Work in this laboratory is supported by the National Science Foundation and the United States Department of Agriculture

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 (2002) in press

The plant hormone auxin regulates many aspects of plant growth and development. Although several auxin biosynthetic pathways have been proposed, none of these pathways has been precisely defined at the molecular level. Here we provide in planta evidence that the two Arabidopsis cytochrome P450s, CYP79B2 and CYP79B3, which convert tryptophan (Trp) to indole-3-acetaldoxime (IAOx) in vitro, are critical enzymes in auxin biosynthesis in vivo. IAOx is thus implicated as an important intermediate in auxin biosynthesis. Plants overexpressing CYP79B2 contain elevated levels of free auxin and display auxin overproduction phenotypes. Conversely, cyp79B2 cyp79B3 double mutants have reduced levels of IAA and show growth defects consistent with partial auxin deficiency. Together with previous work on YUCCA, a flavin monooxygenase also implicated in IAOx production, and nitrilases that convert indole-3-acetonitrile to auxin, this work provides a framework for further dissecting auxin biosynthetic pathways and their regulation.

Y. -Y. Tam and J. Normanly Overexpression of a bacterial indole-3-acetyl-L-aspartic acid hydrolase in Arabidopsis thaliana Physiologia Plantarum (2002) Vol 115:513-522

Transgenic Arabidopsis lines (ecotype Col-0) carrying the Enterobacter agglomerans IaaspH gene under CaMV 35S promoter control were more sensitive to exogenous indole-3-acetyl aspartic acid (IAA-Asp) and metabolized [2'-14C]IAA-Asp more rapidly than control lines. Free IAA, total IAA and IAN levels in independent transgenic lines that accumulated IaaspH mRNA varied insignificantly from control levels, yet IAA-Asp levels were significantly reduced. The transgenic lines were grown in a variety of conditions and subjected to morphometric analysis. All three lines showed statistically significant differences in rosette diameter (in soil), root and hypocotyl length (on agar). These effects were transient in some cases and did not manifest themselves under all growth conditions tried. The two independent lines with single T-DNA insertions had lower seed set compared to control lines.

Brown, D. E., Rashotte, A. M., Murphy, A. S., Taguel, B. W., Peer, W. A, Normanly, J., Taiz, L. and Muday, G. K. Flavonoids act as negative regulators of auxin transport in vivo in Arabidopsis thaliana Plant Physiology, 126 (2): 524-535 (2001).

E. Dolusic, M. Kowalczyk, V. Magnus, G. Sandberg and J. Normanly Biotinylated Indoles as Probes for Indole-Binding Proteins Bioconjugate Chemistry 12:152-162 (2001).

Biotinylated indoles were prepared for application as bifunctional probes for the detection of indole-biding proteins which participate in the life processes of humans, animals, plants, and bacteria. The indole nucleus was functionalized, at ring positions 3, 5, or 6, by attachment of a 2-aminoethyl group, which was then coupled to the carboxyl moiety of biotin, via a spacer composed 3 or 4 concatenated b-alanine residues. The constructs thus obtained were able to inhibit tryptophanase activity, similarly to indole in a concentration-dependent manner. They also bound strongly to lysozyme and wealkly to bovine and human serum albumins, in accordance with the known affinities of these proteins for indole and 3-(2-aminoethyl)indole (tryptamine). The biotin end of the protein-bound bifunctional probes could then be detected by coupling to (strept)avidin conjugated to alkaine phosphatase or horseradish peroxidase, followed by incubation with substrates which are converted by these enzymes to intensely colored or chemiluminescent products.
Y. -Y. Tam, E. Epstein and J. Normanly Characterization of Auxin Conjugates in Arabidopsis. Low Steady-State Levels of Indole-3-Acetyl-Aspartate, Indole-3-Acetyl-Glutamate, and Indole-3-Acetyl-Glucose Plant Physiology (2000) Vol 123:589-595

