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SUPERMAN attenuates positive INNER NO OUTER autoregulation to maintain polar development of Arabidopsis ovule outer integuments

Section of Molecular and Cellular Biology, University of California, Davis, CA 95616, USA

View larger version (135K): [in a new window]   Fig. 1. Expression of P-INO reporter genes in wild type and ino-1 plants. Stages: (A,F,K,P,U,Z) 2-III; (B,G,L,Q,V,AA) 2-IV; (C,H,M,R,W,BB) 2-V; (D,I,N,S,X,CC) 3-I; (E,J,O,T,Y,DD) 4-I. (A-E) Scanning electron micrographs of wild-type ovules. In A a late developing ovule at stage 1 is indicated with an asterisk. (F-J) Composite figures of DIC and confocal INO:GFP fluorescence images and (K-O) respective INO:GFP fluorescence alone in complemented ino-1 ovules. P-INO::INO:GFP::NOS3' fluorescence is detectable only on the abaxial side of the ovule primordium and the abaxial layer of the outer integument. (M,N) Apparent internal localization of INO:GFP is from glancing sections through adjacent surface layers. (P-T) DIC images of wild-type ovules of P-INO::GUS::INO3' transgenic plants stained for GUS activity using 12.5 µg/ml X-gluc. (U-Y) Scanning electron micrographs of ino-1 ovules. (Z-DD) ino-1 ovules stained for P-INO::GUS::INO3' activity using 125 µg/ml X-gluc and visualized using DIC light microscopy. In all panels, the abaxial side (toward the base of the gynoecium) of the ovule is to the left, and adaxial is towards the right. n, nucellus; i, inner integument; f, funiculus; o, outer integument; m, micropyle; arrowhead, outer integument primordium. Scale bar: 25 µm in A-C,F-H,K-M,P-R,U-W,Z-BB; 30 µm in D,I,N,S,X,CC; 50 µm in E,J,O,T,Y,DD.

View larger version (158K): [in a new window]   Fig. 2. Phenotypic effects of ectopic INO expression. (A) Four-week old wild-type plants; leaves are relatively flat. (B) 35S::INO::NOS3' transgene in wild type background; leaves curl towards the abaxial (lower) side. (C) Wild-type flowers comprise four concentric whorls of floral organs: sepals, petals, stamens and a central gynoecium. (D) Flowers of one class of 35S::INO::NOS3' transgenic plants produced supernumary stamens and a gynoecium was absent. se, sepal; p, petal; s, stamen; g, gynoecium. Scale bar: 30 mm in A,B; 1 mm in C,D.

View larger version (113K): [in a new window]   Fig. 3. Expression of P-INO reporter genes in sup-5. Stages: (A,F,K,P) 2-II; (B,G,L,Q) 2-IV; (C,H,M,R) 2-V; (D,I,N,S) 3-I; (E,J,O,T) 4-I. (A-E) Scanning electron micrographs of sup-5 ovules. (F-J) Composite figures of DIC and confocal INO:GFP fluorescence images and (K-O) respective INO:GFP fluorescence alone in sup-5 ovules. Although P-INO::INO:GFP::NOS3' fluorescence is limited to a single cell layer, it expands to the adaxial side of the ovule primordium. (M,N) Apparent internal localization of INO:GFP is from glancing sections through adjacent surface layers. (P-T) DIC images of sup-5 ovules stained for P-INO::GUS::INO3' activity using 12.5 µg/ml X-gluc. In all panels, the abaxial side of the ovule is to the left. n, nucellus; i, inner integument; f, funiculus; o, outer integument; m, micropyle; arrowhead, outer integument primordium. Scale bar: 25 µm in A-C,F-H,K-M,P-R; 30 µm in D,I,N,S; 50 µm in E,J,O,T.

View larger version (143K): [in a new window]   Fig. 4. Effects of P-INO::SUP::INO3' and P-INO::CRC::INO3' transgenes on ovule morphology and reporter gene expression. Stages: (A,F,K,P) 2-III; (B,G) early 2-IV; (C,H,L,Q) 2-IV; (D,I,M,R) 2-V; (N,S) 3-I; (E,J,O,T) 4-I. (A-E) Scanning electron micrographs of ovules from wild-type plants containing the P-INO::SUP::INO3' transgene. (F-J) DIC images of P-INO::SUP::INO3' ovules stained for P-INO::GUS::INO3' activity using 125 µg/ml X-gluc. (K-O) Scanning electron micrographs of ovules from ino-1 plants containing the P-INO::CRC::INO3' transgene. (P-T) DIC images of P-INO::CRC::INO3' ovules from ino-1 plants stained for P-INO::GUS::INO3' activity using 12.5 µg/ml X-gluc. In all panels, the abaxial side of the ovules is to the left. n, nucellus; i, inner integument; f, funiculus; o, outer integument; m, micropyle; arrowhead, outer integument primordium. Scale bar: 25 µm in A-D,F-I,K-M,P-R; 30 µm in N,S; 50 µm in E,J,O,T.

View larger version (111K): [in a new window]   Fig. 5. Stage 4-I ovules from ino-1 plants containing both the P-INO::SUP::INO3' and P-INO::CRC::INO3' transgenes. Scanning electron micrographs of two progeny from a single cross representing the two classes of ovules; (A) sup-like and (B) ino-4-like. Scale bar: 50 µm.

View larger version (9K): [in a new window]   Fig. 6. Model of the transcriptional regulation of INO. Unknown factor ‘X’ initiates transcription from P-INO prior to outer integument initiation. INO maintains and enhances expression from P-INO. SUP blocks the INO autoregulatory loop of incipient expression on the adaxial side of the ovule preventing detectable expression and integument growth in this zone.