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Transcriptional profiles between mp mutant seedlings and transgenics carrying the dexamethasone-inducible GR-bdl protein

ABSTRACT: In order to identify targets of the transcription factor AUXIN RESPONSE FACTOR5 / MONOPTEROS (ARF5/MP), we compared transcriptomes of mp-B4149 mutant seedlings (9 day-old) and seedlings carrying the dexamethasone-inducible version of the MP inhibitor protein BODENLOS (GR-bdl). Without dexamethasone (DEX) treatment, this line is identical to the wild-type, while DEX treatment leads to strong inhibition of ARF-dependent transcription. To remove all endogenous MP-inhibiting Aux/IAA proteins, we treated mp or GR-bdl seedlings during 1 hour with auxin (50 micromolar Indole-3-Acetic Acid), either with or without a pretreatment with 10 micromolar DEX for 1 hour. Genes that are activated by MP are expected to br downregulated in mp seedlings and in the GR-bdl line afer DEX treatment. We used biological duplicates for each of the three treatments. Keywords: Transcriptional profiling of transcription factor targets Overall design: Mp mutant and GR-bdl seeds were germinated on solid 1/2MS media, and transferred to liquid medium after 9 days of horizontal growth. Duplicate cultures were generated for mp mutants, and quadruplicates for the GR-bdl line. 10 micromolar DEX was added to both mp cultures and to two of the GR-bdl cultures, and seedlings were gently shaken during 1 hour. Then, 50 micromolar IAA was added to all 6 cultures and seedlings were incubated with gentle shaking for another hour. Total RNA was isolated from the seedlings using a QIAgen RNeasy kit, and reverse transcription, in-vitro transcription, cRNA labling and fragemntation was done according to established methods (Schmid et al., Nature Genetics [2005] 37, 501-506). Labeled cRNA was hybridized to Arabidopsis ATH1 arrays.

INSTRUMENT(S): [ATH1-121501] Affymetrix Arabidopsis ATH1 Genome Array

ORGANISM(S): Arabidopsis thaliana  

SUBMITTER: Dolf Weijers  

PROVIDER: GSE13881 | GEO | 2010-04-01



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