Article Figures & Data
Figures
- Figure 1.
gar2-1 Reduces DELLA Protein Accumulation.
(A) Representative 21-d-old 35S:gai-GFP, gai, and 35S:gai-GFP gar2-1 plants.
(B) gai-GFP transcripts in 35S:gai-GFP and 35S:gai-GFP gar2-1 plants. UBQ, ubiquitin transcripts (loading control).
(C) gai-GFP fluorescence in primary roots of 35S:gai-GFP or 35S:gai-GFP gar2-1 seedlings (7 d old) treated or untreated with 100 μM GA3 for time shown. Top row, root tips; bottom row, elongation zone.
(D) Representative 42-d-old 35S:GAI-GFP or 35S:GAI-GFP gar2-1 plants.
(E) GAI-GFP fluorescence of 35S:GAI-GFP or 35S:GAI-GFP gar2-1 primary seedling roots treated or untreated with 100 μM GA3 for time shown. Top row, root tips; bottom row, elongation zone.
(F) GFP-RGA fluorescence in pRGA:GFP-RGA or pRGA:GFP-RGA gar2-1 primary seedling roots treated or untreated with 100 μM GA3 for time shown. Top row, root tips; bottom row, elongation zone.
- Figure 2.
sly1gar2-1 Encodes a Mutant Form of SLY1.
(A) Illustration of SLY1 protein showing positions of previously defined F-box, GGF, and LSL domains (McGinnis et al., 2003) and the amino acid substitution conferred by sly1gar2-1.
(B) Representative 30-d-old gai, gai 35S:SLY1, and gai 35S:sly1gar2-1 plants (with wild-type control).
(C) SLY1/sly1gar2-1 transcripts in gai, gai 35S:SLY1, and gai 35S:sly1gar2-1 plants. Endogenous SLY1, endogenous SLY1 transcripts; total SLY1/sly1gar2-1, sum of endogenous SLY1 and 35S:SLY1 (or 35S:sly1gar2-1) transcripts; UBQ, ubiquitin transcripts (loading control).
- Figure 3.
SLY1 Affects Both GAI and RGA Function.
(A) Representative 42-d-old sly1-10, sly1-10 gai-t6, sly1-10 rga-24, and sly1-10 gai-t6 rga-24 plants.
(B) GAI-GFP fluorescence in pGAI:GAI-GFP or sly1-10 pGAI:GAI-GFP primary roots treated (or control) with 100 μM GA3. Top row, root tips; bottom row, elongation zone.
(C) GFP-RGA fluorescence in pRGA:GFP-RGA or sly1-10 pRGA:GFP-RGA primary roots treated (or control) with 100 μM GA3. Top row, root tips; bottom row, elongation zone.
- Figure 4.
Increased SLY1/SLY2 Availability Opposes DELLA Protein Function.
(A) Representative 21-d-old sly1-10, sly1-10 35S:SLY2, and sly1-10 35S:SLY1 plants.
(B) SLY1/SLY2 transcripts in plants in (A). 18S, 18S rRNA loading control.
(C) Germination of seeds (genotypes indicated) in the presence (closed bars) or absence (open bars) of 2 μM PAC. Results are mean ± se of three separate experiments (for each sample, n > 160).
(D) Representative 35-d-old ga1-3 and ga1-3 35S:SLY1 plants, together with SLY1 transcript levels in each. Transgenic SLY1, 35S:SLY1 transcripts; total SLY1, sum of endogenous SLY1 and 35S:SLY1 transcripts; UBQ, ubiquitin transcripts (loading control).
(E) Representative 35-d-old gai and gai 35S:SLY1 plants, together with SLY1 transcript levels in each (see [D]).
- Figure 5.
SLY1 Is the F-Box Subunit of an SCFSLY1 E3 Ubiquitin Ligase.
