Approaches for the Isolation of Arabidopsis adh1 Regulatory Mutants Using Allyl Alcohol Selection

Approaches for the Isolation of Arabidopsis adh1 Regulatory Mutants Using Allyl Alcohol Selection Allyl alcohol, a suicide substrate for the alcohol dehydrogenase enzyme (EC.1.1.1.1), has been frequently used as a negative selection method for the isolation of alcohol dehydrogenase mutants in plants, animals and microorganisms. This approach led to the isolation of mutants that mapped to the ADH gene itself. We attempted to use allyl alcohol selection for the isolation of adh1 regulatory mutants in Arabidopsis. First we selected at plantlet level on ADH1–GUS transgenic plants. This enabled us to use GUS staining to discriminate between structural and regulatory mutants. Allyl alcohol selection of 50000 EMS-treated seeds did not yield any potential mutants. Secondly we selected EMS and γ-ray-treated seeds of a transgenic line transformed with an additional copy of the ADH1 gene including its own promoter. Fifteen allyl alcohol-resistant plants were selected from the mutagenized seed. Genetic analysis of three putative mutants (adr8, adr10, and adr15) indicated that the ADH1-null phenotype was due to monogenic trans-recessive mutations. But treatment with the demethylating agent 5-azacytidine and analysis of methylation levels of the ADH1 gene indicated that these mutant candidates have increased levels of methylation in the promoter and coding region of ADH1. These results suggest that the allyl alcohol resistance of adr8, adr10, and adr15 is due to silencing of ADH1 rather than to a mutation of a regulatory locus. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Russian Journal of Plant Physiology Springer Journals

Approaches for the Isolation of Arabidopsis adh1 Regulatory Mutants Using Allyl Alcohol Selection

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Publisher
Kluwer Academic Publishers-Plenum Publishers
Copyright
Copyright © 2003 by MAIK “Nauka/Interperiodica”
Subject
Life Sciences; Plant Sciences
ISSN
1021-4437
eISSN
1608-3407
D.O.I.
10.1023/B:RUPP.0000003274.04439.d4
Publisher site
See Article on Publisher Site

Abstract

Allyl alcohol, a suicide substrate for the alcohol dehydrogenase enzyme (EC.1.1.1.1), has been frequently used as a negative selection method for the isolation of alcohol dehydrogenase mutants in plants, animals and microorganisms. This approach led to the isolation of mutants that mapped to the ADH gene itself. We attempted to use allyl alcohol selection for the isolation of adh1 regulatory mutants in Arabidopsis. First we selected at plantlet level on ADH1–GUS transgenic plants. This enabled us to use GUS staining to discriminate between structural and regulatory mutants. Allyl alcohol selection of 50000 EMS-treated seeds did not yield any potential mutants. Secondly we selected EMS and γ-ray-treated seeds of a transgenic line transformed with an additional copy of the ADH1 gene including its own promoter. Fifteen allyl alcohol-resistant plants were selected from the mutagenized seed. Genetic analysis of three putative mutants (adr8, adr10, and adr15) indicated that the ADH1-null phenotype was due to monogenic trans-recessive mutations. But treatment with the demethylating agent 5-azacytidine and analysis of methylation levels of the ADH1 gene indicated that these mutant candidates have increased levels of methylation in the promoter and coding region of ADH1. These results suggest that the allyl alcohol resistance of adr8, adr10, and adr15 is due to silencing of ADH1 rather than to a mutation of a regulatory locus.

Journal

Russian Journal of Plant PhysiologySpringer Journals

Published: Oct 17, 2004

References

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