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 gamma-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.