Progress 09/01/08 to 08/31/12
Outputs
OUTPUTS: The main scientific outputs in the final year were: (1) Design and execution of improved drought stress experiments with wild type, vector control, and RNAi phenylalanine hydroxylase (PAH) knockdown Pinus taeda seedlings, to test for drought induction of the PAH gene and for the effect of PAH suppression on drought performance, and to compare PAH expression with the expression of phenylalanine ammonia lyase and of homogentisate (HGA) pathway enzymes. (2) Further analyses of PAH expression in various tissues and environmental conditions. (3) In-depth bioinformatic analyses of EST data to identify the PAH and HGA pathway genes in P. taeda, and to design and test primers for qRT-PCR quantification of their expression. (As the P. taeda genome is not sequenced, detailed transcriptome analysis is the only way to identify gene products.) (4) Sequencing of two alleles of P. taeda gene in order to define its intron-exon structure and natural variation for a definitive publication. PARTICIPANTS: No additions to project personnel and no changes in training. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
The immediate impact of the above work is definition of the structure, diversity, and expression pattern of the P. taeda PAH gene, and of the extent of its coexpression with key genes in phenylpropanoid and homnogentisate synthesis. This work is being summarized in a manuscript targeted for the journal New Phytologist. The longer term impact of the work is to increase awareness in the phenylpropanoid, lignin, and biofuel research communities that gymnosperms have an additional route for tyrosine synthesis (i.e. via hydroxylation of phenylalanine) that appears not to be present in Angiosperm trees or other Angiosperms.
Publications
- No publications reported this period
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Progress 09/01/10 to 08/31/11
Outputs
OUTPUTS: The main scientific outputs were: (1) Characterization of the Pinus taeda gene encoding folate-dependent phenylalanine hydroxylase (PAH); (2) Production and screening of RNAi lines of P. taeda for suppression of PAH gene expression; (3) Partial characterization of the natural expression pattern of P. taeda AAH expression. Postdoctoral O. Frelin was trained in plant genomics and analysis of gene expression. Results were disseminated at the Annual NIFA PIs meeting. A paper on the above work is in preparation. PARTICIPANTS: Oceane Frelin, postdoctoral research associate. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
(1) Characterization of the P. taeda PAH gene. Genomic sequence analysis of the intron/exon structure of the gene revealed exceptionally long intron length. Allelic variation in the promoter region was discovered; this variation could potentially affect expression levels. (2) Production and screening of P. taeda RNAi lines. Multiple independent RNAi lines were screened by quantitative RT-PCR for suppression of PAH expression; promising lines with substantial suppression were identified for further analysis. (3) Expression pattern of P. taeda PAH. Quantitative RT-PCR was used to determine PAH mRNA levels (a) in organs of field grown trees, (b) in greenhouse grown seedlings treated with phenylalanine (which induces PAH expression in bacteria), and (c) in greenhouse grown seedlings subjected to a drought cycle. Significant phenylalanine and drought induction were observed in initial trials. These experiments are being repeated and extended to include RNAi lines in the drought experiment, in order to assess the importance of PAH expression to stress adaptation.
