Progress 01/01/16 to 12/31/17
Outputs
Target Audience:The target audience for this project is other researchers within the field of plant molecular biochemistry, and who study the shikimate pathway or metabolism of compounds derived from the shikimate pathway. The target audience includes those working in acadamia, both at this and other institutions, as well as those in industry. In year 2, this was accomplished through presentation of key resultsat the Gordon Research Conference on Plant Metabolic Engineering, the Purdue Biochemistry Grad/Postdoc Seminar Series, the ASPB Midwest Regional Meeting, the Biochemistry Horizons Symposium, the USDA NIFA Research Fellowship Director's Meeting, and the Purdue Sigma Xi Poster Night. Additionally, as part of a broader effort at scientific education, ahands-on lesson was delivered at Tecumseh Middle School explaining the importance of plant-produced volatile organic compounds and how they are studied. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?PI continued to work under the mentorship of Natalia Dudareva, providing professional development for the PI. This has led to the PI receiving a position as a Research Scientist in the Department of Biochemistry at Purdue University after completion of the postdoctoral fellowship. PI mentored a female undergraduate research assistant. How have the results been disseminated to communities of interest?The results were disseminted by publication in peer-reviewed journals. One manuscript was published in the Plant Journal, with a second manuscript being prepared for imminent submission to Nature Chemical Biolgy. Addionally, key results were presented at the Gordon Research Conference on Plant Metabolic Engineering, the Purdue Biochemistry Grad/Postdoc Seminar Series, the ASPB Midwest Regional Meeting, the Biochemistry Horizons Symposium, the USDA NIFA Research Fellowship Director's Meeting, and the Purdue Sigma Xi Poster Night. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
Impact The shikimate pathway is a critical biosynthetic pathway in plants, with plant function, health, and resistance to both biotic and abiotic stresses all requiring compounds produced from the pathway's product, chorismate. Furthermore, three amino acids essential to the human diet are derived from this pathway. Multiple studies have emphasized the potential for improving agronomic traits through manipulation of the production these amino acids and their derivatives; however, strategies employed for such efforts usually assume the plastids are the sole site of all steps of the shikimate pathway. Therefore, the overall goal of this project is to test for the presence ofcytosolic enzymes forchorismate and subsequent amino acid biosynthesis, and assess the efficacy of metabolic engineering of this pathway. The results obtained in this project demonstrate that the "plastid only"assumption is faulty. Genetic manipulations have led to the discovery that a complete shikimate pathway is present in the plant cytosol, and that deregulating the first step is sufficient to increase metabolic flux through the entire pathway. The research has also revealed that alterations in this pathway may impact other metabolic processes, demonstratinginterplay between this pathway and other key metabolic pathways. Finally, candidate genes for the required enzymes have been identified, including those responsible for the completion of the cytosolic post-chorismate phenylalanine-biosynthetic pathway.This new fundamental knowledge of plant metabolism will allow formulation of better rational metabolic engineering strategies for improvement of agronomic traits such as nutritional value, biotic and abiotic stress resistance, yield enhancement, and biofuel production. Accomplishments towards Research Objective 1 As attempts to identify the gene responsible for the cytosolic DAHP synthase from the model species petunia were unsuccessful, an alternative strategy was employed using spinach, which has broader available genomic resources. The enzyme activity was detected in crude extracts from this species and partially purified by sequential chromatography steps. The final step resulted in a well-resolved peak of activty across multiple fractions. Proteomic analysis of fractions with activity identified peptides that map to 1334 spinach proteins, of which 5were identified as candidates based on correlation of peptide abundance profiles with enzyme activity across fractions andprotein size. Three of the proteins were successfully expressed as soluble proteins in E. coli, and were found to not have activity. The remaining two candidates could not be expressed in E coli, and attempts were instead made to test for their ability to complement S cerevisae DAHPS null mutants. This identified a single previously uncharacterized gene, annotated as a beta-galactosidase, as the likely DAHPS-encoding gene. Additional confirmation of this identification is being pursued prior to publication of these results. The knowledge gained under this objective will be of value to the broader plant science community as the committed step in a previously unknown pathway, and as a potential target for genetic manipulation or herbicide/pesticide strategies. Accomplishments towards Research Objective 2 Untargeted Metabolite Analysis Stable transgenics overexpressing a feedback-insensitive DAHPS in the cytosol had previously been shown to have elevated levels of aromatic amino acids, with unexpected perturbations in other classes of compounds. To better assess the global effect of altering flux through the cytosolic shikimate pathway, an untargeted metabolite analysis was perfored by LC-QTOF. Of the 1581 detected metabolites, 161 showed statistically altered abundances in transgenics relative to wild-type: 59 were increased, and 102 were decreased. Identification of these compounds by querying metabolite databases with the exact masses revealed that amino acids, quinolines, indoles, and flavanoids were generall increased in the transgenics, while terpenoids were generally