Background and Training
PhD: California Institute of Technology
Postdoctoral training: The Whitehead Institute for Biomedical Research
Regulation of signaling metabolites and cellulosic biofuel feedstock development
We use a variety of plant systems including the model flowering plant, Arabidopsis thaliana, tobacco, rice, and the model grass, Brachypodium distachyon to characterize homeostasis mechanisms (i.e. how levels are regulated within the cell) for metabolites that function as signaling compounds. For example, we use stable isotope labeling to examine the regulation of growth and development by the signaling molecule indole-3-acetic acid (IAA), also known as auxin. We are developing analytical tools (primarily high throughput sample prep, quantitative metabolite profiling, and fluorescence-based cell sorting) and mutants that are disrupted in some aspect of metabolite regulation in order to characterize IAA homeostasis. Mass spectrometry features prominently in our experimental approach. Our long-term goal is to understand how auxin biosynthetic pathways interact with biosynthetic pathways for other important signaling molecules such as cytokinins, gibberellins, salicylic acid, ethylene, jasmonic acid, abscisic acid and brassinolide and to apply this knowledge to the development and optimization of cellulosic biofuel feedstocks such as Switchgrass.
Figure 1. IAA biosynthetic pathways identified for fungi (yeast biochemical pathways map from yeastgenome.org) and plants (AraCyc, (Niyogi, Last et al. 1993; Pagnussat, Yu et al. 2005; Woodward and Bartel 2005; Normanly 2009; Graindorge, Giustini et al. 2010 ; Tzin and Galili 2010) including pathways that appear to be specific to indole glucosinolate (IG) producing plant species. Abbreviations next to arrows refer to genes that have been confirmed (or in the case of SAL1 are proposed) to function in these pathways (green italics; plant genes, orange; yeast orange underlined; U. maydis). Dashed arrows indicate the lack of an enzyme activity or gene identified for that particular step in the pathway. Genes with mutant alleles that we will use in this project are described in Table 1. Compound names are spelled out once with abbreviations given in () except PhP; phenylpyruvate and CADP; carboxyphenylamino-1’-deoxyribulose-5’-phosphate. The aromatic amino acid transferase that converts prephenate to arogenate in plants has been identified as MEE17 and PAT (also AAT) and corresponds to At2g22250 (Pagnussat, Yu et al. 2005; Graindorge, Giustini et al.2010). A number of Arabidopsis genes with either arogenate dehydratase or prephenate dehydratase (or both) activities have been identified (Huang, Tohge et al. 2010; Rao, Hunter et al. 2010; Tzin and Galili 2010).
Graindorge, M., Giustini, C., Jacomin, A. C., Kraut, A., Curien, G., Matringe, M. 2010 Identification of a plant gene encoding glutamate/aspartate-prephenate aminotransferase: The last homeless enzyme of aromatic amino acids Febs Letters 584:4357-4360
Huang, T., Tohge, T., Lytovchenko, A., Fernie, A. R., Jander, G. 2010 Pleitotropic physiological consequences of feedback-insensitive phenylalanine biosynthesis in Arabidopsis thaliana, The Plant Journal 63:823-835
Normanly, J. Approaching cellular and molecular resolution of auxin biosynthesis and metabolism in Cold Spring Harbor Lab Perspectives: Auxin Signaling, eds. M. Estelle, D. Weijers, K. Ljung, O. Leyser 2:a001594 (2009)
Niyogi, K. K., Last, R. L., Fink, G. R., Keith, B. 1993 Suppressors of trp1 fluorescence identify a new Arabidopsis gene, TRP4, encoding the anthranilate synthase b subunit, The Plant Cell 5:1011-1027
Pagnussat, G. C., Yu, H.-J., Ngo, Q. A., Rajani, S., Mayalagu, S., Johnson, C. S., Capron, A., Xie, L.-F., Ye, D., Sundaresan, V. 2005 Development 132:603-614.
Tzin, V., Galili, G. 2010 The biosynthetic pathways for shikimate and aromatic amino acids in Arabidopsis thaliana in The Arabidopsis Book, ed. Georg Jander, The American Society of Plant Biologists http://www.bioone.org/doi/full/10.1199/tab.0132
Woodward, A. W. and B. Bartel (2005). "Auxin: regulation, action, and interaction." Ann. Bot. 95: 707-735.
How does auxin trigger virulence traits in plants?
