Progress 03/01/11 to 02/28/16
Outputs
Target Audience:Scientists with interest in the regulation of metabolic pathways leading toward various isoprenoids (largest class of plant natural products). Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?Graduate student working on this project graduated in July, 2016 (Brenton Poirier). How have the results been disseminated to communities of interest?Period since annual report (09/30/2015): one manuscript submitted. Entire funding period (2011-2016): two papers published and one manuscript submitted. IMPORTANT! The progress reported here and mansucripts submitted were primarily funded through a grant by the National Science foundation. The Hatch funds were instrumental to generate preliminary data for follow-up research that formed the foundation for submitting competitive proposals to federal funding agencies. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
It was our goal to characterize the functions of putative regulators of phytosterol biosynthesis (originally termed RMI1 and RMI2). Over the course of this study, we recognized that the primary function of these genes was in apical meristem function. A summary of the findings is below (does not include results presented in previous progress reports): The subgroup IIIB basic Helix-Loop-Helix (bHLH) transcription factors SCREAM/ICE1 and SCREAM2 have well-characterized roles in the terminal differentiation of stomatal guard cells in Arabidopsis thaliana. Here we report on the characterization of the functional roles of the remaining members of sub-group IIIB, bHLH093 and bHLH061. Previously, delayed flowering has been reported for mutants of bHLH093, termed NFL (NO FLOWERING IN SHORT DAY). It was demonstrated that the phenotype of nfl mutants could be rescued by exogenous application of gibberellin (GA) or by crossing with a della quadruple mutant, indicating that NFL functions upstream of GA signaling in promoting flowering in short days. T-DNA insertion mutants were obtained to further characterize the functions of bHLH093, as well as bHLH061. bhlh093/bhlh061 double mutants fail to produce a primary inflorescence shoot and display greater phenotypic severity than bhlh093 single mutants. An ultrastructural characterization of tissues surrounding the shoot apical meristem (SAM) prior to flowering indicates that the SAM in bhlh093/bhlh061 mutants terminates prior to the establishment of an inflorescence meristem, and the delay in flowering is due to structural defects that develop in tissues surrounding the SAM prior to flowering. As previously reported for bhlh093 mutants, the transition to flowering is restored in bhlh093/bhlh061 with GA application to the apex. We also demonstrated that GA application is sufficient to prevent structural defects that develop in the tissues surrounding the SAM. An in-depth investigation of the effects of light-intensity and photoperiod revealed that bhlh093/bhlh061 mutants also display delayed flowering under long day growth conditions with higher light intensity, and the severity of the phenotype correlates with length of photoperiod and light intensity. Our results indicate that bHLH093 and bHLH061 function redundantly in GA-mediated transition to flowering under both short and long day growth conditions.
Publications
- Type:
Journal Articles
Status:
Submitted
Year Published:
2016
Citation:
Poirier BC, Feldman MJ, Lange BM (2016) bHLH093/NFL and bHLH061 are Required for Shoot Apical Meristem Function in Arabidopsis thaliana. PLOS One, submitted.
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Progress 10/01/14 to 09/30/15
Outputs
Target Audience:Scientists with interest in the regulation of metabolic pathways leading toward various isoprenoids (largest class of plant natural products). Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?Graduate student working on this project moved on to take up a position as postdoctoral associate at the Donald Danforth Center for Plant Sciences in St. Louis, MO. How have the results been disseminated to communities of interest?Two original publications in excellent international journals. What do you plan to do during the next reporting period to accomplish the goals?Publish manuscript on basic helix-loop-helix protein with important functions in mediating developmental growth that is controlled by gibberellins (isoprenoid hormones). Publish manuscript reporting on regulation of isoprenoid pathways in different organs of Arabidopsis.
Impacts
What was accomplished under these goals?