decreased. This information will be critical in forming rational metabolic engineering strategies targeting the cytosolic shikimate pathway. Targeted Metabolite Analysis Targeted metabolite analysis of lignin, sesquiterpenes, chlorophyll, and anthocyanins were performed. Lignin, a structural cell wall polymer derived from phenylalanine, was unexpectedly found to be slightly decreased in transgenic lines. This unexpect result likely reflects ananticipated regulation, warranting further investigation. Sesquiterpenes and chlorophyll were selected for analysis as they are both terpenoid-derived, and untargeted analysis revealed that terpenoids may be detrimentally effected in the transgenics; however, neither sesquiterpenes norchlorophyll were statistically altered in transgenics relative to wild-type. Contrary to preliminary data, anthocyanins were increased to varying degrees of statistical significant in the tissues tested. This result is consistent with the finding of increased flavanoids in the untargeted analysis, and is likely due to the increased availability of their shared phenylalanine precursor. Additional accomplishments towards research goal. Completion of the cytosolic post-chorismate phenylalanine biosynthetic pathway Based on the theprocedure for nonaqueous fractionation developed for subcellular localization of enzymes during the previous reporting period, a cytosolic distribution was identified for the phenylalanine-biosynthetic enzymes chorismate mutase (CM) and prephenate dehydratase (PDT) in petunia flowers. A potential cyctosolic CM (PhCM2) had previously been identified in this species. Downregulation of PhCM2 by RNAi decreased production of phenylalanine and its derived volatiles. Flux modeling, performed in collaboration with the laboratory of John Morgan (Purdue), demonstrated that the decreased volatile formation was due primarily to adecrease in cytosolic phenylalaninebiosynthesis, thereby demonstrating for the first time the functional role of PhCM2 in this pathway. A 5'-RACE experiment on genes for the plastidial arogenate dehydratase isoforms that have PDT activity in petunia flowers revealed an alternative transcript of PhADT3 that encodes a protein lacking a plastidial transit peptide, designated PhADT3S. Enzyme assays performed on recombinantly produced PhADT3S confirm that this truncated protein retains PDT activity, and immunopurification combined proteiomic analysis demonstrated the presence of this short isoformin planta. It is likely that this enzyme is at least partially responsible for the observed cytosolic PDT activity. Analysis of additional candidate phenylalanine biosynthetic enzymes Transient RNAi downregulation of the previously identified DHQ synthase (designated PhDHQS2) resulted in a significant decrease in emission of phenylalanine-derived volatiles, thereby demonstrating its involvement in phenylalanine biosynthesis. To determine subcellular localization,PhDHQS2-GFP fusion proteins were expressed in Nicotiana benthamiana and observed by confocal microscopy. A clear colocalization of the GFP fluorescence was observed with chlorophyll autofluorescence, demonstrating that this enzyme is involved in the plastidial shikimate pathway, not the cytosolic pathway as hypothesized. Nonetheless, this represents identification of a new enzyme of the plastidial shikimate pathway. A 5'-RACE experiment performed on the known plastidial EPSP synthase isoforms of Petunia flowers revealed the presence of a transcript severly truncated at the 5' end. Although the protein encoded by this short transcript lacks the native plastid-targeting peptide of the full-length protein, it is unclear whether the short isoform retains the relevant enzymatic activity, and therefore further study is warranted.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2017
Citation:
Lynch JH, Orlova I, Zhao C, Guo L, Jaini R, Maeda H, Akhtar T, Cruz?Lebron J, Rhodes D, Morgan J, Pilot G. Multifaceted plant responses to circumvent Phe hyperaccumulation by downregulation of flux through the shikimate pathway and by vacuolar Phe sequestration. The Plant Journal. 2017 Dec 1;92(5):939-50.
- Type:
Journal Articles
Status:
Other
Year Published:
2018
Citation:
Lynch, J.H., Qian, Y., Guo, L., Rhodes, D., Morgan, J.A., and Dudareva, N. Completion of the cytosolic phenylalanine biosynthetic pathway in plants. Pending Submission to Nature Chemical Biology
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Progress 01/01/16 to 12/31/16
Outputs
Target Audience:The target audience for this project is other researchers within the field of plant molecular biochemistry, and who study the shikimate pathway or metabolism of compounds derived from the shikimate pathway. The target audience includes those working in acadamia, both at this and other institutions, as well as those in industry. In year 1, this was accomplished through presentation of key results to members of this institution. Changes/Problems:The expected timeline that was proposed anticipated the identification of the gene for the cytosolic DAHP synthase, and its partial biochemical purification, by the end of year 1. Although multiple candidates have been identified and are undergoing testing, a positive identification has not yet been achieved. To ensure the success of this objective, the enzyme will be purified from a second species (spinach) and identified by the proteomics facility. This is not expected to substantially alter the outcome from that originally proposed. What opportunities for training and professional development has the project provided?Attended an intensive, 2-day training at Gerstel US applications lab in Baltimore Maryland. The course covered use of integrated accesories for GC-MS systems, including the versatile MPS autosampler and accessories for liquid, headspace and SPME sample injection, as well as Thermal Desorption and related applications. Attended the "Grant & Proposal Writing Workshop," led by Dr Peter Dunn of Purdue University. How have the results been disseminated to communities of interest?
