Seed development in wild-type and mnt mutant plants. (A-J) Cleared seeds imaged with differential contrast optics; (A-E) wild-type and (F-J) mnt seeds at similar stages of embryogenesis. Scale bar: 100 μm. (K) CLSM images of Feulgen-stained seeds at 5 dap. Scale bars: 20 μm in embryo and chalazal endosperm; 25μ m in peripheral endosperm.

Ovule development in wild-type and mnt mutant plants. (A-F) Early ovule development in wild type (A,C,D) and mnt (B,E,F). (A,B) SEMs of stage 1-II ovules. (C,E) Sections of stage 2-III ovules showing megaspore mother cell (arrow) and initiating inner integument (^*). (D,F) SEMs of later stage 2 ovules showing integuments beginning to extend over nucellus. (G,H) Sections of mature wild-type (G) and mnt (H) ovules. The arrow in (H) shows an extra cell layer. Scale bars: 10 μm in A-F; 20 μm in G,H. (I-K) Comparison of number of cells (I), total length of integument (J) and mean size of cells (K) in the abaxial oi2 and ii1' layers of wild-type and mnt integuments. Error bars: s.e.m. n=4 wild type, 8 mnt.

Comparison of seeds generated by wild-type and mnt mutant plants. (A-D) Seeds from pollinations where all secondary shoots were allowed to set seed; (E-H) seeds from pollinations where secondary shoots were removed (see text). (A,E) [wild type × wild type]; (B,F) [wild type × mnt]; (C,G) [mnt × mnt]; (D,H) [mnt × wild type]. Scale bar: 250 μm. (I) Comparison of seed weights following the two treatments. Error bars: s.e.m.

Floral and vegetative phenotypes of mnt. (A) Alignment of buds from wild-type and mnt inflorescences, approximately stage 12-16. In each part of the figure the bottom row of buds shows the top row partially dissected. (B) Lengths of floral organs in stage 13 flowers. (C) Nail varnish casts of epidermis from carpel valve (left) and medial sepal (right) of wild-type and mnt stage 13 flowers. In each pair the wild type is on the left. (D) Transverse sections of inflorescence stem between nodes 2 and 3 from the base: (top) wild-type, (bottom) mnt. (E) Largest leaf from a wild-type (left) and mnt mutant (right) rosette. Scale bars: 1 mm in A; 50μ m in C; 200 μm in D, left; 100 μm in D, right. G, gynoecium (excluding stigma); P, petal; Se, medial sepal; St, long stamen.

Mapping and sequencing MNT. (A) mnt mutations were mapped to BAC MTG10 on chromosome V. Figures below the marker names show the number of recombination events in 1578 chromosomes scored. (B) Scoring of an allelism test between mnt mutants and Salk_108995 T-DNA insertion mutants by floral phenotype. (C) Scoring of complementation by floral and seed phenotype. mnt + ARF2=T1 progeny of an mnt mutant transformed with the wild-type ARF2 gene and flanking genomic DNA. This construct restored wild-type floral (left) and seed phenotypes (right) to the mutant. (D) The ARF2 gene with the positions of the mnt/arf2-9 and Salk_108995/arf2-8 mutations marked. (E) Alignment of a fragment of wild-type ARF2 cDNA with the mutated region in mnt/arf2-9 showing a 4-bp deletion. (F) Alignment of the N-terminal portion of the wild-type ARF2 and mutant mnt/arf2-9 proteins, showing an early frameshift and stop codon. (G) The ARF2 protein marked with the position of the stop codon generated by the mnt/arf2-9 lesion. DBD, DNA binding domain; MR, variable middle region; III and IV, domains involved in dimerization with other ARFs or with Aux/IAAs (Liscum and Reed, 2002).

Expression of nuclear-localized GFP under control of the ARF2 promoter. (A) Expression in ovules; SEMs show ovules at similar stages. (B) Expression in floral organs.