1) Misexpression of the AtNPC1 - 1 and AtNPC1 - 2 genes leads to altered sphingolipid metabolism, growth impairment, and male reproductive defects in a hemizygous Arabidopsis thaliana (L.) double-mutant population. Abolishing the expression of both gene copies has lethal effects. Niemann-Pick disease type C1 is a lysosomal storage disorder caused by mutations in the NPC1 gene. At the cellular level, the disorder is characterized by the accumulation of storage lipids and lipid trafficking defects. The Arabidopsis thaliana genome contains two genes (At1g42470 and At4g38350) with weak homology to mammalian NPC1. The corresponding proteins have 11 predicted membrane-spanning regions and contain a putative sterol-sensing domain. The At1g42470 protein is localized to the plasma membrane, while At4g38350 protein has a dual localization in the plasma and tonoplast membranes. A phenotypic analysis of T-DNA insertion mutants indicated that At1g42470 and At4g38350 (designated AtNPC1-1 and AtNPC1-2, respectively) have partially redundant functions and are essential for plant reproductive viability and development. Homozygous plants impaired in the expression of both genes were not recoverable. Plants of a hemizygous AtNPC1-1/atnpc1-1/atnpc1-2/atnpc1-2 population were severely dwarfed and exhibited male gametophytic defects. These gene disruptions did not have an effect on sterol concentrations; however, hemizygous AtNPC1-1/atnpc1-1/atnpc1-2/atnpc1-2 mutants had increased fatty acid amounts. Among these, fatty acid α-hydroxytetracosanoic acid (h24:0) occurs in plant sphingolipids. Follow-up analyses confirmed the accumulation of significantly increased levels of sphingolipids (assayed as hydrolyzed sphingoid base component) in the hemizygous double-mutant population. Certain effects of NPC1 misexpression may be common across divergent lineages of eukaryotes (sphingolipid accumulation), while other defects (sterol accumulation) may occur only in certain groups of eukaryotic organisms. 2) In plants, two spatially separated pathways provide the precursors for isoprenoid biosynthesis. We generated transgenic Arabidopsis (Arabidopsis thaliana) lines with modulated levels of expression of each individual gene involved in the cytosolic/peroxisomal mevalonate and plastidial methylerythritol phosphate pathways. By assessing the correlation of transgene expression levels with isoprenoid marker metabolites (gene-to-metabolite correlation), we determined the relative importance of transcriptional control at each individual step of isoprenoid precursor biosynthesis. The accumulation patterns of metabolic intermediates (metabolite-to-gene correlation) were then used to infer flux bottlenecks in the sterol pathway. The extent of metabolic cross talk, the exchange of isoprenoid intermediates between compartmentalized pathways, was assessed by a combination of gene-to-metabolite and metabolite-to-metabolite correlation analyses. This strategy allowed the selection of genes to be modulated by metabolic engineering, and we demonstrate that the overexpression of predictable combinations of genes can be used to significantly enhance flux toward specific end products of the sterol pathway. Transgenic plants accumulating increased amounts of sterols are characterized by significantly elevated biomass, which can be a desirable trait in crop and biofuel plants.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2015
Citation:
Feldman M.J., Poirier B.C., Lange B.M. (2015) Misexpression of the Niemann-Pick disease type C1 (NPC1)-like protein in Arabidopsis causes sphingolipid accumulation and reproductive defects. Planta 242, 921-933.
- Type:
Journal Articles
Status:
Published
Year Published:
2015
Citation:
Lange I., Poirier B.C., Herron B.K., Lange B.M. (2015) Comprehensive assessment of transcriptional regulation facilitates metabolic engineering of isoprenoid accumulation in Arabidopsis. Plant Physiol. 169, 1595-1606.
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Progress 10/01/13 to 09/30/14
Outputs
Target Audience:
Nothing Reported
Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided? One graduate student (Brenton Poirier) is supported by project WPN000606 funds and received training in advanced molecular biological, genetic and biochemical techniques. How have the results been disseminated to communities of interest? Presentation at seminar series (Michigan State University). Manuscript submitted to international scientific journal. What do you plan to do during the next reporting period to accomplish the goals? Complete studies on the functions of two basic-Helix-Loop-Helix transcription factors on the development of apical meristem (area at the tip of a plant shoot responsible for vertical growth). The study had been initiated because we had preliminary correlative evidence that these transcription factors may play roles in the regulation of phytosterol biosynthesis. However, follow-up studies have now revealed unrelated, but still highly important, functions in controlling growth.
Impacts
What was accomplished under these goals?
In February, 2014, we submitted a manuscript reporting on the effects of impaired expression of the Niemann-Pick type disease type C1-like genes in the model plant Arabidopsis. Reviewers requested substantial revisions, which required additional experimentation. These experiments have now been completed and a revised version of the manuscript has been submitted to PLANTA. TITLE: Misexpression of the Niemann-Pick disease type C1 (NPC1)-like protein in Arabidopsis causes lipid accumulation and reproductive defects ABSTRACT: Niemann-Pick disease type C1 is a lysosomal storage disorder caused by mutations in the NPC1 gene. At the cellular level, the disorder is characterized by the accumulation of storage lipids and lipid trafficking defects. The Arabidopsis thaliana genome contains two genes (At1g42470 and At4g38350) with weak homology to mammalian NPC1. The corresponding proteins have 11 predicted membrane-spanning regions and contain a putative sterol-sensing domain. The At1g42470 protein is localized to the plasma membrane, while At4g38350 protein has a dual localization in the plasma membrane and the tonoplast. A phenotypic analysis of T-DNA insertion mutants indicated that At1g42470 and At4g38350 (designated AtNPC1-1 and AtNPC1-2, respectively) have partially redundant functions and are essential for plant reproductive viability and development. Homozygous plants impaired in the expression of both genes were not recoverable and hemizygous AtNPC1-1/atnpc1-1/atnpc1-2/atnpc1-2 plants were severely dwarfed. Gene disruptions did not have an effect on sterol concentrations. However, hemizygous AtNPC1-1/atnpc1-1/atnpc1-2/atnpc1-2 mutants over-accumulated hydrolyzable fatty acids. Among these fatty acids was a-hydroxytetracosanoic acid (h24:0), which is a signature constituent of sphingolipids. Follow-up analyses confirmed dramatically high levels of sphingoid base in the hemizygous double mutant. The evidence presented here suggests that NPC1 misexpression leads to faulty sphingolipid metabolism, which may contribute to reproductive defects in hemizygous mutants and has apparent lethal effects when the expression of all gene copies is abolished.
