Progress 09/01/08 to 08/31/09
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, one postdoctoral fellow was supported: Bala Krishna P. Venkata, (Keck Graduate Institute, Claremont, CA)(until 1-2009). He performed site-directed mutagenesis, Agrobacterium-mediated transformation, and analysis of mutant phenotypes. Three research technicians were supported by the funding: (1) Cindy C. Cordova, (Keck Graduate Institute, Claremont, CA)(until 3-2009). She worked on growth and harvesting of Pisum sativum, and mutant characterization of the START domain mutants.(2) Hanh T. Nguyen (Kansas State University)(6-2009 to 8-2009). She worked on mutant characterization of START domain mutants. (3) 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. 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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Progress 09/01/07 to 08/31/08
Outputs
OUTPUTS: The goal of this project, which utilizes Arabidopsis and the crop species Pisum sativum, is to investigate a putative link between sterol metabolism and cell differentiation in plant development. The first objective of this project is a metabolic analysis of sterol composition of developing seeds as well as stems, shoots, flowers, and leaves. Pisum sativum tissues were harvested and sterol contents were determined by GC-MS. The data reveal that immature seeds contain the greatest levels of sterols, due to high amounts of sitosterol and campesterol. The relative composition of these major sterols remained constant throughout embryogenesis, in contrast to a published analysis of Brassica campestris floral meristems, where there is a shift from high cholesterol to high sitosterol content as flowers mature. In developing seeds, small changes are observed, such as a decrease in minor sterols of the cycloartenol branch during mature stages, including a reduction in 24-methylenecycloartenol. Significant differences are observed in sterol profiles leaves versus stems, and cotyledons versus radicle, consistent with tissue-specific expression of sterols. The second objective 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 Arabidopsis GLABRA2 (GL2) transcription factor, which acts in a complex regulatory circuit controlling epidermal patterning, is being investigated. In previous work, transgenic lines carrying GL2:EYFP were 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 affects protein but not transcript levels, suggesting that the START domain is required for stable transcription factor levels, perhaps via ligand binding. The START deletion protein is found in the nucleus. Thus, the START domain is not strictly required for sub-cellular localization. Missense mutants that alter charged residues within the predicted ligand-binding pocket (K338L, R384L, E385L, and R390L) result in reduced levels of protein expression concomitant with defects in epidermal cell differentiation. A gl2 R384L E385L transgenic line that expresses high levels of the mutant protein will be used in suppressor screens, with the aim of identifying novel components. In addition, a chemical genetics screen is being initiated to elucidate the molecular mechanisms underlying START domain function. A fluorescent reporter construct for GL2 transcription factor function utilizes the target gene Phospholipase Dz1. With duel expression of GL2:EYFP and PLDz1pro::PLDz1:TFP, both the transcription factor and its target can be visualized in vivo. Although the 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 sterol signaling networks that act during embryogenesis of flowering plants. PARTICIPANTS: Kathrin Schrick is the PI for this project. She is the manager of the project and performed a subset of the experiments, including most of the confocal laser scanning microscopy. Bala Krishna P. Venkata is the Postdoctoral Fellow that is working on this project. He has carried out the site-directed mutagenesis, real-time PCR and Western blotting to characterize the GL2 START domain mutants. Cindy Cordova is a research assistant who helped with growth and harvesting of the Pisum sativum samples. She has also helped with genotyping GL2 START domain mutants. Shozo Fujioka is a collaborator at the RIKEN (Japan) who has performed the GC-MS analysis of sterol extracts from the Pisum sativum samples. Darleen DeMason (University of California, Riverside) has helped with advice for growing two crops of Pisum sativum in the Pomona College greenhouse. TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
The focus of this project is to determine the connection between sterol metabolism and sterol signal transduction pathways in plants. This project is categorized as fundamental research, as it tests scientific hypotheses and provides basic knowledge in the field of plant developmental biology. The model system Arabidopsis is being used in this project, in addition to the crop Pisum sativum. Both species are dicotyledonous plants, yet the knowledge gained is expected to be translatable to monocot crops. Towards the first objective, investigation of sterol composition of developing pea embryos has resulted in the finding that sterol levels are very high in young stages. Further, the analysis has led to the identification of candidate sterols that act in signaling. 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. Such conceptual breakthroughs have potential for critical advances in applied research. The results are providing important information about the molecular mechanisms underlying development in plants. Moreover, key differences in transcription factor function in plants versus animals are being revealed. 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
- No publications reported this period
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