowledge gained will provide a fundamental basis for future technological applications in the field of agriculture. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
In this project, the crop Pisum sativum (garden pea) and Arabidopsis thaliana were utilized as model systems to investigate the molecular connections between sterol metabolism and cell differentiation in the embryonic development of flowering plants. The first objective was a metabolic analysis of sterol composition of developing seeds as well as other green tissues of the garden pea. GC-MS analysis revealed that immature seeds contain the highest levels of sterols, mainly sitosterol, campesterol, and isofucosterol. Statistical regression models indicated that the natural log of seed fresh weight was a predictor of declining sterol contents during embryogenesis. High concentrations of sterols were also correlated with elevated mRNA expression of sterol biosynthesis genes during early stages of seed development in Arabidopsis. Although the relative composition of most sterols remained constant throughout seed development, especially high concentrations of isofucosterol, the direct precursor to sitosterol, were found in the youngest seeds. Conversely, especially low levels of stigmasterol, a derivative of sitosterol, were detected in early stages. Significant differences were observed in sterol profiles of leaves versus stems, and cotyledons versus radicles, consistent with tissue-specific expression of sterols. Overall, the findings point towards coordinated gene expression of sterol biosynthesis enzymes in regulatory networks underlying embryonic development of flowering plants. The second objective of this project was the functional characterization of a candidate sterol-binding domain, the steroidogenic acute regulatory (StAR)-related lipid transfer (START) domain from homeodomain leucine-zipper transcription factors. The Arabidopsis GLABRA2 (GL2) transcription factor, which acts in a complex regulatory circuit controlling epidermal patterning, was investigated. Transgenic lines carrying GL2:EYFP were shown to rescue the gl2 null mutant. Utilizing the GL2:EYFP construct as a tool, the predicted function of the START as a regulatory module was investigated. Deletion of the START domain affected protein but not transcript levels, suggesting that the START domain is required for stable transcription factor levels, perhaps via ligand binding. The GL2 mutant protein that lacks the START domain is found in the nucleus, suggesting that this domain is not required for sub-cellular localization. Missense mutants that alter charged residues within the predicted ligand-binding pocket (K338L, R384L, E385L, and R390L) resulted in reduced levels of protein expression concomitant with defects in epidermal cell differentiation. Although the putative ligand for the GL2 START domain is not yet known, this project was set up to test a model in which the transcription factor is regulated by a positive feedback loop that requires a functional START domain. The long-term outcomes and impacts of both objectives of this project was to define and further characterize the sterol signaling networks that act during embryogenesis and seed development of flowering plants.
Publications
- Kathrin Schrick, Cindy Cordova, Grace Li, Leigh Murray, Shozo Fujioka. 2011. A dynamic role for sterols in embryogenesis of Pisum sativum. Phytochemistry 72: 465-475.
- Kathrin Schrick, Sunitha Shiva, James C. Arpin, Nicole Delimont, Giorgis Isaac, Pamela Tamura, Ruth Welti. 2012. Steryl glycoside and acyl steryl glycoside analysis of Arabidopsis seeds by electrospray ionization tandem mass spectrometry. Lipids 47: 185-193.
- Kumari, S., Shridhar, S., Singh, D., Priya, P., Schrick, K., and Yadav, G. 2012. The role of lectins and HD-Zip transcription factors in isoprenoid based plant stress responses. Proceedings of Indian National Science Academy (accepted).