Publications
- Type:
Journal Articles
Status:
Under Review
Year Published:
2014
Citation:
Feldman M.J., Poirier B.C., Lange B.M. (2014) Misexpression of the Niemann-Pick disease type C1 (NPC1)-like protein in Arabidopsis causes lipid accumulation and reproductive defects. Planta, revised manuscript submitted.
- Type:
Conference Papers and Presentations
Status:
Other
Year Published:
2014
Citation:
: Invited presentation: Comprehensive assessment of transcriptional regulation enables knowledge-based engineering of isoprenoid pathways in Arabidopsis Seminar Series of the Department of Plant Biology, Michigan State University, February 17, 2014
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Progress 01/01/13 to 09/30/13
Outputs
Target Audience: This project focuses on unraveling the regulation of the central pathways that provide precursors for the biosynthesis of isoprenoids, the largest class of plant natural products. This should be of interest for all scientists involved in studying plant metabolism. While the activities involve primarily fundamental research, there are several potential outcomes that have broader applicability. For example, we recently demonstrated that enhanced flux toward sterols leads to dramatic increases in plant biomass. Our research thus has applied aspects and should be of interest for both breeders, metabolic engineers and decision makers in the agricultural and bioenergy fields. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided? One graduate student (Brenton Poirier) is supported by project WPN00606 funds and receives training in advanced molecular biological, genetic, and biochemical techniques. How have the results been disseminated to communities of interest? Through publications in international journals. What do you plan to do during the next reporting period to accomplish the goals? Project (1) Analysis of seed levels of sterols, carotenoids, chlorophylls and tocopherols in Arabidopsis transgenic lines with varying levels of expression of genes involved in the early steps of isoprenoid biosynthesis. Submission of one manuscript to an international journal is planned for 2014. Project (2) Complete analysis of plants defective in the expression of a regulatory gene that regulates stem length in Arabidopsis. We have recently demonstrated that the encoded regulator protein is required for responses to the plant hormone, gibberellin. Submission of one manuscript to an international journal is planned for 2014.
Impacts
What was accomplished under these goals?
We are using two complementary approaches to evaluate the regulation of the biosynthesis of isoprenoids (a class of metabolites with various essential functions in plants): (1) generation and analysis of a population of transgenic Arabidopsis lines with varying levels of expression of 13 different genes involved in generating the universal intermediates of isoprenoid biosynthesis; and (2) testing the functions of several candidate genes putatively involved in the regulation of isoprenoid biosynthesis, including those that encode transcriptional regulators and proteins with hypothesized roles in isoprenoid transport in Arabidopsis. Accomplishments: Project (1) A manuscript reporting on the impact of modulating flux through cytosolic and plastidial pathways leading to different isoprenoid end products has been submitted to "The Plant Cell", the mostly highly ranked journal in the plant sciences. ABSTRACT: In plants, two spatially separated pathways provide the precursors for isoprenoid biosynthesis. We have generated transgenic Arabidopsis thaliana lines with modulated levels of expression of each individual gene involved in the cytosolic/peroxisomal mevalonate (MVA) and plastidial methylerythritol phosphate (MEP) pathways. By assessing the correlation of transgene expression levels with isoprenoid marker metabolites (gene-to-metabolite correlation), we have determined the relative importance of transcriptional control at each individual step of isoprenoid precursor biosynthesis. The accumulation patterns of metabolic intermediates (metabolite-to-gene correlation) were then employed to infer flux bottlenecks in the sterol pathway. The extent of metabolic crosstalk - the exchange of isoprenoid intermediates between compartmentalized pathways - was assessed by a combination of gene-to-metabolite and metabolite-to-metabolite correlation analyses. We demonstrate that the overexpression of predictable combinations of genes can be used to significantly increase flux toward specific end products of the sterol pathway. Transgenic plants accumulating increased amounts of sterols are characterized by significantly elevated