Amide-linked indole-3-acetic acid (IAA) conjugates constitute approximately 90% of the IAA pool in the dicot Arabidopsis thaliana, while ester-linked conjugates and free IAA account for approximately 10% and 1%, respectively when whole seedlings are measured. We show here that IAA-Asp, IAA-Glu and IAA-Glc are present at low levels in Arabidopsis. Nine-day old wild type Arabidopsis seedlings yielded 17.4 ± 4.6 ng per g FW IAA-Asp and 3.5 ± 1.6 ng per g FW IAA-Glu, and IAA-Glc was present at 7-17 ng per g FW in 12-day old wild type seedlings. Total IAA content in 9-day old Arabidopsis seedlings was 1200 ± 178 ng per g FW, so these three IAA conjugates together made up only 3% of the conjugate pool throughout the whole plant. We detected less than wild type levels of IAA-Asp and IAA-Glu (7.8 ± 0.4 ng per g FW and 1.8 ± 0.3 ng per g FW, respectively) in an Arabidopsis mutant that accumulates conjugated IAA. Our results are consistent with IAA-Asp, IAA-Glu and IAA-Glc being either minor, transient or specifically localized IAA metabolites under normal growth conditions and bring into question the physiological relevance of IAA-Asp accumulation in response to high concentrations of exogenous IAA.
J. Normanly and B. Bartel Redundancy as a way of life: IAA metabolism Current Opinion in Plant Biology (1999) Vol 2:207-213

Plants have evloved elaborate systems for regulating cellular levels of indole-3-acetic acid (IAA). The redundancy of this network has complicated the elucidation of IAA metabolism, but molecular geneticstudies and precise analytical methods have begun to expose the circuitry. It is now clear that plants synthesize, inactivate and catabolize IAA by multiple pathways, and multiple genes can encode a particular enzyme within a pathway. A number of these genes are now cloned, which greatly facilitates the future dissection of IAA metabolism.

B. Quirino, J. Normanly and R. M. Amasino Diverse Range of gene activity during Arabidopsis thaliana leaf senescence includes pathogen-independent induction of defense-related genes Plant Molecular Biology (1999) Vol 40:267-278

Nitrilases are able to convert indole-3-acetonitrile to IAA in vitro and nitrilase genes are upregulated during senescence in Arabidopsis. At the same time, free IAA levels increase two-fold in senescing leaves while IAN and conjugated IAA levels drop two-fold.

Y. -Y. Tam and J. Normanly Determination of indole-3-pyruvic acid levels in Arabidopsis thaliana by gas chromatography-selected ion monitoring-mass spectrometry Journal of Chromatography A (1998) Vol 800:101-108

A rapid and simple method is described for the determination of indole-3-pyruvic acid (IPA) levels in Arabdiopsis thaliana by gas chromatography-selected ion monitoring-mass spectrometry (GC-SIM-MS). The method includes derivatization of IPA with hydroxylamine in the crude extract, followed by ethyl acetate partitioning, solid-phase extraction with C18 resin, reversed-phase high-performance liquid chromatography (HPLC), and GC-SIM-MS. Three derivatizing reagents were tested; these were penafluorbenzylhydroxylamine, pentafluorophenylhydrazine, and hydroxylamine. Hydroxylamine proved to be the mot useful, as the IPA-oxime was easiest to purify from plant extracts and was the most stable. IPA was qauantified in Arabidopsis seedlings ranging in age from 5-12 days; levels varied from 4 to 13 ng/g, peaking at 7-9 days. © 1998 Elsevier Science B. V.

J. Normanly, P. Grisafi, G. R. Fink and B. Bartel Arabidopsis mutants resistant to the auxin effects of indole-3-acetonitrile are defective in the nitrilase encoded by the NIT1 gene The Plant Cell (1997) Vol 9:1781-1790

Indole-3-acetonitrile (IAN) is a candidate precursor of the plant growth hormone indole-3-acetic acid (IAA). We demonstrated that IAN has auxinlike effects on Arabidopsis seedlings and that exogenous IAN is converted to IAA in vivo. We isolated mutants with reduced sensitivity to IAN that remained sensitive to IAA. These mutants were recessive and fell into a single complementation group that mapped to chromosome 3, within 0.5 centimorgans of a cluster of three nitrilase-encoding genes, NIT1, NIT2, and NIT3. Each of the three mutants contained a single base change in the coding region of the NIT1 gene, and the expression pattern o f NIT1 is consistent with the IAN insensitivity observed in the nit1 mutant alleles. The half-life of IAN and levels of IAA and IAN were unchanged in the nit1 mutant, confirming that Arabidopsis has other functional nitrilases. Overexpressing NIT2 in transgenic Arabidopsis caused increased sensitivity to IAN and faster turnover of exogenous IAN in vivo. ©1997 American Society of Plant Physiologists