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Progress 05/01/09 to 04/30/10
Outputs
OUTPUTS: The aim of this project is to investigate a putative link between sterol metabolism and cell differentiation in plant growth and development using Arabidopsis and the garden pea, Pisum sativum as model systems. The first objective is to conduct a metabolic profiling of sterol composition in developing seeds. GC-MS data revealed the occurrence of 25 steroid compounds in 30 tissue samples, including two developmental series of twelve seed stages and ten pod stages. Immature seeds exhibited the greatest concentrations of sterols. Regression models indicated that the natural log of seed or pod fresh weight was a consistent predictor of declining sterol content during embryonic development. Although total sterol levels were reduced in mature embryos, the composition of sitosterol and campesterol remained relatively constant. In mature seeds, a significant decrease in isofucosterol was observed, as well as minor changes such as increases in cycloartenol and campesterol derivatives. Notably, the highest levels of isofucosterol, a precursor to sitosterol, occurred in young seeds and flower buds, tissues that contain rapidly dividing cells. The highest levels of stigmasterol, a derivative of sitosterol, were found in fully-differentiated leaves while all seed stages exhibited low levels of stigmasterol. The observed differences in sterol content were correlated to mRNA expression data for sterol biosynthesis genes from Arabidopsis. The second objective of this project is the functional characterization of a candidate sterol-binding domain, the steroidogenic acute regulatory (StAR)-related lipid transfer (START) domain from homeodomain leucine-zipper transcription factors. The focus of this work is the GLABRA2 (GL2) transcription factor of Arabidopsis. A GL2:EYFP fusion, expressed under the native promoter was shown to rescue a gl2 null mutant. Site-directed mutagenesis and deletion of the START domain of GL2 was used to address its presumed function as a regulatory module. Deletion of the START domain affected protein but not transcript levels, suggesting that the START domain is required for stable transcription factor levels. The mutant protein was detected in the nucleus. Thus, the START domain is not strictly required for sub-cellular localization. A series of missense mutants that alter charged residues within the predicted ligand-binding pocket resulted in reduced levels of protein expression concomitant with defects in epidermal cell differentiation. A novel trichomeless phenotype was in a START domain mutant that expresses unusually high levels of the protein. To further characterize the mutant, a transcriptome experiment was performed. Although the putative ligand for the GL2 START domain is not yet known, this work tests a model in which its activity is regulated by a positive feedback loop via binding to its START domain. The long-term aim of both objectives of this project is to define and characterize sterol signaling networks that act during embryogenesis of flowering plants. PARTICIPANTS: During the reporting period, two research technicians were supported by the funding: (1) Hanh T. Nguyen (Kansas State University)(6-2009 to 8-2009). She worked on mutant characterization of START domain mutants. (2) Achira Mukhopadhyay, research technician (Kansas State University)(1-2010 to 8-2010). She performed mutagenesis and suppressor screens. In addition, three undergraduate students were supported: (1) Meghan Herde, undergraduate (Kansas State University)(10-2009 to 5-2010) (2) Shawna Cikanek, undergraduate (Kansas State University)(10-2009 to present), and Preston Stephens, undergraduate (Kansas State University)(10-2009 to 12-2009). The undergraduates are helping with routine work. In addition, there was a collaboration with Grace Li (Ph.D. student) and Dr. Leigh Murray (Professor) of the Department of Statistics at Kansas State University. Li and Murray aided with statistical regression analysis of the Pisum sativum sterol profiling data. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
In this project, the overall goal is to elucidate the molecular link between sterol metabolism and putative sterol signal transduction pathways in higher plants. This project is categorized as fundamental research, as it tests scientific hypotheses and provides basic knowledge in the area of plant growth and development. The model systems Arabidopsis thaliana and Pisum sativum are being utilized in this work. Both species are dicotyledonous plants, yet the knowledge gained is expected to be translatable to monocot crops as well. Towards the first objective, investigation of sterol composition of developing pea seeds has resulted in the finding that sterol concentrations are highest in young embryos in comparison to all other tissues examined. The analysis has led to the identification of candidate sterols that are expressed specifically in rapidly dividing cells and cells undergoing cell-type differentiation. The findings, which have been submitted to the journal Phytochemistry, implicate the coordinated expression of sterol biosynthesis enzymes in gene regulatory networks underlying the embryonic development of flowering plants. In the second objective, characterization of the START domain, a putative lipid/sterol binding domain in plant transcription factors, is providing basic knowledge about the function of this domain. Our results indicate that the putative ligand-binding domain is essential for the function of the transcription factor. The level at which this domain functions has been determined to be post-transcriptional, consistent with ligand binding. A semi-dominant mutation in this domain is being utilized to reveal novel components that interact with this transcription factor. Thus, the data are providing important information about the molecular mechanisms underlying transcription factor function during development in higher plants. The primary impact of this work will be in the form of conceptual breakthroughs in the mechanistic understanding of crosstalk between lipid metabolism and transcriptional regulation in plants. Progress in this area will enhance economic opportunities for agricultural production by revealing a novel signaling mechanisms in plants, whose long term impact will be to improve nutrition and health and enhance the environment. Moreover, this work has already resulted in metabolic data for a major crop, Pisum sativum, which is among the four important cultivated legumes world-wide. Peas are propagated exclusively from seed, warranting the need to enhance our fundamental knowledge of embryogenesis in this crop. This research is in response to pertinent priorities in the Program in Growth and Development, foremost being developmental pathways leading to the formation of vegetative or reproductive structures, and characterization of cellular structures or processes that are crucial for plant development. In addition, in the course of this project, one postdoctoral fellow, and several undergraduate students are receiving educational training and will gain valuable laboratory skills in the field of biotechnology.
Publications
- Seth DeBolt, Wolf-Rudiger Scheible, Kathrin Schrick, Manfred Auer, Fred Beisson, Volker Bischoff, Pierrette Bouvier-Nave, Andrew Carroll, Kian Hematy, Yonghua Li, Jennifer Milne, Meera Nair, Hubert Schaller, Chris Somerville and Marcin Zemla. 2009. Mutations in UDP-glucose:sterol-glucosyltransferase in Arabidopsis cause transparent testa phenotype and suberization defects in seeds. Plant Physiology 151: 78-87.
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