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.
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.
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.
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)
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.