biomass, which is often a desirable trait in crop plants. Project (2) A manuscript reporting on the analysis of Arabidopsis lines defective in the expression of a gene hypothesized to be involved in sterol transport will be submitted to "Planta" in early 2014. ABSTRACT: Niemann-Pick type C1 disease is a lysosomal storage disorder caused by mutations in the NPC1 gene. At the cellular level, the disorder is characterized by the accumulation of storage lipids and lipid trafficking defects. The Arabidopsis thaliana genome contains two genes (At1g42470 and At4g38350) with weak homology to mammalian NPC1. The corresponding proteins have 11 predicted membrane-spanning regions and contain a putative sterol sensing domain. The At1g42470 protein is localized to the plasma membrane, while At4g38350 protein has a dual localization in the plasma membrane and the tonoplast. A phenotypic analysis of T-DNA insertion mutants indicated that At1g42470 and At4g38350 (designated AtNPC1-1 and AtNPC1-2, respectively) have partially redundant functions and are essential for plant viability and development. Homozygous plants impaired in the expression of both genes were not recoverable and AtNPC1-1/atnpc1-1/atnpc1-2/atnpc1-2 plants were severely dwarfed. Gene disruptions did not have an effect on sterol concentrations. However, members of a segregating AtNPC1-1/atnpc1-1/atnpc1-2/atnpc1-2 population accumulated decreased amounts of saturated (16:0) and increased levels of highly unsaturated (16:3 and 18:3) fatty acids. In leaf tissue hydrolyzates of AtNPC1-1/atnpc1-1/atnpc1-2/atnpc1-2 hemizygous plants, but not in wild-type controls, an accumulation of long-chain alpha-hydroxy fatty acids was observed, indicating that NPC1 deficiency in plants may affect sphingolipid metabolism or transport.
Publications
- Type:
Journal Articles
Status:
Submitted
Year Published:
2014
Citation:
Lange I., Herron B.K., Poirier B.C., Rios-Estepa R., Lange B.M. (2014) Comprehensive assessment of transcriptional regulation enables knowledge-based engineering of isoprenoid pathways in Arabidopsis. Plant Cell, submitted.
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Progress 01/01/12 to 12/31/12
Outputs
OUTPUTS: In this project we are using two complementary approaches to evaluate the regulation of the biosynthesis of isoprenoids (a class of metabolites with various essential functions in plants): (1) we are generating and analyzing a large number of transgenic Arabidopsis lines with varying levels of expression of 13 different genes involved in generating the universal intermediates of isoprenoid biosynthesis; (2) we are testing the functions of several candidate genes putatively involved in the regulation of isoprenoid biosynthesis, including those that encode transcriptional regulators and proteins with hypothesized roles in isoprenoid transport in Arabidopsis. I am sharing the information about progress made as part of our CRIS project on my laboratory website (http://public.wsu.edu/~lange-m/index.html) and via the annual CRIS report. PARTICIPANTS: Max Feldman, graduate student (graduated in May 2012)
Brenton Poirier, graduate student (started on project in July 2012)
Iris Lange, Research Technologist III TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: See Outputs/Impacts.
Impacts
The data acquisition for subproject 1 was completed in 2012. This includes determining the transcript levels of the transformed gene, measuring the concentrations of signature isoprenoids (sterols, carotenoids, and chlorophylls), and characterizing the phenotype of each transgenic line. We are currently assessing if additional experiments are required to develop a manuscript for publication in a scientific journal. We are expecting to submit a manuscript later this year (2013). The overall goal of this project is to gain a quantitative understanding of the regulatory importance of each individual gene in the pathways providing isoprenoid precursors. This knowledge enables future efforts aimed at purposely altering the accumulation levels of specific isoprenoids, which may lead to plants with improved fitness. During the experiments, as part of subproject 2, to characterize a putative positive regulator of the biosynthesis of sterols (which constitute one important class of isoprenoids), we came to the conclusion that the transcription factor under investigation did not appear to be involved directly in the regulation of this pathway. The more likely role is in the development of meristems (areas of active cell division and growth). These activities are close to completion and we are expecting to submit a manuscript for publication later this year (2013).