Publications
- No publications reported this period
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Progress 09/01/09 to 08/31/10
Outputs
OUTPUTS: The main scientific outputs were: (1) Definition of the roles of two key pterin biosynthesis genes in bacteria; (2) Identification and characterization of a unique folate-dependent phenylalanine hydroxylase in pine and other non-flowering plants, and production and screening of transgenic pine lines harboring phenylalanine hydroxylase RNAi and GUS fusion constructs. Postdoctorals A. Pribat and A. Lara-Nunez were trained in pterin and folate biochemistry and analysis. The results were disseminated in appropriate professional journals (Journal of Bacteriology and The Plant Cell) and via presentations at scientific conferences. In addition, The Plant Cell article was featured in an 'In Brief' editorial article ('Introducing Aromatic Amino Acid Hydroxylases from Plants', Farquharson, K.L., The Plant Cell 2010 22: 3192.) PARTICIPANTS: Postdoctorals Anne Pribat and Aurora Lara-Nunez were trained in pterin and folate biochemistry and analysis. Research on bacterial pterins was conducted in collaboration with Jesse F. Gregory JF 3rd and Valerie de Crecy-Lagard (University of Florida). Research on phenylalanine hydroxylase was carried out in collaboration with the groups of Ralf Reski (University of Freiburg, Germany) and Lloyd Sumner (Samuel Roberts Noble Foundation, Ardmore, OK). TARGET AUDIENCES: The target audience for information on bacterial pterins is the research community that explores bacterial pterin and folate metabolism, which are intimately involved in antibiotic resistance. The target audience for information on plant phenylalanine hydroxylase is the research community working on lignin and biomass production and engineering. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
(1) Bacterial pterin synthesis. Tetrahydromonapterin is a major pterin in Escherichia coli and is hypothesized to be the cofactor for phenylalanine hydroxylase (PhhA) in Pseudomonas aeruginosa, but neither its biosynthetic origin nor its cofactor role has been clearly demonstrated. A comparative genomics analysis implicated the folX and folM genes in tetrahydromonapterin synthesis. folX encodes dihydroneopterin triphosphate epimerase, which interconverts dihydroneopterin and dihydromonapterin triphosphates. folM encodes a short-chain dehydrogenase/reductase known to have dihydrofolate and dihydrobiopterin reductase activity. The roles of FolX and FolM were tested experimentally first in E. coli, which lacks PhhA and in which the expression of P. aeruginosa PhhA plus the recycling enzyme pterin 4a-carbinolamine dehydratase, PhhB, rescues tyrosine auxotrophy. This rescue was abrogated by deleting folX or folM and restored by expressing the deleted gene from a plasmid. The folX deletion selectively eliminated tetrahydromonapterin production. Purified FolM showed high, NADPH-dependent dihydromonapterin reductase activity. These results were substantiated in P. aeruginosa by deleting tyrA (making PhhA the sole source of tyrosine) and folX. The tyrA strain was, as expected, prototrophic for tyrosine, whereas the tyrA folX strain was auxotrophic. As in E. coli, the folX deletant lacked tetrahydromonapterin. Collectively, these data establish that tetrahydromonapterin formation requires both FolX and FolM, that tetrahydromonapterin is the physiological cofactor for PhhA. (2) Folate-dependent phenylalanine hydroxylase. Tetrahydropterin-dependent aromatic amino acid hydroxylases (AAHs) are known from animals and microbes but not plants. A survey of genomes and ESTs revealed AAH-like sequences in gymnosperms, mosses, and algae. Analysis of AAH cDNAs from Pinus taeda, Physcomitrella patens, and Chlamydomonas reinhardtii indicated that the encoded proteins form a distinct clade within the AAH family. These proteins were shown to have phenylalanine hydroxylase activity by functional complementation of an E. coli tyrosine auxotroph and by enzyme assays. The P. taeda and P. patens AAHs were specific for phenylalanine, required iron, showed Michaelian kinetics, and were active as monomers. Uniquely, they preferred 10-formyltetrahydrofolate as cofactor and, consistent with preferring a folate cofactor, retained activity in complementation tests with tetrahydropterin-depleted E. coli strains. Targeting assays in Arabidopsis mesophyll protoplasts using green fluorescent protein fusions, and import assays with purified pea chloroplasts, indicated chloroplastic localization. Ablating the single AAH gene in P. patens caused accumulation of phenylalanine and caffeic acid esters. These data show that plants have functional plastidial AAHs, establish an unprecedented electron donor role for a folate, and uncover a novel link between folate and aromatic metabolism.
Publications
- Pribat, A., Blaby, I.K., Lara-Nunez, A., Gregory, J.F. 3rd, de Crecy-Lagard, V., and Hanson, A.D. (2010) FolX and FolM are essential for tetrahydromonapterin synthesis in Escherichia coli and Pseudomonas aeruginosa. Journal of Bacteriology 192(2): 475-482.
- Pribat, A., Noiriel, A., Morse, A.M., Davis, J.M,. Fouquet, R., Loizeau, K., Ravanel, S., Frank, W., Haas, R., Reski, R., Bedair, M., Sumner, L.W., and Hanson, A.D. (2010) Nonflowering plants possess a unique folate-dependent phenylalanine hydroxylase that is localized in chloroplasts. The Plant Cell 22(10): 3410-3422.