Nothing Reported
What do you plan to do during the next reporting period to accomplish the goals?Research Objective 1 Additional candidate genes for the cytosolic DAHP synthase will be screened The remaining cytosolic DAHP synthase candidates will be screened by heterologous expression. If the enzyme is not positively identified by this screen, candidates which did not properly express in the E coli system (for example, those which produced inclusion bodies) will be subcloned into a yeast expression vector, and expressed in S. cerevisiae. Though more cumbersome to use, eukaryotic expression systems can help produce active eukaryotic proteins even when such efforts fail in E coli. The cytosolic DAHP synthase will be purified from spinach To help identify candidate genes, the cytosolic DAHP synthase will be purified from an alternative source, and subjected to proteomic analysis. Spinach is a prime candidate for the source organism, as it it has previously been reported has having a particularly high DAHP synthase activity under conditions specific for the cytosolic isoform. Homology between candidates identified in the new species and candidates already identified in petunia will help to focus efforts on those with the highest probability of being the desired protein. Research Objective 2 Targeted metabolite profiling will be completed on stable transgenic plants The effect of increased flux through the cytosolic shikimate pathway will be studied in detail through analysis of the products of known downstream pathways. Following final confirmation of the level transgene expression, targeted analysis of phenylpropanoids and their derivatives will be completed. Compounds to be analyzed include monolignols, phenylpropanoid volatiles, and flavanoids/anthocyanins. Feeding with stable isotope labeled intermediates, in conjunction with metabolic modeling, will be completed to determine the relative contributions of the cytosolic and plastidial pathways to production of these compounds. An ongoing collaboration with Dr John Morgan of Purdue University, a recognized expert in plant metabolic modeling, will ensure the success of the latter experiments. Comparative untargeted metabolic analysis will be completed During the preliminary targeted metabolite analysis completed so far, the aquired GC-MS and LC-MS chromatograms revealed dramatic changes in the abundance of multiple unidentified compounds, thereby demonstrating effects of the transgene on unanticipated pathways. Characterization of these effects will be required to determine the metabolic engineering potential of this pathway. Therefore a collaboration has been initiated with Dr Bruce Cooper of the Bindley Bioscience Metabolomic Profiling Facility at Purdue University. With his assistance, a detailed untargeted metabolic profiling will be completed for comparative analysis of the transgenic and control plants, to produce a detailed observation of altered metabolite abundances. The use of a newly acquired Q-TOF mass spectrometer, in conjunction with available MS libraries, will enable identification of the unknown compounds. Where possible, relevant targeted followup experiments will be completed on effected pathways to determine the biochemical basis for the alterations.
Impacts
What was accomplished under these goals?
Impact The shikimate pathway is a critical biosynthetic pathway in plants, with plant function, health, and resistance to both biotic and abiotic stresses all requiring compounds produced from the pathway's product, chorismate. Furthermore, three amino acids essential to the human diet are derived from this pathway. Multiple studies have emphasized the potential for improving agronomic traits through manipulation of the production these amino acids and their derivatives; however, strategies employed for such efforts usually assume the plastids are the site of all steps shikimate pathway. The results obtained thus far demonstrate that this assumption is faulty. Genetic manipulations have led to the discovery that a complete shikimate pathway is present in the plant cytosol, and that deregulating the first step is sufficient to increase metabolic flux through the entire pathway. The research has also revealed the interplay between this pathway and other key metabolic pathways. Finally, candidate genes for the required enzymes are being identified. This new fundamental knowledge of plant metabolism will allow formulation of better rational metabolic engineering strategies for improvement of agronomic traits. Accomplishments towards Research Objective 1 Partial purification and proteomic analysis has led to identification of candidate DAHP synthase genes The protein extraction procedure from the preliminary results was repeated in large scale. When the protein was subjected to anion exchange chromatography, the target enzyme activity was found to elute as a well-resolved peak in recovered fractions. The fractions defining the peak were subjected to proteomic analysis by the laboratory of Dr Mark Hall, which generated peptide abundance profiles for each of the fractions. Of 675 distinct proteins identified, 17 were identified as candidates based on abundance profiles that correlate with the observed activity, predicted subcellular localization, and expression profiles in our RNAseq developmental dataset. The genes for the final candidates are being systematically cloned from petunia cDNA, and subcloned into bacterial expression vectors for heterologous expression, and the resulting protein screened