Publications
- No publications reported this period
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Progress 01/01/11 to 12/31/11
Outputs
OUTPUTS: Phytosterols are important structural components of plasma membranes in all eukaryotes and have vital functions as lipid hormones. More recently, evidence has emerged that phytosterols play roles in meristem organization, vascular patterning, and signaling pathways involving plant hormones such as auxin and ethylene. Phytosterols have been used successfully and safely for several decades to lower plasma cholesterol levels. Many margarines, butters, breakfast cereals and spreads are now enriched with plant-derived sterols and their esters. Over the last decade, considerable progress has been made in characterizing the properties of the individual enzymes involved in plant sterol biosynthesis. However, due to the complexity of the sterol pathway, current knowledge of its regulation is still fragmentary. We have used various bioinformatic approaches to identify candidate genes putatively involved in sterol pathway regulation. We have begun with follow-up experiments to test for the functions of these candidate genes. The main goal of the proposed activities is to further characterize the roles of what appear to be, based on substantial preliminary data, the best candidate genes for regulators of sterol biosynthesis. The characterization of the regulation of phytosterol pathway regulation is integral to the understanding of many different aspects of plant development. Our proposed activities will also provide the experimental resources that enable future molecular breeding and metabolic engineering efforts targeted at manipulating phytosterol biosynthesis. Eventually, an improved understanding of phytosterol pathway regulation will contribute positively to many areas of agriculture, forestry, and natural resource management. The translation of our discoveries into applications in the field will be assisted by the combination of basic and applied research conducted in my laboratory. PARTICIPANTS: Max Feldman, graduate student TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Based on a series of bioinformatic analyses we identified two classes of candidate genes with potential involvement in the regulation of the sterol pathway in the model plant Arabidopsis: transcription factors of the basic helix-loop-helix class (we termed the two candidate genes "Regulator of Membrane Integrity (RMI)" 1 & 2) and proteins with sterol sensing domains (we termed the two candidate genes "Niemann-Pick type C (NPC)" 1 & 2). Mutants in rmi1 are dwarfs that have reduced sterol and fatty acid levels, show membrane deficiencies, and exhibit defects in reproduction. A complementation experiment provided evidence that these phenotypic characteristics are due to mutations in RMI1. We have also generated RMI1 overexpression lines and are currently performing microarray experiments to evaluate the effects of unnatural RMI1 expression levels in knock-out mutants and overexpressors on the Arabidopsis transcriptome. A manuscript reporting on these findings is in preparation. Npc1/2 double mutants show a severe visible phenotype (dwarf). Interestingly, this phenotype was not reflected in deficiencies in sterols or fatty acids. However, we observed deficiencies in reproduction that are now being further investigated.
Publications
- No publications reported this period
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Progress 01/01/10 to 12/31/10
Outputs
OUTPUTS: The overall goal of our activities is to evaluate the potentially rate-limiting roles of various genes/enzymes involved in plant terpenoid biosynthesis. This year the focus was on completing experiments to assess transgenic lines with either over-expression or co-suppression of genes involved in the early steps of terpenoid biosynthesis. Constructs for the expression of 13 different genes (representing both the cytosolic mevalonate and the plastidial methylerythritol phosphate pathway) had been transformed into Arabidopsis. At least 15 independent transgenic lines had been regenerated for each gene construct (> 195 homozygous lines). These lines (three replicates each) were now characterized with regard to transgene expression levels, the amounts of plastidial and cytosolic metabolic end products, and general plant fitness. We are currently analyzing this vast amount of data and I am expecting to submit a manuscript to a high impact journal in 2011 (target: Nature Biotechnology). 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
By evaluating the correlation of gene expression levels with isoprenoid marker metabolites (chlorophylls, carotenoi ds, phytosterols and tocopherols) and biometric traits, we are providing a quantitative measure of the relative importance of transcriptional control at each step of these pathways. Our results are laying the groundwork for knowledge-based metabolic engineering efforts aimed at modulating flux through isoprenoid biosynthesis. The transgenic lines described here are also a highly valuable resource for generating transgenic lines in which the expression levels of two or more genes are tweaked for the overaccumulation of specific isoprenoid end products.
Publications
- No publications reported this period
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Progress 01/01/09 to 12/31/09
Outputs
OUTPUTS: The overall goal of our activities is to evaluate the potentially rate-limiting roles of various genes/enzymes involved in plant terpenoid biosynthesis. Using Arabidopsis as a model system, we have been approaching this project from two different angles: (1) experimental perturbations of pathway flux and global analysis of pathway regulation; (2) analysis of transgenic lines with either elevated or reduced expression levels of genes involved in the early steps of terpenoid biosynthesis. (1.1) We performed experiments to assess the impact of high light treatments on the sterol pathway in Arabidopsis. Seedlings were grown in Petri dishes under continuous light intensities of 67, 81, 98, 108, 141 or 163 microE m-2 s-1. Aerial parts were harvested at 14 days after germination, and sterols were extracted and analyzed by GC-MS. Interestingly, the amount of total sterols did not change significantly under the different light intensities. However, several sterol pathway intermediates (cycloartenol, 24-methylenecycloartanol, and avenasterol) accumulated in a light intensity-dependent manner. A manuscript reporting on our findings has been submitted to Nature Biotechnology (main focus of this paper is the utility of using metabolomics to understand conditional and genotypic variance in Arabidopsis). (1.2) Using a gene network discovery approach, we identified two genes encoding closely related helix-loop-helix (HLH) transcription factors putatively involved in the regulation of Arabidopsis sterol biosynthesis. Knock-out mutants impaired in the expression of these HLH genes and overexpressors were analyzed for various traits, and we now have conclusive evidence for the relevance of the encoded HLH proteins in controlling sterol biosynthesis. In 2010, we are planning to write up a manuscript reporting on our findings. (2.1) Populations of transgenic lines with modulated expression levels of genes involved in 13 different steps of terpenoid biosynthesis have been generated. At least 10 individual lines were analyzed for each transgenic population with regard to growth characteristics (5 different parameters), transgene expression levels, and sterol, chlorophyll and carotenoid levels. This massive amount of information is currently being compiled and we are planning to complete a manuscript within 2010. PARTICIPANTS: Not relevant to this project. TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
The characterization of the regulation of terpenoid biosynthesis is integral to the understanding of many different aspects of plant development. Our activities will also provide the experimental resources that enable future molecular breeding and metabolic engineering efforts targeted at manipulating terpenoid biosynthesis. The translation of our discoveries into applications in the field will be assisted by the combination of basic and applied research conducted in my laboratory.
