Biology Reference
In-Depth Information
of WRKY2 by ABA treatment. Our results suggest that WRKY2 transcription
factor mediates seed germination and postgermination developmental arrest by
ABA.
Methods
Plant Material and Growth Conditions
The Arabidopsis thaliana ecotypes Columbia, Wassilewskija and Landsberg erecta
were used throughout this study. Seeds of the different genotypes of Arabidopsis
thaliana were harvested from plants of the same age and stored for 5 weeks in
the dark at 4°C. Seeds were surface-sterilized with 10% bleach and washed three
times with sterile water. Sterile seeds were suspended in 0.1% agarose and plated
on MS medium plus 1% sucrose. ABA (mixed isomers, Sigma) was added to the
medium where indicated. Plates were routinely kept for 3 days in the dark at 4°C
to break dormancy (stratification) and transferred thereafter to a tissue culture
room under constant light at 22°C. Seeds of abi5-1, abi3-1, aba2-3 and aba3-1
were obtained from the Arabidopsis Biological Resource Center (ABRC) (Alonso
et al., 2003).
Identification of the WRKY2 T-DNA Insertion Mutants
The wrky2-1 mutant (Salk_020399), obtained from the Arabidopsis Biological
Resource Center (ABRC) (Alonso et al., 2003), contains a T-DNA insertion in
the promoter of the WRKY2 gene, while wrky2-2 mutant (Sail_739_F05) is a
gift of Dr. Zhixiang Chen (Department of Botany and Plant Pathology, Purdue
University, West Lafayette, Indiana, USA). Homozygous plants of the wrky2-1
mutant were identified by two PCRs. In the first PCR, a pair of gene-specific
primers designed to anneal outside of the T-DNA insertion were used, which
in case of homozygosity does not produce a band of the predicted size (negative
selection): forward primer 5'-ATCGTCATCATCTTCACCATTT-3' and reverse
primer 5'-AACTGAAATCCTCAGTTCCGT-3'. In the subsequent PCR, the
T-DNA border primer (5'-AAACGTCCGCAATGTGTTAT-3') in combination
with forward primer in the first PCR. To confirm the nature and location of
the T-DNA insertion, the PCR products were sequenced. To remove addition-
al T-DNA loci or mutations from the mutants, backcrosses to wild-type plants
were performed and plants homozygous for the T-DNA insertion were again
identified.
