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First published online April 10, 2009
doi: 10.1242/10.1242/dev.025932

Development 136, 1509-1517 (2009)
Published by The Company of Biologists 2009
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A gain-of-function mutation in IAA18 alters Arabidopsis embryonic apical patterning

Sara E. Ploense, Miin-Feng Wu, Punita Nagpal and Jason W. Reed*

Department of Biology, University of North Carolina at Chapel Hill, Coker Hall, Chapel Hill, NC 27599, USA.


Figure 1
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  		Fig. 1. Morphology of iaa18-1 seedlings and iaa18-1/IAA18 P35S:MP/ARF5 plants. (A) Wild-type Landsberg erecta (Ler, left), homozygous iaa18-1 (center) and iaa18-1/IAA18 (right) adult plants. (B) Seven-day-old Ler seedling. (C-H) Seven-day-old iaa18-1/ seedlings of different phenotypic classes. (C) iaa18-1/IAA18 dicot. (D) Asymmetric dicot. (E) Fused cotyledons. (F) Monocot. (G) Rootless. (H) Unopened cotyledons. (I) Twelve-day-old mp-CSH1 seedling. (J) Twelve-day-old mp-CSH1 iaa18-1 seedling. (K) Twelve-day-old mp-CSH1 iaa18-1/IAA18 seedling. (L) Four-week-old mp-CSH1 iaa18-1 plant. Scale bars: 1.8 mm. (M-P,S,T) Whole shoots of adult plants. (M) P35S:MP/ARF5 plant with wild-type appearance. (N) P35S:MP/ARF5 plant with apical terminal flowers (arrowhead). (O,P,S) iaa18-1/IAA18 P35S:MP/ARF5 plants lacking curled leaves and with a range of heights. (T) iaa18-1/IAA18 plant with curled leaves. (Q,R) Inflorescence termini of P35S:MP/ARF5 (Q) and iaa18-1/IAA18 P35S:MP/ARF5 (R) plants. Scale bars: 2.5 mm. The pin-formed inflorescence and terminal flower phenotypes conferred by P35S:MP/ARF5 in the Landsberg erecta background were generally weaker than those described in the Columbia ecotype (Hardtke et al., 2004Go).

 

Figure 2
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  		Fig. 2. Expression of IAA18:GUS fusion constructs in transgenic plants. (A) IAA18:GUS fusion constructs. White boxes, IAA18 exons; black boxes, GUS gene; *, iaa18-1 mutation. (B) Expression level of IAA18 in wild-type, iaa18-1 and IAA18:GUS seedlings after mock treatment or after 40 minutes of treatment with 10 µM 1-NAA. (C) Expression levels of IAA18, iaa18NT:GUS and IAA18:GUS genes either untreated (dry) or after immersion in water (pH 7.4) or 50 mM MES (pH 5.4). In B and C, signals from RNA blot hybridizations of the indicated genes were digitized and normalized to signals from β-tubulin, and then each value was normalized to the untreated wild-type value from the first experiment. Error bars indicate s.d. of normalized measurements from four (IAA18 and iaa18-1) or two (IAA18:GUS) blots. (D-H) Six-day-old seedlings stained with X-gluc. (D) IAA18NT:GUS (left) and iaa18-1NT:GUS (right). (E,G) IAA18:GUS in root (E) and shoot (G) meristems. (F,H) iaa18-1NT:GUS in root (F) and shoot (H) meristems. In E and F, asterisks mark the level of the quiescent center and arrowheads indicate the point at which cells begin to elongate. Scale bars: 190 µm.

 

Figure 3
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  		Fig. 3. Embryo phenotypes and expression of IAA18, IAA18:GUS and iaa-18NT:GUS in wild-type and mutant embryos. (A,B) X-gluc stained IAA18NT:GUS (A) and iaa18-1NT:GUS (B) ovules at midglobular stage. (C,D) Mid-heart stage wild-type (C) and iaa18-1 (D) embryos. Arrowhead in D indicates overgrowth of cells at the cotyledon margin of the mutant embryo. (E,F) Mature wild-type (E) and iaa18-1 monocot (F) embryos. (G-M,A') X-Gluc-stained iaa18-1NT:GUS embryos. (N-R) In situ hybridization of wild-type embryos using an IAA18 probe. (G,N) Early globular stage. (H,O) Late globular stage. (I,P,A') Transition stage. (J,Q) Mid heart stage. (K,R) Late heart stage. (L) Torpedo stage. (M) Mature embryo. (S) axr6-1 IAA18:GUS embryo. About one-quarter of F2 embryos on axr6-1/AXR6 IAA18:GUS/- F1 plants stained, but fewer did in subsequent generations, suggesting possible IAA18:GUS transgene silencing in the axr6-1 background. (T) axr1-13 IAA18:GUS embryo. Fewer than 1% of axr1-13 IAA18:GUS embryos stained, perhaps because AXR1 has an additional paralog (Dharmasiri et al., 2007Go). (U) iaa18-1 iaa18-1NT:GUS heart stage embryo. (V) axr6-1 iaa18-1NT:GUS heart stage embryo. (W) axr1-13 iaa18-1NT:GUS heart stage embryo. (X,Y) mp-CSH1 iaa18-1NT:GUS early globular and transition stage embryos. (Z,B') mp-CSH1 nph4-1 iaa18-1NT:GUS heart stage embryos. A' and B' show top-down views of the embryo shoot apex. Mutant embryos were staged according to size compared with wild-type embryos. Scale bars: 10 µm. Unless indicated otherwise, X-Gluc stained embryos shown have the iaa18-1NT:GUS construct.

 

Figure 4
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  		Fig. 4. Expression of PIN1:GFP in wild-type and iaa18-1 embryos. (A-C) Wild-type (A) and iaa18-1/IAA18 (B,C) early globular stage embryos. Arrowhead in (C) indicates misaligned periclinal cell division planes in adjacent L1 cells. (D-F) Wild-type (D) and iaa18-1/IAA18 (E,F) mid-globular stage embryos. Arrowhead in F indicates an ectopic periclinal cell division. (G-I) Wild-type (G) and iaa18-1/IAA18 (H,I) transition stage embryos. Small arrows in I indicate apparent polarity of PIN1:GFP localization away from the cotyledon primordium on the right. (J,K) Wild-type (J) and iaa18-1/IAA18 (K) torpedo stage embryos. Arrowhead in K indicates a cell layer lacking fluorescence in the mutant embryo. (A-H) Optical sections (1 µm) through the central apical-basal axis of the embryo. (B,C,E,F) Adjacent sections through the same iaa18-1/IAA18 early and mid-globular embryos, respectively. (I) A z-stack projection of the same iaa18-1/IAA18 embryo as in H. (J,K) Z-stack projections of 16 1 µm sections through the central apical-basal axis of the embryo. Wild-type and mutant embryos were fixed, stained with DAPI and photographed in parallel using identical settings. (A-H) Overlays of fluorescent and DIC images. The PIN1:GFP lines used have been described previously (Heisler et al., 2005Go) (see Table S4 in the supplementary material).

 

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