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First published online 15 October 2003
doi: 10.1242/dev.00812

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A contradictory GLABRA3 allele helps define gene interactions controlling trichome development in Arabidopsis

¹ Department of Plant Biology, University of Minnesota, St Paul, MN 55108-1095, USA
² College of Biological Sciences Imaging Center, University of Minnesota, St Paul, MN 55108-1095, USA

View larger version (152K): [in a new window]   Fig. 1. Comparison of shapeshifter (sst), Col wild-type and gl3-1 leaf trichomes. (A-C) Stereomicroscopic images of young leaves of Col, homozygous sst and heterozygous sst plants, respectively. Scale bars: 0.5 mm. (D-O) Scanning electon micrographs of mature trichomes. (D) Col. (E-J) Homozygous sst leaves. Note the size of D compared with G and H; scale bars in D-L 50 µm. (K) gl3-1 and (L) sst x gl3-1 F1 leaf trichomes. (M-O) Closeup of Col trichome, papillaeless sst trichome, sst trichome with papillae, respectively. (M-O) Scale bars: 25 µm.

View larger version (186K): [in a new window]   Fig. 2. Molecular complementation of gl3-1 and gl3-sst mutants. Young leaves of (A) gl3-1 and (C) gl3-sst mutants. (B,D) Leaves of (B) gl3-1 and (D) gl3-sst plants transformed with pEMYB5::GFP-cGL3 construct, Scale bar: (A,B) 0.25 mm; (C,D) 0.5 mm.

View larger version (170K): [in a new window]   Fig. 3. SEM analysis of developing trichomes on Col, gl3-sst and gl3-1 leaves. (A,D,G) Stage 2 trichomes on Col, gl3-sst and gl3-1 leaves, respectively. (B,E,H) Stage 3-4 trichomes on Col, gl3-sst, and gl3-1 leaves, respectively. (C,F) Stage 5 trichomes on Col and gl3-sst leaves, respectively. Scale bar: (A,B,D-H) 10 µm; (C) 25 µm.

View larger version (69K): [in a new window]   Fig. 4. Comparison of Col, gl3-sst and gl3-1 nuclei. (A-C) Fluorescence images of DAPI-stained Col, gl3-sst, and gl3-1 leaf trichomes. (D-F) Maximum projections of confocal images of trichome nuclei expressing the nuclear envelope-localized N7 GFP fusion gene: (D) Col, (E,F) two different gl3-sst trichome nuclei. Scale bar: (A-C) 50 µm; (D-F) 25 µm.

View larger version (69K): [in a new window]   Fig. 5. Visualization of interphase chromatin in Col and gl3-sst epidermal cell nuclei. Confocal microscopy of Col and gl3-sst plants containing a chromosomally integrated Lac operator array and a constitutively active 35S::GFP-Lac I transgene. (A,B) Trichome nuclei from Col and gl3-sst plants, respectively. (C,D) Guard and neighbor cell nuclei on leaf epidermis of Col and gl3-sst plants, respectively. The pairs of images shown in A and B, and in C and D are maximum projections of confocal images captured at the same magnifications and laser intensities.

View larger version (17K): [in a new window]   Fig. 6. Protein interactions with GL3 versus gl3-sst. Comparison of protein interactions using a yeast two-hybrid assay between GL3 and gl3-sst with (A) GL1, (B) TTG1, (C) TRY. D shows the ability of TRY to compete for the GL1 binding sites of GL3. Using the pBridge vector (Clontech), a third protein (TRY) under the control of a methionine-repressible promoter was expressed in a yeast interaction assay comparing the interactions of GL3 or gl3-sst with GL1. The assay was performed at varying methionine concentrations (0 µM, 15 µM, 30 µM, and 125 µM). The GL3 and gl3-sst proteins were compared as GAL4 activation domain (AD) fusions whereas GL1, TTG1 and TRY were expressed as GAL4 binding domain fusions (BD) except in D where TRY was expressed as a free protein (no AD or BD domain). The samples were normalized by using OD550 readings. The strength of the interaction was determined by using ß-galactosidase activity as measured using an ONPG assay.

View larger version (71K): [in a new window]   Fig. 7. Nuclear localization of GL1, GL3 and TRY proteins. (A,D,G,J) Light microscopic images of stage 5 (D) and stage 6 mature (A,G,J) trichomes; (C,F,I,L) the same specimens imaged with fluorescence microscopy to visualize GFP; (B,E,H,K) a merged images of the first and third columns. (A-C) Leaf trichome on gl3-1 plant expressing the pEMYB5::GFP-cGL3 construct. (D-F) Leaf trichome on gl1 plant expressing pEATML1::GFP-cGL1 construct. (G-I) Leaf trichome on Col plant expressing pETRY::GFP-cTRY. (J-L) Leaf trichome on untransformed control Col leaf (arrow indicates the nucleus). Scale bar: (A-F) 25 µm; (G-L) 50 µm.