During embryogenesis, the architecture of the plant and the food reserves for
seed germination are established. Abscisic acid (ABA) regulates seed
development and dormancy. It controls genes involved in stress responses.
ABA-responsive basic leucine zipper (bZIP) transcription factors are identified by
interaction with ABA responsive cis-regulatory elements. The transcription factor
ABI5 is one of these. It regulates gene expression during embryogenesis and in
response to ABA. An ABA-insensitive mutant, abi5-6, exhibits no gross
morphological defects other than the effect on seed germination in the presence
of ABA. Thus, microarray analysis was employed to search for molecular
phenotypes. We used cDNA microarrays to analyze ABA regulated gene
expression and the role of ABI5 in seedlings. 310 genes were identified as
ABI5/ABA regulated genes. 161 of these genes were regulated by ABI5, and
134 of ABI5-regulated genes were co-regulated by ABA. Only a small number of
genes altered expression in both Pro35S:ABI5 and abi5-6 genetic backgrounds
indicating the preferential binding of the bZIP protein dimers to specific promoter
sequences. To determine the optimal platform for identifying ABI5-regulated
genes in seeds, a cDNA microarray, the Agilent Arabidopsis Oligo microarray,
and the Affymetrix ATH1 arrays were tested. Cross platform comparisons
utilized 4,518 genes present on all three platforms. The best correlation was
between the Agilent and the Affymetrix results. Furthermore, the Affymetrix results correlated best with qRT-PCR validation data for selected genes. A small
number of genes including AtCOR413 pm-1 showed a consistent expression
pattern across the three platforms. A robust ABRE cis-regulatory element was
identified in the promoter of AtCOR413 pm-1. Further studies showed binding of
ABI5 to the promoter of AtCOR413 pm-1 by Electrophoretic Mobility Shift
Assays (EMSA) and validated the expression of ABI5 and AtCOR413 pm-1 in
abi5-6 seeds by qRT-PCR and RNA gel blot analysis. Transactivation assays
using AtCOR413 pm-1 promoter:GUS fusions in Arabidopsis dry seed and
seedlings revealed ABI5 acts as a negative regulator for AtCOR413 pm-1 in dry
seeds, while other proteins may play major roles in regulating responses to ABA
and low temperature (LT) in seedlings.
| Identifer | oai:union.ndltd.org:tamu.edu/oai:repository.tamu.edu:1969.1/ETD-TAMU-2552 |
| Date | 15 May 2009 |
| Creators | Hur, Jung-Im |
| Contributors | Thomas, Terry L |
| Source Sets | Texas A and M University |
| Language | en_US |
| Detected Language | English |
| Type | Book, Thesis, Electronic Dissertation, text |
| Format | electronic, application/pdf, born digital |
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