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
Abstract
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.
Abstract
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.
Abstract
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.
Abstract
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
Abstract
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
Abstract
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)
Abstract
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.