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Progress 09/01/08 to 08/31/09
Outputs
OUTPUTS: The principle scientific outputs were (i) cDNA clones and expression constructs of phenylalanine hydroxylases, (i) training of postdoctoral A. Pribat in pterin biochemistry, and (iii) new information, particularly the unexpected finding that certain important bacteria have a novel bypass step in pterin biosynthesis, mediated by the enzyme PTPS-III. As this novel enzyme occurs in various pathogens, PTPS-III proteins are potential targets for novel antifolate compounds. This finding was therefore disseminated in an appropriate professional journal (Journal of Bacteriology). PARTICIPANTS: Postdoctoral Anne Pribat was trained in pterin biochemistry and bacterial genetics. Research on subcellular localization of PAH proteins was conducted in collaboration with Stephane Ravanel (CNRS-CEA-INRA-Universite Joseph Fourier Grenoble I, Grenoble, France). Construction of moss PAH knockouts was carried out in collaboration with Ralf Reski (University of Freiburg, Freiburg, Germany) and metabolomic analysis of the knockouts was done in collaboration with Lloyd Sumner (Plant Biology Division, The Samuel Roberts Noble Foundation, Ardmore, OK). TARGET AUDIENCES: The target audience for the information on the novel enzyme PTPS-III is the research community that explores new antimicrobial drug targets. PROJECT MODIFICATIONS: A minor excursion was made to adequately document discovery of the novel PTPS-III enzyme.
Impacts
(1) Pterin-dependent phenylalanine hydroxylase (PAH). cDNAs encoding PAH were cloned and sequenced from pine (Pinus taeda), moss (Physcomitrella patens) and also Chlamydomonas, and complementation evidence was obtained for their function. Recombinant pine and moss PAH proteins were produced and purified, and shown to have PAH activity with tetrahydrobiopterin as electron donor. Green fluorescent protein fusion constructs of pine and moss PAH were made and pilot tests of transient expression in Arabidopsis protoplasts were conducted (in collaboration with the group of S. Ravanel) to obtain evidence for subcellular location. Multiple independent PAH knockout lines were made in moss (in collaboration with the group of R. Reski) and initial metabolomic analyses were made (in collaboration with the group of L. Sumner). (2) A novel pterin synthesis enzyme, PTPS-III. In the course of our work on pterin metabolism relating to phenylalanine hydroxylase we made an unexpected and important discovery that represents a significant change in knowledge. We therefore pursued this discovery in sufficient depth to produce a high-quality publication. Specifically, dihydroneopterin aldolase (FolB) catalyzes conversion of dihydroneopterin to 6-hydroxymethyldihydropterin (HMDHP) in the classical folate biosynthesis pathway. However, we found that folB genes are missing from the genomes of certain bacteria from the phyla Chloroflexi, Acidobacteria, Firmicutes, Planctomycetes, and Spirochaetes. Almost all of these folB-deficient genomes contain an unusual paralog of the tetrahydrobiopterin synthesis enzyme 6-pyruvoyltetrahydropterin synthase (PTPS) in which a glutamate residue replaces or accompanies the catalytic cysteine. A similar PTPS paralog from the malaria parasite Plasmodium falciparum is known to form HMDHP from dihydroneopterin triphosphate in vitro and has been proposed to provide a bypass to the FolB step in vivo. Bacterial genes encoding PTPS-like proteins with active-site glutamate, cysteine, or both residues were accordingly tested together with the P. falciparum gene for complementation of the Escherichia coli folB mutation. The P. falciparum sequence and bacterial sequences with glutamate or glutamate plus cysteine were active; those with cysteine alone were not. These results demonstrate that PTPS paralogs with an active-site glutamate (designated PTPS-III proteins) can functionally replace FolB in vivo. Recombinant bacterial PTPS-III proteins, like the P. falciparum enzyme, mediated conversion of dihydroneopterin triphosphate to HMDHP, but other PTPS proteins did not. Neither PTPS-III nor other PTPS proteins exhibited significant dihydroneopterin aldolase activity. Phylogenetic analysis indicated that PTPS-III proteins may have arisen independently in various PTPS lineages. Consistent with this possibility, introducing a glutamate residue into the active site of a PTPS protein conferred incipient activity in the growth complementation assay, and replacing glutamate with alanine in a PTPS-III protein abolished complementation.
Publications
- Pribat, A., Jeanguenin, L., Lara-Nunez, A., Ziemak, M.J., Hyde, J.E., de Crecy-Lagard, V., and Hanson, A.D. (2009) 6-Pyruvoyltetrahydropterin synthase paralogs replace the folate synthesis enzyme dihydroneopterin aldolase in diverse bacteria. Journal of Bacteriology 191(13): 4158-4165.
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