for DAHP synthase activity. To date, 8 candidates have been screened by this method and found to not possess the target activity. Two additional proteins expressed as inclusion bodies and thus were not suitible for enzyme assay; if screening the remaining genes does not identify the cytosolic DAHP synthase, those which expressed as inclusion bodies will be expressed in eukaryotic systems. Accomplishments towards Research Objective 2 Overexpression in petunia supports presence of cytosolic shikimate pathway The E coli DAHP synthase AroG that had been rendered insensitive to feedback regulation by a point mutation, and which naturally lacks a plastidial targeting peptide, was transiently overexpressed in flowers of Petunia hybrida, Cv Mitchell. Headspace scent collections revealed that total Phe-derived volatile emission was increased by 58% in samples overexpressing the transgene (p<0.05), with five of the 10 individual compounds analyzed having statistically significant (p<0.05) increased emission rates. Content of all three of the aromatic amino acids was found to be increased by expression of the transgene. Therefore, new knowledge was obtained that the complete pathway for all three of these amino acids must be present in the cytosol. Stimulation of cytosolic shikimate pathway has unintended consequences The observed increase in flux through the cytosolic shikimate pathway created a significant draw on central carbon metabolism, as citrate, fumarate, and succinate were all found to be decreased. It is unclear what the implications of this draw on central carbon metabolism may be, but will be addressed in stable transgenic lines. During analysis of organic acid content, less anthocyanins were observed in extracts of flowers overexpressing the DAHP synthase. This is opposite what was expected, as Phe (which was found to be elevated) serves as a precursor to anthocyanin production. This has raised the intriguing possibility that stimulation of the enzyme HCT, which occurs immediately following the branchpoint to anthocyanins, is being stimulated by elevated levels of shikimate, as has previously been proposed in lignin biosynthesis. Such "feed forward" would draw more flux down the metabolic branch that competes with anthocyanin production. Consistent with this, isoeugenol, which relies on HCT for its synthesis, was the volatile showing the highest apparent increase in emission. This hypothesis is being followed up on. The knowledge generated under this finding will help guide future metabolic engineering efforts. Stable lines overexpression AroG in the cytosol have been developed in Arabidopsis and petunia Arabidopsis thaliana, ecotype Columbia, has been transformed with the gene for overexpression of AroG in the cytosol. Six independent lines showing highest expression of the transgene were selected for analysis. Tissue from the homozygous T3 generation is currently being analyzed. Preliminary analysis shows that all six lines have an increase in Phe and Trp content, but not Tyr. Stable transformation of Petunia hybrida, cv multiflora, was completed by leaf disc transformation. Cv multiflora was chosen in place of Cv. Mitchell to better study the apparent anthocyanin deficient phenotype observed in transient overexpression, as Cv. Mitchell is white while Cv. Multiflora is light pink due to higher anthocyanin content. More than 50 individual plantlets survived antibiotic selection, and are currently being propogated for analysis. The generation of these stable transgenic lines will facilitate detailed metabolic analyses required to accomplish the ultimate goals of this project. Additional accomplishments towards research goal. A procedure for nonaqueous fractionation has been developed for subcellular localization of enzymes. In nonaqueous fractionation, tissues are flash-frozen, lyophyilized, and then fractionated on a density gradient made of organic solvents. Classically, the distribution of metabolites in the density gradient is correlated with the distribution of markers for subcellular organelles, and thus the subcellular distribution of those metabolites can be calculated. I have adapted this procedure to identify the subcellular distribution of enzymatic activities in petunia flowers. For validation of the procedure, the subcellular location of two known proteins of Phe metabolism were determined: prephenate aminotransferase and phenylalanine ammonia lyase. The procedure correctly assigned the two activities to the plastids and cytosol, respectively. The procedure will be used to test for cytosolic localization of shikimate pathway enzymatic activities. Such a new method is necessary as it is difficult to discern between true cytosolic proteins and contaminents of lysed organelles when performing traditional aqueous fractionation, and will therefore be of use not only for this project, but also for other researchers analyzing enzymatic activies localized to multiple locations within the cell. Additional candidates for cytosolic aromatic amino acid biosynthetic enzymes have been identified Candidate genes for cytosolic enzymes of the shikimate pathway and Trp and Phe biosynthetic pathways have been identified, including dehydroquinate synthase, dehydroquinate dehydratase, shikimate dehydrogenase, chorismate mutase, anthranilate synthase, and prephenate dehydratase. This identification is necessary to determining the molecular basis for the newly confirmed cytosolic shikimate pathway.
Publications
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