(A) Relatedness tree of ASK sequences. ASK subunits interacting with SLY1 or SLY2 are indicated (+, positive interaction; −, no detectable interaction; +/−, weak interaction).
(B) Representative yeast two-hybrid experiments: A and B, no detectable interaction; C to F, detectable interaction.
(C) Schematic indication of SLY1 derivative proteins (used in [D]; see also Figure 2A). SLY1ΔF lacks an N-terminal portion of SLY1 containing the conserved F-box sequence (F), whereas SLY1ΔC lacks a C-terminal portion of SLY1 (GGF and LSL domains; McGinnis et al., 2003).
(D) Quantitation of the interactions between ASK1 or ASK2 and SLY1, SLY1ΔF, or SLY1ΔC. Results are shown as mean and se of at least three independent experiments.
(E) Immunoblot assay of proteins coimmunoprecipitated by an anti-Myc antibody or in crude extracts from plants expressing 35S:Myc-ASK2 or 35S:Myc-AtCUL1 transgenes (or wild-type control).
(F) GFP-RGA fluorescence in pRGA:GFP-RGA or pRGA:GFP-RGA 35S:RNAi-ASK1 primary roots treated (or control) with 100 μM GA3. Top row, root tips; bottom row, elongation zone.
(G) GAI-GFP fluorescence in pGAI:GAI-GFP or pGAI:GAI-GFP 35S:RNAi-ASK1 primary roots treated (or control) with 100 μM GA3. Top row, root tips; bottom row, elongation zone.
- Figure 6.
The LSL Domain of SLY1 Interacts with GAI, gai, and RGA.
(A) Quantitation of the interactions between GAI and SLY1 or SLY1-derivative proteins lacking F-box, GGF, or LSL domains (McGinnis et al., 2003; Figure 2A). Results are shown as mean and se of three independent experiments.
(B) In vitro interaction of 35S-Met–labeled gai, GAI, or RGA with immobilized GST-SLY1. GST-SLY1ΔLSL lacks the LSL domain (Figure 2A).
- Figure 7.
sly1gar2-1 Has Increased Affinity for DELLA Proteins.
(A) Immunoblot assay of SLY1/sly1gar2-1 levels in wild-type, gai, and gai sly1gar2-1 plants (with tubulin loading control).
(B) Immunoblot assay of E. coli–expressed proteins displaying specific interaction with gai. Anti-GST provides an input protein control. Anti-GAI provides control for amount of immobilized gai (subsequently released from beads by boiling).
(C) Immunoblot assay of proteins coimmunoprecipitated by control immobilization substrate (A) (see Methods) or immobilized gai (B) from wild-type or gai sly1gar2-1 plant extracts. Anti-tubulin provides input protein control. Anti-GAI provides control for amount of immobilized gai (subsequently released from beads by boiling).
(D) Immunoblot assay of proteins from sly1-10 or gai-t6 sly1-10 plants specifically interacting with GST-SLY1 or GST-sly1gar2-1. Anti-GST and anti-GAI antibodies provide input controls.
(E) Immunoblot assay (anti-GAI antibody) of gai proteins from ethanol-induced alcA:gai plants (gai) together with gai incubated with CIP or denatured CIP (de-CIP).
(F) Immunoblot assay of specific interactions between native (treated with denatured CIP) or CIP-treated gai (CIP) and immobilized GST-SLY1 or GST-sly1gar2-1. The anti-GAI antibody provided input protein control. Anti-SLY1 provides control for amount of immobilized SLY1/sly1gar2-1 (subsequently released from beads by boiling).
Additional Files
Supplemental Data
Files in this Data Supplement:
- Supplemental Data
- Supplemental Figure 1 - GAR2 maps in the vicinity of SLY1. A 0.53 cM region of chromosome 4 was found to contain GAR2 and further refinement of map position-identified BACs T19F6 or T22A6 (carries SLY1) as containing GAR2. BAC clone overlaps and nucleotide position on chromosome 4 are as shown.