Publications
- No publications reported this period
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Progress 01/01/08 to 12/31/08
Outputs
OUTPUTS: The overall goal of our activities is to evaluate the potentially rate-limiting roles of various genes/enzymes involved in plant terpenoid biosynthesis. Using Arabidopsis as a model system, we have been approaching this project from two different angles: (1) experimental perturbations of pathway flux; (2) analysis of transgenic lines with either over-expression or co-suppression of genes involved in the early steps of terpenoid biosynthesis. (1) Perturbations (1.1) To assess the role of redox control in regulating terpenoid biosynthesis at the post-transcriptional level, we performed an experiments in which Arabidopsis seedlings were treated with the plant hormone abscisic acid (which is known to affect the redox status of plant cells). We found evidence that abscisic acid (ABA) up-regulates its own biosynthesis via the terpenoid pathway. We also observed that the accumulation of alpha-tocopherol, which is also derived from the terpenoid pathway, was increased by ABA. The genes encoding enzymes of the tocopherol pathway were coordinately up-regulated by ABA. The results of this project were reported in a paper published recently in a very good journal (Phytochemistry). (1.2) To assess the role of light in terpenoid pathway regulation, we subjected Arabidopsis seedlings to different light conditions. Under low light conditions, sterol precursor supply was the primary factor limiting sterol pathway flux. However, under high light conditions, several sterol pathway intermediates accumulated, indicating that additional sterol pathway-specific steps had become rate-limiting. A manuscript reporting on our findings is currently in preparation. (2) Populations of transgenic lines with modulated expression levels of genes involved in early steps of terpenoid biosynthesis were generated. Completed: 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR), 1-deoxy-D-xylulose 5-phosphate synthase, 1-deoxy-D-xylulose 5-phosphate reductoisomerase, 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate reductase. In progress: mevalonate kinase, phosphomevalonate kinase, mevalonate diphosphate decarboxylase, 2-C-methyl-D-erythritol 2,4-cyclodiphosphate synthase. Main findings: We detected increased sterol levels in 11 transgenic lines. For example, total sterols were increased >2-fold over appropriate wild-type controls in an HMGR overexpression line. In accordance with the role of sterols and derived brassinosteroids in controlling cell expansion, plant biomass was significantly higher in the transgenic lines than in wild-type controls. Both transgenic lines also contained vastly increased levels of certain sterol pathway intermediates, indicating that additional sterol pathway-specific enzymes have rate-limiting roles. Based on sterol profiles obtained with activities under (1.2) and (2) we hypothesize that two enzymes might play rate-limiting roles in sterol biosynthesis: sterol methyl oxidase and sterol C24 reductase. We are currently studying the enzymology and regulation of these enzymes. PARTICIPANTS: Not relevant to this project. TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
The characterization of the regulation of terpenoid biosynthesis is integral to the understanding of many different aspects of plant development. Our proposed activities will also provide the experimental resources that enable future molecular breeding and metabolic engineering efforts targeted at manipulating terpenoid biosynthesis. Another important outcome of our work is the code that we develop for our mathematical models, which we deposit with online repositories, from where it can be downloaded and tested by the scientific community. Eventually, an improved understanding of terpenoid biosynthesis will contribute positively to many areas of agriculture, forestry, and natural resource management. The translation of our discoveries into applications in the field will be assisted by the combination of basic and applied research conducted in my laboratory.
Publications
- Ghassemian, M., J.Lutes, H.Chang, I.Lange, W.Chen, T.Zhu, X.Wang, and B.M.Lange. 2008. Abscisic acid-induced modulation o metabolic and redox control pathways in Arabidopsis thaliana. Phytochemistry. 69:2899-2911.
