Article Figures & Data
Figures
- Figure 1.
Effect of the rga and spy Mutations on GA4 mRNA Levels.
Shown are autoradiographs of RNA blots containing 15 μg of total RNA isolated from different GA biosynthetic and signal transduction mutants, as labeled. (−) or (+) GA3 indicates that the RNA samples were isolated from untreated seedlings or seedlings treated with GA3 for 8 hr, respectively. The blots were hybridized with a radiolabeled antisense GA4 RNA probe and then reprobed with the oligonucleotides corresponding to the 18S rDNA sequence. The numbers under the blots indicate the relative amounts of GA4 mRNA after normalization using 18S rRNA as a loading control. The value of untreated Ler was arbitrarily set at 1.0.
- Figure 2.
The GFP-RGA Fusion Rescues the Phenotype Caused by the rga Mutation.
The phenotype of a transgenic plant (rga-24/ga1-3 background) that was homozygous for the 35S::GFP-RGA fusion gene was compared with the phenotypes of ga1-3 and rga-24/ga1-3. All plants were 50 days old.
- Figure 3.
Fluorescence in the Root of Transgenic rga/ga1-3 Plants Expressing the GFP-RGA Protein.
Shown are overlays of fluorescent and bright-field images generated by confocal laser microscopy. Exclusive nuclear localization of GFP-RGA is seen in a region of a root behind the tip in the elongation zone (A) and in a single root hair cell with a fluorescent nucleus (B).
- Figure 4.
Effects of GA and PAC Treatment on the RGA Promoter–Expressed GFP-RGA Protein.
Roots of transgenic plants (Ler background) expressing the RGA promoter::GFP-RGA fusion were observed using confocal laser microscopy. Shown are the fluorescent images of root tips that were untreated (Control), treated with 100 μM GA3 for 2 hr (+GA), or incubated with 100 μM PAC and 0.01% Tween 20 for 48 hr (+PAC).
- Figure 5.
Effect of GA Treatment on the GFP-RGA Protein Expressed by the Cauliflower Mosaic Virus 35S Promoter.
Roots of transgenic plants (rga/ga1-3 background) expressing the 35S::GFP-RGA fusion were observed by using confocal laser microscopy. Shown are three-dimensional projections of the fluorescent images of root tips either untreated (top) or treated with 100 μM GA3 (bottom) for the times indicated.
- Figure 6.
Immunoblot Analysis of GFP-RGA Levels.
The blots contained 50 μg of total protein extracted from Ler and transgenic seedlings carrying either the 35S::GFP-RGA (top) or the RGA promoter::GFP-RGA (bottom) fusion gene. Lane C, water-treated control. The times after GA or PAC treatment were as labeled. A rat anti-GFP antiserum and a peroxidase-conjugated goat anti-rat IgG were used as primary and secondary antibodies, respectively. The arrows indicate the GFP-RGA fusion protein (91 kD). The additional lower band in all lanes represents nonspecific background protein because it is present in Ler as well.
- Figure 7.
GA Treatment Reduces the Level of the Endogenous RGA Protein.
The blot contained 25 μg of total protein extracted from seedlings of Ler and mutant plants as labeled. The leaves of the ga1-3 plants were treated (+) or not treated (−) with GA3 for 2 hr. Lane C, 2 ng of Ni column–purified 65-kD His-tagged RGA protein. A rabbit anti-RGA antiserum and a goat anti-rabbit IgG were used as primary and secondary antibodies, respectively. The extra upper band in each lane represents nonspecific background protein because it is present in rga-24 as well.
- Figure 8.
Proposed Role of RGA and GAI in GA Homeostasis.
In the ground (GA-deficient) state, RGA and GAI would repress GA signaling. After the synthesis of bioactive GAs, RGA and GAI would be inactivated (presumably by proteolysis), leading to the induction of GA response. The GA signaling pathway then would reduce bioactive GAs through the inhibition of GA biosynthesis and the induction of GA catabolism. An environmental or endogenous signal would keep the level of bioactive GAs above the homeostatic mean and allow for GA-stimulated growth and development. After the input signal stopped, the system would return to its basal level. Arrows and T-bars indicate positive and inhibitory effects, respectively.
Tables
GA ga1-3 rga-2/ga1-3 spy-9/ga1-3 Ler rga-2/GA1 spy-9/GA1 GA53 Undetectable Undetectable Undetectable 4.2 3.9 6.5 GA44 Trace Trace Trace 1.6 0.7 2.7 GA19 1.6 3.2 2 6.9 10.4 8.4 GA20 0.1 0.2 0.1 0.3 0.4 0.3 GA1 0.1 Trace 0.1 0.4 0.4 0.5 GA29 0.1 0.1 0.1 0.3 0.3 0.3 GA8 2.4 0.4 Trace Trace 1.9 Trace GA12 Trace 0.9 0.5 44.4 42.0 56.2 GA15 0.7 0.6 0.6 7.2 8.3 10.4 GA24 Trace Trace Trace 40.6 41.3 46.0 GA9 Trace Trace Trace 2.3 2.5 1.7 GA4 Undetectable Undetectable Undetectable 22.5 18.6 15.3 GA34 0.4 0.3 0.4 6.4 5.5 4.3 a Values are in ng of GA/g dry weight. Trace indicates <0.1 ng of GA/g dry weight. Undetectable indicates that no corresponding peak was detected by GC-SIM. The GAs in the top half of the table are part of the early 13-hydroxylation
pathway: GA53→GA44→GA19→GA20→GA1→GA8.
↓
GA29
Those in the bottom half are part of the non-13-hydroxylation pathway: GA12→GA15→GA24→GA9→GA4→GA34. GA1 and GA4 are bioactive GAs, whereas the other GAs are either precursors or deactivated GAs (GA29, GA8, and GA34).