J. Normanly Auxin Metabolism Physiologia Plantarum (1997) Vol 100:431-442

Auxin metabolism encompasses transport, conjugation, deconjugation, conversion, and catabolism. The balance between auxin metabolism and biosynthesis determines the actual level of the hormone in a given cell and consequently plays an important role in many developmental processes from seed germination to fruit ripening. Mass spectrometry used in conjunction with stable isotope labeling studies has enabled comprehensive examination of auxin biosynthesis and turnover along witht the identification of many auxin conjugates. It appears that the conjugate moiety may signal the metabolic fate (e.g. storage and eventual hydrolysis to free hormone, or catabolism). Recently identified auxin-metabolizing enzymes are encoded by gene families which vary in specificity for auxin metabolites. The expression patterns of these genes will reveal a great deal about the mechanics of auxin metabolism. ©Physiologia Plantarum ISSN 0031-9317

N. Ilic, J. Normanly, and J. D. Cohen Quantification of free plus conjugated indole-3-acetic acid in Arabidopsis requires correction for the non-enzymatic conversion of indolic nitriles Plant Physiology (1996) Vol 111:781-788

The genetic advantages to the use of Arabidopsis mutants for the study of auxin metabolism have previously been partially offset by the complexity of indolic metabolism in this plant and the lack of proper methods. To address some of these problems, we developed isotopic labeling methods to determine amounts and examine the metabolism of indolic compounds in Arabidopsis. Isolation and identification of endogenous indoleacetonitrile (IAN) [a possible precursor of the auxin indoleacetic acid (IAA)] was carried out under mild conditions, thus proving its natural occurrence. We describe here the synthesis of [13C]labeled IAN and its utility in the GC-MS quantification of endogenous IAN levels. We also quantified the non enzymatic conversion of IAN to IAA under conditions used to hydrolyze IAA conjugates. [13C]Labeled IAN was employed to asses the contribution of IAN to measured IAA following hydrolysis of IAA conjugates. We studied the stability and breakdown of the indolic glucosinolate glucobrassicin, which is known to be present in Arabidopsis. This is potentially an important concern when using Arabidopsis for studies of indolic chemistry, since levels of indolic auxins and auxin precursors are well below the levels of the indolic glucosinolates. We found that under conditions of extraction and base hydrolysis, formation of IAA from glucobrassicin was negligible. ©1996 American Society of Plant Physiologists

J. Normanly, J. P. Slovin, and J. D. Cohen Update on hormones: Rethinking auxin biosynthesis and metabolism Plant Physiology,107:323-329 (1995)

Review article
This review focuses on the new and more complex picture of IAA biosynthesis that has emerged as a result of recent experiments. ©1995 American Society of Plant Physiologists

J. Normanly, J. D. Cohen, and G. R. Fink Arabidopsis thaliana auxotrophs reveal a tryptophan-independent biosynthetic pathway for indole-3-acetic acid Proc. Natl. Acad. Sci., 90:10355-10359 (1993)

We used tryptophan auxotrophs of the dicot Arabidopsis thaliana (wall cress) to determine whether tryptophan has the capacity to serve as a precursor to the auxin, indole-3-acetic acid (IAA). Quantitative gas chromatography-selected ion monitoring-mass spectrometry (GC-SIM-MS) revealed that the trp2-1 mutant, which is defective in the conversion of indole to tryptophan, accumulated amide- and ester-linked IAA at levels 38-fold and 19-fold, respectively, above those of wild type. Tryptophan and free IAA were isolated from the trp2-1 mutant grown in the presence of [15N]anthranilate and [2H5]tryptophan, and the relative 15N and 2H5 enrichments of tryptophan and IAA were determined via GC-SIM-MS. The 15N enrichment of tryptophan, 13%+/- 4%, was less than the 15N enrichment of the IAA pool, 39% +/- 4%; therefore, IAA biosynthesis occurs via a tryptophan-independent pathway. The amount of 2H5 incorporated by the plant into IAA from tryptophan (95+/- 4%) was low and only slightly above the level of spontaneous, nonenzymatic conversion of [2H5]tryptophan to [2H5]IAA. These results show that the dicot Arabidopsis is similar to the monocot Zea mays in that the major route of IAA biosynthesis does not occur through tryptophan.

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