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Progress 01/01/07 to 12/31/07
Outputs
The overall goal of this project is to evaluate the role of shared enzymatic control in the regulation of the cytosolic mevalonate (MVA) and the plastidial mevalonate-independent (nonMVA) pathways of isoprenoid biosynthesis in plants, using Arabidopsis thaliana as a model. During the years 2005 and 2006 the genes encoding all 15 enzymes involved in these pathways have been cloned and plants have been transformed with constructs for overexpressing these genes. In 2007, we completed the selection of 225 homozygous lines (15 genes x 15 independent transformants per transgene). We have grown up populations of plants representing 15 independent transgenic lines for each of two selected genes (1-deoxy-D-xylulose 5-phosphate synthase (DXS) and 1-hydroxy-2-methyl-2-butenyl-4-phosphate reductase (HDR); both encode enzymes of the non-MVA pathway). Biometric parameters (timing of growth stages, rosette radius, length of bolt, and seed weight), transgene mRNA abundance (by
real-time PCR) and the levels of marker isoprenoids (were determined) (chlorophylls and carotenoids by LC-MS, sterols and tocopherols by GC-MS). To evaluate if a correlation between the expression of a transgene and the pool sizes of marker isoprenoids exists, parametric (ANOVA and Pearson's rank-order) and non-parametric (Kendall's and Spearman's rank-order) correlation analyses were performed using the statistical package "R". The combined transcript-to-metabolite correlation data generated with all 15 sets of transgenic lines will allow us to calculate, using the statistical methods listed above, correlation coefficients that are direct indicators of the relevance of each individual gene involved in the MVA and nonMVA pathways for controlling flux at the transcriptional level. There was a very weak positive correlation of DXS transgene levels with chlorophylls (R2 = 0.27; ? = 0.13), whereas no correlation with phytosterol levels was detectable. These results indicated that DXS
might exert only marginal control, at the transcriptional level, over the nonMVA pathway. In contrast, HDR transgenic lines showed transgene expression levels of 1.4 to 5.6-fold above wild-type controls, which correlated positively with chlorophyll concentrations (R2 = 0.48; ? = 0.008), whereas carotenoid and phytosterol levels were similar in control and transgenic lines. Some of our transgenic lines showed dramatic phenotypic differences when compared to controls. For example, transgenic lines expressing cDNAs corresponding to the nonMVA pathway enzymes 1-deoxy-D-xylulose 5-phosphate reductoisomerase, 2C-methyl-D-erythritol-2,4-cyclodiphosphate synthase and HDS all exhibit dwarfing, early senescence and sterility (Fig. 7), reminiscent of the phenotype of a T-DNA knock-out line impaired in the expression of the MVA pathway enzyme 3-hydroxy-3-methylglutaryl-CoA reductase. This raises the intriguing question if regulatory crosstalk between these spatially separated pathways may play a
role in their coordination. We are now in a unique position to address this issue.
Impacts
The regulation of isoprenoid biosynthesis, poorly understood, particularly at the level of precursor supply, is and our results will thus facilitate investigations in diverse areas of biology. I anticipate that the transgenic lines that we are generating will be usefully integrated into the programs of other researchers in the plant biology community. Eventually, an improved understanding of isoprenoid pathway regulation will contribute positively to many areas of agriculture, forestry, and natural resource management. The translation of our discoveries into applications in the field will be assisted by the combination of basic and applied research conducted in my laboratory. I maintain close contacts with industry and commodity groups. I also have a strong commitment to educational and outreach activities. My research group of 6 men and 4 women includes 2 undergraduate students who receive training in laboratory techniques and the application of the scientific method.
I am also teaching an Advanced Topics in Plant Biochemistry class, which constitutes one of the most comprehensive natural products biochemistry offerings of any U.S. graduate program in plant biology. As part of this course graduate and undergraduate students are involved in the development of an online educational resource (similar to a textbook) covering hot topics in natural products biochemistry
Publications
- Rios-Estepa, R., and B.M.Lange. 2007. Experimental and mathematical approaches to modeling plant metabolic networks. Archives of Biochemistry and Biophysics. 68:2351-2374.
- Fiehn, O., L.Sumner, S.Rhee, J.Ward, J.Dickerson, B.M.Lange, G.Lange, U.Roessner, R.Last, and B.Nikolau. 2007. Minimum reproting standards for plant biology context information in metabolomics studies. Metabolomics 3:195-201.