Plant-associated microorganisms, ranging from symbionts to pathogens also produce IAA, either to facilitate a symbiotic interaction or as a result of the complex signaling interactions that occur between a pathogen and the plant host, presumably to subvert host defense mechanisms. A wide variety of plant-associated microorganisms have multiple routes for synthesizing IAA. While pathways producing IAA from tryptophan (Trp) have been described in species ranging from microbes to humans, there is abundant evidence for Trp-independent IAA biosynthesis in plants and more recently in yeast, but no genes have been identified for this alternate pathway. We are working to identify and characterize the genes and chemical intermediates involved in Trp-independent IAA synthesis in Arabidopsis and yeast. This project builds upon the observations that IAA is imported into yeast where it triggers virulence traits such as substrate adhesion and filamentation presumably via complex signaling interactions.
Figure 2. The edge of a patch of the S. cerevisiae, which is able to filament in the presence of IAA but not in its absence.
Size bar = 10 µM
Patil, R. A., Kolewe, M. E., Normanly, J., Walker, E. L., Roberts, S. C. Taxane biosynthetic pathway gene expression in Taxus suspension cultures with different bulk paclitaxel accumulation patterns – a molecular level approach to understand variability in paclitaxel accumulation Biotechnol J. 2011 Nov 18. doi: 10.1002/biot.201100183. [Epub ahead of print] [PubMed]
Nonhebel, H. M., Yuan, Y., Al-Amier, H., Pieck, M., Akor, E., Ahamed, A., Cohen, J.D., Celenza, J., Normanly, J., Redirection of Trp metabolism in tobacco by ectopic expression of an Arabidopsis indolic glucosinolate biosynthetic gene, Phytochemistry 72:37-48 (2011) [Pubmed]
Barkawi, L.S., Tam, Y.-Y., Tillman, J. A., Normanly, J., Cohen, J. D., A High Throughput Method for the Quantitative Analysis of Auxins, Nature Protocols 5: 1609-1618 (2010) [Pubmed]
Prusty Rao, R., Hunter, A., Kashpur, O., Normanly, J. Aberrant synthesis of indole-3-acetic acid in Saccharomyces cerevisiae triggers morphogenic transition, a virulence trait of dimorphic pathogenic fungi, Genetics 185: 211-220 (2010). [Pubmed]
Normanly, J. Approaching cellular and molecular resolution of auxin biosynthesis and metabolism in Cold Spring Harbor Lab Perspectives: Auxin Signaling, eds. M. Estelle, D. Weijers, K. Ljung, O. Leyser, doi: 10.1101/cshperspect.a001594 (2009).
Kramer, E.M., Lewandowski, M., Beri, S., Bernard, J., Borkowski, M. Burchfield, L. A., Mathisen, B., Normanly, J. Auxin gradients are associated with polarity changes in trees, Science 320:1610 (2008). [Pubmed]
Barkawi, L., Tam, Y., Tillman, J., Calio, J., Al-Amier, H., Emerick, M., Normanly, J., Cohen, J., A high-throughput method for the quantitative analysis of indole-3-acetic acid and other auxins from plant tissue, Analytical Biochemistry 372: 177-188 (2008). [Pubmed]
Calio, J., Tam, Y.Y., and Normanly, J. Auxin Biology and Biosynthesis in Recent Advances in Phytochemistry: Integrative Plant Biochemistry, ed, J. Romeo, Elsevier, vol 40: 287-305 (2006).
Ljung,K., Hull, A., Celenza, J., Yamada, M., Estelle, M., Normanly, J., and Sandberg, G. Sites and regulation of auxin biosynthesis in Arabidopsis roots.Plant Cell, 17:1090-1140 (2005). [Pubmed]
Celenza, J.L., Quiel, J.A., Smolen, G.A., Merrikh, H., Silvestro, A., Normanly, J., and Bender J. The Arabidopsis ATR1 Myb transcription factor controls indolic glucosinolate homeostasis.Plant Physiology, 137:253-262 (2005) [Pubmed]
Normanly, J, Sovin, JP, and Cohen, JD Auxin Metabolism in Plant Hormones: Biosynthesis, Signal Transduction, Action! 3rd edition. P.J. Davies, ed. Kluwer Academic Publishers: Dordrecht, The Netherlands. pp 36-62 (2004)
Zhao, Y., Hull, A. K., Gupta, N., Goss, K. A., Alonso, J., Ecker, J. R., Normanly, J., Chory, J., and Celenza, J. L. Trp-dependent auxin biosynthesis in Arabidopsis: involvement of cytochrome P450s CYP79B2 and CYP79B3. Genes and Development 16:3100-3112 (2002). [Pubmed]
Tam, Y. Y., and Normanly, J. Overexpression of a bacterial indole-3-acetyl-L-aspartic acid hydrolase in Arabidopsis thaliana Physiologia Plantarum 115:513-522 (2002). [Pubmed]