[Arabidopsis] Auxin overproduction in iaaH transformants

Danielle Marcos marcos at botany.utoronto.ca
Mon Jun 26 08:57:20 EST 2006


I'm posting the helpful response that I got to my query in the hopes=20
that it will help out others. Thanks again to Prof. Cohen.  If I=20
receive any more responses, I will post those too.  - Danielle Marcos


Danielle:=A0 The IAM system is not an absolute selection since=20
Arabidopsis does contain low levels of IAM (see below) and a functional=20=

hydrolase.=A0 Depaneding on how you set up your experiment you will see=20=

bigger or smaller differences between control and iaaH transformed=20
materials. Hope this helps, Jerry=A0=A0=A0=A0=A0=A0=A0=A0 Planta. 2002=20=

Nov;216(1):155-61.
Occurrence and formation of indole-3-acetamide in Arabidopsis thaliana.
  Pollmann S, Muller A, Piotrowski M, Weiler EW.
Lehrstuhl fur Pflanzenphysiologie, Fakultat fur Biologie,=20
Ruhr-Universitat Bochum, 44780 Bochum, Germany.
An HPLC/GC-MS/MS technique (high-pressure liquid chromatography in=20
combination with gas chromatography-tandem mass spectrometry) has been=20=

worked out to analyze indole-3-acetamide (IAM) with very high=20
sensitivity, using isotopically labelled IAM as an internal standard.=20
Using this technique, the occurrence of IAM in sterile-grown=20
Arabidopsis thaliana (L.) Heynh. was demonstrated unequivocally. In=20
comparison, plants grown under non-sterile conditions in soil in a=20
greenhouse showed approximately 50% higher average levels of IAM, but=20
the differences were not statistically significant. Thus, microbial=20
contributions to the IAM extracted from the tissue are likely to be=20
minor. Levels of IAM in sterile-grown seedlings were highest in imbibed=20=

seeds and then sharply declined during the first 24 h of germination=20
and further during early seedling development to remain below 20-30=20
pmol g(-1) fresh weight throughout the rosette stage. The decline in=20
indole-3-aetic acid (IAA) levels during germination was paralleled by a=20=

similar decline in IAM levels. Recombinant nitrilase isoforms 1, 2 and=20=

3, known to synthesize IAA from indole-3-acetonitrile, were shown to=20
produce significant amounts of IAM in vitro as a second end product of=20=

the reaction besides IAA. NIT2 was earlier shown to be highly expressed=20=

in developing and in mature A. thaliana embryos, and NIT3 is the=20
dominantly active gene in the hypocotyl and the cotyledons of young,=20
germinating seedlings. Collectively, these data suggest that the=20
elevated levels of IAM in seeds and germinating seedlings result from=20
nitrilase action on indole-3-acetonitrile, a metabolite produced in the=20=

plants presumably from glucobrassicin turnover.

Phytochemistry 62 (2003) 293=96300
Molecular cloning and characterization of an amidase from Arabidopsis=20
thaliana capable of converting indole-3-acetamide into the plant growth=20=

hormone, indole-3-acetic acid
  Stephan Pollmann, Daniel Neu, Elmar W.Weiler *

Abstract
Acylamidohydrolases from higher plants have not been characterized or=20
cloned so far. At AMI1 is the first member of this enzyme
  family from a higher plant and was identified in the genome of=20
Arabidopsis thaliana based on sequence homology with the=20
catalyticdomain
  sequence of bacterial acylamidohydrolases, particularly those that=20
exhibit indole-3-acetamide amidohydrolase activity.
AtAMI 1 polypeptide and mRNA are present in leaf tissues, as shown by=20
immunoblotting and RT-PCR, respectively. At AMI1 was
  expressed from its cDNA in enzymatically active form and exhibits=20
substrate specificity for indole-3-acetamide, but also some
  activity against l -asparagine.The recombinant enzyme was=20
characterized further. The results show that higher plants have=20
acylamidohydrolases
  with properties similar to the enzymes of certain plant-associated=20
bacteria such as Agrobacterium-, Pseudomonasand
Rhodococcus -species, in which these enzymes serve to synthesize the=20
plant growth hormone, indole-3-acetic acid, utilized by the
  bacteria to colonize their host plants.As indole-3-acetamide is a=20
native metabolite in Arabidopsis thaliana, it can no longer be ruled
  out that one pathway for the biosynthesis of indole-3-acetic acid=20
involves indole-3-acetamide-hydrolysis by At AMI1.


At 01:41 PM 6/23/2006, you wrote:
Hello,

  I'm currently working with Arabidopsis Col-0 plants transformed with=20=

an inducible indoleacetamide hydrolase (iaaH) transgene.=A0 The iaaH =
gene=20
encodes an enzyme that catalyzes the production of auxin (in the form=20
of indole acetic acid or IAA) from indoleacetamide (IAM).=A0 Col-0 =
plants=20
do not produce IAM. Therefore, only plants expressing iaaH that are=20
supplied with IAM should overproduce auxin, while plants expressing=20
iaaH that are not supplied with IAM should not.=A0 However, I observe=20
phenotypes consistent with auxin overproduction in plants expressing=20
iaaH both in the presence and absence of auxin.=A0 Has anyone else had a=20=

similar experience?=A0 Is there any way of avoiding this?

  Thanks,

  Danielle Marcos
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