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Progress 01/01/06 to 12/31/06
Outputs
Using the BioPathAt tool developed in my laboratory we showed that several metabolic pathways (including the nonMVA pathway but not the MVA pathway) are coordinately up-regulated in etiolated seedlings treated with red light. However, gene expression patterns did not allow us to distinguish between red light and far-red light treated seedling populations (patterns were almost identical), whereas metabolite profiling unambiguously identified pathways that were differentially regulated (Ghassemian et al., 2006). In a collaborative effort with Dr. Browse's laboratory, at Washington State University we investigated the role, based upon transcriptional profiling, of various metabolic pathways (including those generating mono- and sesquiterpenes) in Arabidopsis thaliana stamens. The utilization of the BioPathAt tool developed in my laboratory enabled us to determine which metabolic pathways were activated by a jasmonic acid treatment. In addition, the BioPathAt tool was
used to visualize gene expression patterns on metabolic pathway maps to illustrate which genes within a particular pathway appeared to be coregulated at the transcriptional level (Mandaokar et al., 2006). In 2005, we generated transgenic lines of Arabidopsis thaliana in which the expression levels of genes involved in the early steps of isoprenoid biosynthesis were modulated (13 genes were PCR-amplified and constructs which allowed for the selection of transgenic lines using three different selection markers were generated: total: 39 constructs). In 2005 and 2006, we performed 7 individual transformation events per construct and collected a total of 273 (39 x 7) T0 seed populations. In 2006, seeds were germinated on plates under selection pressure, 10 different plants per line were transferred onto soil and seeds were collected from these plants (total: 2730 (10 x 273) T1 lines). Seedlings from the T1 seed generation were grown to select for a 3 : 1 survivor to non-survivor rate (as
expected for single copy gene insertions following Mendelian genetics) and seeds from selected survivor plant (T2) lines were collected. This process has been completed for all lines representing 4 of the 13 genes (total of 840 T1 lines segregated). Homozygous T2 lines are then identified by a 100 % germination rate of their seeds under selection pressure (currently underway).
Impacts
The development of the BioPathAt tool in 2004 and 2005 has proved to be highly valuable in discovering which metabolic pathways are differentially expressed in wild-type and mutant plants, how these pathways are affected by environmental treatments, and which role they play in plant development. It is expected that our own work and collaborations with other investigators will continue to make use of this bioinformatic tool. In 2006, two papers that utilize the BioPathAt tool were published in international journals. In order to assess the extend to which pathway flux is regulated at the transcriptional level, we are generating transgenic Arabidopsis thaliana lines in which the steady-state transcript levels of particular genes involved in the MVA and nonMVA pathways are modulated, and we are evaluating how differences in mRNA abundance correlate with changes in the levels of isoprenoid end products. Such experiments are providing the framework for follow-up efforts
aimed at increasing the amounts of specific isoprenoid end products in transgenic plants with altered expression levels of combinations of genes. Although A. thaliana will be used as an experimental model system, the results obtained as part of the proposed activities will have broader applicability for improving plant productivity and enabling the production of plant-based pharmaceuticals derived from isoprenoid biosynthetic pathways.
Publications
- Mandaokar, A.D., B. Thines, B. Shin, B.M. Lange, G. Choi, Y.J. Koo, Y.J. Yoo, Y.D. Choi, G. Choi, and J.A. Browse. 2006. Transcriptional Regulators of Stamen Development in Arabidopsis Identified by Transcriptional Profiling. The Plant Journal : for Cell and Molecular Biology. 46:984-1008.
- Ghassemian, M., J. Lutes, J. Tepperman, H. Chang, T. Zhu, X. Wang, P. Quail, and B.M. Lange. 2006. Integrative analysis of transcript and metabolite profiling data sets to evaluate the regulation of biochemical pathways during photomorphogenesis.. Archives of Biochemistry and Biophysics. 448:45-59.
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Progress 01/01/05 to 12/31/05
Outputs
This project aims at providing insight into the mechanisms underlying the regulation of enzymes involved in isoprenoid biosynthesis (particularly the cytosolic MVA and the plastidial nonMVA pathways) in Arabidopsis thaliana by integrating transgenic with gene expression/metabolite profiling and mathematical modeling approaches.
Impacts
Thirteen genes of the MVA and nonMVA pathways in Arabidopsis were cloned, inserted into three different Gateway cassettes, the constructs transformed into Agrobacterium, and Arabidopsis plants transformed using the floral dip method. We are currently in the process of obtaining 90 independent transgenic lines. A tool for the visualization of post-genomic data sets (microarrays, proteomics and metabolomics) directly in metabolic maps (incl. all pathways of isoprenoid biosynthesis) has been developed and published (Lange and Ghassemian, 2005). We showed that several metabolic pathways (including the nonMVA pathway but not the MVA pathway) are coordinately up-regulated in etiolated seedlings treated with red light. However, gene expression patterns did not allow distinguishing red light and far-red light treated seedling populations, whereas metabolite profiling unambiguously identified pathways that were differentially regulated. Treatment of Arabidopsis seedlings with
abscisic acid, a plant hormone, resulted in the modulation of a network of genes/metabolites involved in redox control (incl. genes/metabolites involved in and derived from the nonMVA pathway).
Publications
- Lange B.M., Ghassemian M. (2005) Comprehensive post-genomic data analysis approaches integrating biochemical pathway maps. Phytochemistry 66, 413-451.
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