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doi: 10.1242/10.1242/dev.00577

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Modes of intercellular transcription factor movement in the Arabidopsis apex

¹ Plant Biology Laboratory, The Salk Institute for Biological Studies, La Jolla, CA 92037, USA
² Department of Biology, University of California San Diego, La Jolla, CA 92093, USA
³ Department of Plant and Microbial Biology, University of California, Berkeley, CA 94720, USA
⁴ Department of Biochemistry and Cell Biology, State University of New York, Stony Brook, NY 11794-5215, USA
⁵ Department of Molecular Biology, Max Planck Institute for Developmental Biology, D-72076 Tübingen, Germany

View larger version (111K): [in a new window]   Fig. 1. Movement of TVCVMP:GFP, 2xGFP and NLS:2xGFP. Confocal images of GFP fluorescence in inflorescence apices (A-C) and leaf epidermis (D-F) of 2-week-old ML1::TVCVMP:GFP (A,D), ML1::2xGFP (B,E) and ML1::NLS:2xGFP (C,F) transgenic plants. Inset in A shows that TVCVMP:GFP RNA is restricted to the L1, as detected by in situ hybridization. In D-F, the GFP fluorescence channel is overlaid with the transmissible light channel. TVCVMP:GFP is found in all cells in the inflorescence apex (A), and it is associated with the cell wall in a punctate pattern (D). 2xGFP forms a gradient of six to ten cells in the apex, with the highest concentration in L1 (B). It is located in both the nucleus and the cytoplasm (E). NLS:2xGFP can move efficiently only one cell layer from the L1 in the apex (C), and it appears mostly nuclear (F).

View larger version (58K): [in a new window]   Fig. 4. Western blots of whole-cell extracts from 2-week-old transgenic seedlings of ML1::2xGFP and ML1::NLS:2xGFP (A), ML1::AP1:GFP and ML1::GFP:AP1 (B), and ML1::GLFY, ML1:LFY:GFP and ML1::GFP:LFY (C) probed with anti-GFP antibody. Extract of wild-type Col-0 seedlings was used as a negative control in A. Blots were deliberately overexposed to reveal the presence of any minor bands. No major degradation products in the form of single GFP were found. Bands detected at higher molecular weight in the 2xGFP and GLFY lanes probably represent dimers formed during the extraction procedure. In B, GFP:AP1 bands are marked with an arrow and the minor single GFP band in GFP:AP1 is marked with an arrowhead.

View larger version (75K): [in a new window]   Fig. 2. Absence of movement of a functional AP1:GFP fusion. The C-terminal AP1:GFP fusion can rescue ap1-15 mutant flowers to near wild type when expressed from the AP1 promoter (A), and leads to the development of bracts subtending flowers when expressed in the L1 of wild type (C). The N-terminal fusion GFP:AP1 does not rescue ap1-15 mutant flowers when expressed from the AP1 promoter (B). Confocal images of GFP fluorescence in inflorescence apices of 2-week-old ML1::AP1:GFP (D,E) and ML1::GFP:AP1 (F) plants are shown. AP1:GFP does not move from the L1 (D; optical section through the shoot apex), and is tightly associated with nuclei (E; tangential section through the L1 of a floral primordium). By contrast, GFP:AP1 is largely cytoplasmic and moves into deeper tissues layers from the L1 (F).

View larger version (153K): [in a new window]   Fig. 3. Movement of LFY-GFP fusions. Confocal images of GFP fluorescence in inflorescence apices (A,B,D) and leaf epidermis (C) of 2-week-old ML1::GLFY (A,C), ML1::GFP:LFY (B) and ML1::LFY:GFP (D) transgenic plants. GLFY moves several cell layers into the underlying tissue from the L1 in the apex (A). GFP:LFY shows a similar gradient from the L1 as GLFY (B). The signal from LFY:GFP, which has the longest moving range, appears `fuzzy' because of its higher cytoplasmic localization (D). All three fusions are localized to both the nucleus and cytoplasm in leaf epidermal cells, and bright spots are sometimes found along the cell wall with GLFY (shown in C).

View larger version (118K): [in a new window]   Fig. 5. Restricted lateral protein movement in the Arabidopsis shoot apex. Confocal images of GFP fluorescence for inflorescence meristems with surrounding young floral primordia from AG intron*::NLS:2xGFP (A,D), AG intron*::2xGFP (B,E) and AG intron*::GLFY (C,F) plants. The confocal images have been overlaid with images from the transmitted-light channel for orientation only. Note that the confocal images are optical sections, which is not true for the transmitted-light images. The mutated AG sequences in the reporters activate expression in the shoot apical meristem and the center of young flowers. Close-up views of stage 3 flowers reveal discrete lateral boundaries of the GFP signal (D-F). No lateral movement is obvious in any of the three cases.

View larger version (75K): [in a new window]   Fig. 6. Rescue of lfy-12 mutant flowers by LFY and GLFY expression in the center of the floral primordia. A wild-type Arabidopsis flower (A) and a shoot-like structure that replaces a flower in lfy-12 mutants (B) are shown. Both AG intron*::LFY and AG intron*::GLFY can rescue the stamens and carpels of lfy-12 mutant flowers (C,D), but only LFY rescues petals completely (arrow in C).

View larger version (70K): [in a new window]   Fig. 7. Movement of truncated LFY from the L1. (A) Diagram of GFP:LFY and the three truncated forms of the GFP:LFY fusion protein. GFP-coding sequence is shown in green and LFY-coding sequence in light blue, along with restriction sites used to make the deletions: B, BamHI; P, PstI; X, XbaI; S, StyI; H, HindIII. (B-D) Confocal images of GFP fluorescence in inflorescence apices of 2-week-old plants. All three forms of truncated GFP:LFY move from the L1 to the inner tissue layers. GFP:LFY1 is mostly located in the nucleus (B), whereas GFP:LFY2 appears to be largely cytoplasmic (C). Both can move several cell layers from the L1. GFP:LFY3 is mainly found in the form of large aggregates, but can still move one to two cell layers from the L1 (D).

View larger version (104K): [in a new window]   Fig. 8. Movement of mutant GLFY fusions from the L1. (A) Diagram of fusions of GFP to weak alleles lfy-2 (P240L) and lfy-20 (N306D), and intermediate alleles lfy-3 (T244M) and lfy-9 (R331K) in the GLFY background. (B-E) Confocal images of GFP fluorescence in inflorescence apices of 2-week-old plants. All fusion proteins can move from the L1 into interior layers, although to different degrees.