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First published online 15 April 2009
doi: 10.1242/dev.032177

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DORNRÖSCHEN is a direct target of the auxin response factor MONOPTEROS in the Arabidopsis embryo

Melanie Cole^1,*, John Chandler^1,*, Dolf Weijers², Bianca Jacobs¹, Petra Comelli¹ and Wolfgang Werr^1,

¹ Institute of Developmental Biology, University of Cologne, Gyrhofstrasse 17, 50931 Cologne, Germany.
² Laboratory of Biochemistry, Wageningen University, Dreijenlaan 3, 6703 HA Wageningen, The Netherlands.

View larger version (115K): [in this window] [in a new window]   Fig. 1. Dynamics of DRN expression in the embryo and germinating seedlings. (A) Schematic of the DRN::GFP transcriptional and DRN::DRN-GFP translational fusions. (B-D) DRN::GFP expression in the apical cell of the early embryo (B) and restriction to the apical cell lineage in 4-cell embryos (C) (subtending non-expressing suspensor cells are outlined in B and C) or 8-cell embryos (D). (E) Cell autonomy of the DRN-GFP fusion protein at the 8-cell stage. (F-I) Comparison of DRN::GFP and DRN::DRN-GFP expression patterns in early (F,G) and late (H,I) globular stage embryos. The focus of the DRN-GFP fusion protein into the apical hemisphere presumably relates to the stability of the chimeric protein relative to that of the GFP marker. (J,K) Focussing of DRN::GFP expression to the prospective cotyledons at the late globular (J) and early heart (K) stage. (L,M) Expression patterns of the transcriptional DRN::GFP (L) and translational DRN::DRN-GFP (M) fusion in torpedo-stage embryos. Note DRN promoter activity in the SAM, which is absent during the heart stage (compare with K). (N) Lateral expression of DRN::GFP in a single cell extending laterally from the L1 layer at the position of an incipient leaf primordium in the torpedo-stage embryo. Inset, close-up of the SAM region showing a single GFP-expressing cell at the lateral flank. (O) A similar occurrence in the vegetative SAM of a two-leaf stage seedling. The frontal cotyledon has been removed and the first two leaf primordia are emerging on both sides of the SAM. The large cells above the leaf primordia belong to the remaining cotyledon at the back. Note DRN::GFP activity in the L1 layer of the leaf primordia (O) or the cotyledons (N). Scale bars: 20 µm.

View larger version (69K): [in this window] [in a new window]   Fig. 2. Functional contribution of AuxREs in the DRN upstream and downstream region. (A) Position of AuxREs upstream or downstream of the DRN coding region, which is replaced by GFP in the DRN::GFP construct. Canonical AuxREs are indicated by capital letters (A,B,C,D,E) in the wild type; mutated AuxREs are indicated by crosses in the individual constructs depicted beneath and denoted by lowercase letters. (B) DR5::GFP expression in late heart-stage embryos showing auxin concentration/perception maxima in the tips of the cotyledons and in the embryonic root. (C-F) Comparison of GFP expression obtained with the three AuxRE mutated constructs depicted in A relative to the non-mutated DRN::GFP transcriptional fusion at different stages of embryo development: (C) globular stage, (D) early heart stage, (E) late heart/early torpedo stage, (F) late torpedo stage. Note the absence of DRN::GFP expression in cotyledon tips later than heart stage (E or F) when upstream elements are mutated (constructs abc or abcde), which contrasts with the marking of the prospective cotyledons in D.

View larger version (53K): [in this window] [in a new window]   Fig. 3. Molecular and genetic interactions between MP and DRN. (A-C) DRN::GFP expression pattern in wild-type embryos (A) as compared with in the mp-U55 (B) or arf5-1 (C) mutant backgrounds. Note GFP activity in the SAM in both wild-type and mutant embryos, but the absence of expression in the tips of cotyledons in both mp mutant backgrounds. (D,E) Downregulation of DRN::GFP in the SAM of early heart-stage wild-type embryos (D) as compared with a continuous stripe of DRN::GFP activity from the cotyledons through the SAM in mp-U55 mutant embryos (E). (F,G) MP::MP-GFP expression in early (F) or late (G) heart-stage embryos showing a broad expression domain in apical and basal regions, including the vasculature. (H) Schematic of the DRN gene showing the upstream region and transcriptional unit (box), AuxRE positions (ovals) and the position of PCR amplicons in the promoter or coding region. (I) Results of two independent ChIP experiments performed with nuclei from immature siliques containing heart-stage embryos. The relative levels of PCR amplicons are depicted, as estimated by real-time PCR and normalized to the DRN ORF amplicon. AuxRE-B and AuxRE-C, which lie proximal to the DRN transcription start site, are selectively amplified. By contrast, chromatin templates spanning the distal AuxRE-A or the noARE amplicons are present at levels similar to those for the DRN ORF. Bars indicate the variation in three independent real-time PCR replicates performed for each ChIP experiment. Scale bars: 20 µm.

View larger version (35K): [in this window] [in a new window]   Fig. 4. Genetic interactions between mp and drn mutant alleles. (A) The mp basal domain defect, (B) the absence of the seedling root and presence of a single cotyledon and (C) the pin-like inflorescence phenotype. For frequencies see Table 2.

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