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First published online 4 October 2006
doi: 10.1242/dev.02602

Development 133, 4211-4218 (2006)
Published by The Company of Biologists 2006
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Arabidopsis microRNA167 controls patterns of ARF6 and ARF8 expression, and regulates both female and male reproduction

Miin-Feng Wu, Qing Tian* and Jason W. Reed{dagger}

University of North Carolina at Chapel Hill, Department of Biology, CB #3280, Coker Hall, Chapel Hill, NC 27599-3280, USA.


Figure 1
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  		Fig. 1. ARF6 and ARF8 mRNA expression patterns. (A-I,L) Sections of wild-type (A-F), mARF6 (G-I) and arf6-2 (L) flowers hybridized with an antisense ARF6 probe. (J,K,M) Sections of wild-type (J), mARF8 (K) and arf8-3 (M) flowers hybridized with an antisense ARF8 probe. (A) Longitudinal section of inflorescence. Arrows indicate vasculature. (B) Longitudinal section of stage 9 flower. Arrow indicates ARF6 expression in the medial ridge of carpels. (C) Longitudinal section of a stage 12 flower. Arrow indicates stamen filament vasculature; arrowhead indicates nectary. (D) Cross section of a stage 9 flower gynoecium. Arrow indicates the medial ridge of carpels. (E) Stage 2-II ovule. Arrow indicates funiculus; arrowhead indicates the placental region. (F) Stage 3-I ovule. Arrow indicates funiculus. (G) Cross section of a stage 9 flower bud. Arrow indicates anther vasculature. (H,I) Stage 2-III (H) and stage 3 (I) ovules. Arrows indicate the integument and nucellus regions. (J) Stage 2-II ovule. Arrow indicates funiculus; arrowhead indicates the placental region. (K) Stage 3-I ovule. Arrow indicates integuments; arrowhead indicates nucellus. ch, chalaza; fu, funiculus; nu, nucellus. Scale bars: 60 µm in A-C,G; 30 µm in D-F,H-M.

 

Figure 2
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  		Fig. 2. Expression patterns of gARF6, mARF6, gARF8 and mARF8 protein:GUS fusions, and MIR167 promoter::GUS fusions. (A-E) gARF6:GUS. (F-J) mARF6:GUS. (K,M) gARF8:GUS. (L,N) mARF8:GUS. (O-T) PMIR167a::GUS. (U,V) PMIR167b::GUS. (W,X) PMIR167c::GUS. (Y) PMIR167d::GUS. (A,B) gARF6:GUS staining patterns in stage 11 (A) and stage 13 (B) flowers. (C-E) gARF6:GUS expression in stage 2-III (C), stage 3-I (D) and stage 4-I (E) ovules. Arrows indicate funiculus. (F,G) mARF6:GUS expression in stage 11 (F) and stage 13 (G) flowers. (H-J) mARF6:GUS expression in ovules. Ovule development stages in H,I,J are equivalent to those in C,D,E, respectively. (K) gARF8:GUS staining in a stage 13 flower. Arrow indicates stamen filament expression. (L) mARF8:GUS staining in a stage 13 flower. (M) gARF8:GUS staining in a stage 4-I ovule funiculus (arrow). (N) mARF8:GUS expression in a stage 4-I ovule. Arrow indicates reduced outer integument growth. (O) PMIR167a::GUS expression in a stage 13 flower. (P-R) PMIR167a::GUS in stage 1-II (P), stage 2-III (Q) and stage 3-IV (R) ovules. (S,T) PMIR167a::GUS expression in floral stage 10 (S) and stage 13 (T) anthers. (U,V) PMIR167b::GUS expression in a stage 13 flower (U) and a stage 4-I ovule (V). (W,X) PMIR167c::GUS expression in a stage 13 flower (W) and a stage 4-I ovule (X). (Y) PMIR167d::GUS expression in a stage 13 flower. Scale bars: 0.3 mm in A,B,F,G,K,L,O,U,W,Y; 12 µm in C-E, H-J,M,N,P,R,V,X; 30 µm in Q,S,T.

 

Figure 3
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  		Fig. 3. Effects of overexpressing MIR167 genes. (A) Sequences of miR167 target sites on ARF6 and ARF8 mRNA, and the mutated target sites of mARF6 and mARF8. The Watson-Crick base pairings to the miR167 sequence are shown. {Delta}G (kcal/mol) was calculated by Mfold (Zuker, 2003Go). Mutated nucleotides are in lower case. (B) Phenotypes of plants overexpressing four different MIR167 genes. From left to right: wild type (WT), arf6/+ arf8, arf6 arf8, P35S::MIR167a (a), P35S::MIR167b (b), P35S::MIR167c (c) and P35S::MIR167d (d). (C) Stage 13 flowers of P35S::MIR167 plants. Genotypes are the same as in B. (D) Northern blot analyses of MIR167-overexpressing transgenic plant flowers. U6 snRNA and ß-tubulin are included as loading controls. Numbers beneath lanes indicate relative transcript levels normalized to loading controls.

 

Figure 4
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  		Fig. 4. ARF6 expression, and flower and ovule phenotypes of mARF6 plants. (A) Northern blot analysis of ARF6 transcript levels in wild-type, arf6-2, P35S::ARF6, and individual mARF6 and gARF6 transgenic plant flowers. The transcript of P35S::ARF6 is shorter because it lacks the 5' and 3' UTRs. Arrow indicates the ARF6 transcript. Numbers beneath lanes indicate relative ARF6 transcript levels normalized to the ß-tubulin loading control. (B) Wild-type, gARF6, mARF6-I and mARF6-II flowers. Arrows indicate indehiscent anthers. (C-E,G-I) Wild-type (C-F) and mARF6-II (G-J) stage 2-IV (C,G) and stage 4-I (D,E,H,I) ovules. Arrows in C,D,G and H indicate outer integuments. (K) Stage 4-I mARF6-I ovule. (L) Stage 4-I mARF6-III ovule; asterisk indicates exposed embryo sac. (F,J,M) Wild-type (F,M left), arf6 arf8 (M middle) and mARF6-II (J,M right) gynoecia (M) and ovules (F,J) after pollination with the pollen-specific reporter LAT52:GUS pollen (Johnson et al., 2004Go). (N,O) Embryos of gARF6 (N) and mARF6-II (O) plants 7 days after pollination with wild-type pollen. Arrowhead in O indicates arrested embryo. fu, funiculus; ii, inner integument; oi, outer integument; nu, nucellus. Scale bars: 0.3 mm in B,M; 12 µm in C,D,F,G,H,J-L,N,O; 20 µm in E,I.

 

Figure 5
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  		Fig. 5. Anther phenotypes of mARF6 plants. (A) Anther from a stage 13 wild-type flower. c, connective cells; v, vascular bundle; sp, septum; st, stomium. (D) Anther from a stage 13 mARF6-II flower. (B,E) Enlarged views of anther vascular bundles and surrounding connective cells from A and D, respectively. (C,F) DR5::GUS staining patterns in wild-type (C) and mARF6-I (F) stage 13 flowers. Scale bars: 30 µm in A,D; 12 µm in B,E; 0.3 mm in C,F.

 

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