Article Figures & Data
Figures
- Figure 1.
The sln1-1 Mutant Allele.
(A) Five-day-old seedlings homozygous for SLN1 or sln1-1.
(B) Scheme of SLN1/SLN1 showing the site of the mutation in sln1-1. Numbers represent amino acid positions in SLN1 (1 indicates the start Met, and 618 indicates the final Pro).
(C) Seedling phenotypes and protein gel blot analysis of SLN1 and sln1-1 plants. The seedlings were grown at 20°C for 5 days with or without 100 μM GA3. Proteins then were extracted from seedlings, and 15 μg of total protein per lane was loaded and probed with anti-GAI antibodies (see Methods). The arrow indicates SLN1; an additional, nonspecific band served as a loading control.
- Figure 2.
The Proteasome Pathway Mediates GA-Induced SLN1 Protein Degradation.
Extracts were prepared from 5-day-old SLN1 seedlings, which had been treated for 2 h with GA3 and 1% DMSO with or without protease or proteasome inhibitors (Pefabloc SC [P. Sc], MG115, MG132, or proteasome inhibitor I). Extracts from water-treated SLN1 seedlings were used as a positive control. Total protein (15 μg/lane) was loaded and probed with anti-GAI antibodies. The arrow indicates SLN1. The strong bottom band in all lanes represents the nonspecific background protein described for Figure 1, and the weak lower bands may indicate SLN1 degradation products.
- Figure 3.
GA-Promoted Leaf Extension Growth Requires Proteasome-Dependent GA-Mediated SLN1 Destabilization.
Seedlings were germinated and grown for 3 days on water. The length of the first leaf (from leaf tip to seed) of each seedling was measured, and treatment (combinations of water, GA, and MG132 as shown) was begun. Twelve hours after the initiation of treatment, the length of the first leaf was measured again. The results shown are mean differences (n = 30; error bars represent standard errors) between the first and second measurements.
- Figure 4.
Effect of Protein Phosphatase and Protein Kinase Inhibitors on the GA-Induced Degradation of SLN1.
(A) Effect of protein phosphatase inhibitors on GA-induced SLN1 degradation. Extracts were prepared from 5-day-old SLN1 seedlings treated for 2 h with GA3 with or without OA or SV. Water-treated SLN1 seedlings were used as a positive control. The arrow indicates the SLN1 protein. Additional nonspecific bands served as loading controls.
(B) Effect of protein kinase inhibitors on GA-induced SLN1 degradation. Extracts were prepared from 5-day-old SLN1 seedlings treated for 2 h with GA3 and 1% DMSO with or without Tyrophostin B46 (AG555), PP2, genistein, or staurosporine. Extracts also were prepared from control SLN1 seedlings treated with water and 1% DMSO. Total protein (15 μg/lane) was loaded and probed with anti-GAI antibodies. The arrow indicates the SLN1 protein. The bottom band in all lanes represents a nonspecific background protein as described for Figure 1.
- Figure 5.
Scheme of SLN1 Function in GA Signaling.
Bioactive GA interacts with the membrane-associated GA receptor (not shown), thus activating signal transduction via second messengers, perhaps mediated by protein phosphorylation or dephosphorylation (GA signal). SLN1 is in the nucleus, in an active form that represses GA responses (e.g., SLN1 represses the accumulation of transcripts that encode GAmyb, as shown in [A]). The arrival of the GA signal causes the modification of active SLN1 into a form that is destroyed via a proteasome-dependent mechanism (B), resulting in the activation of GAmyb transcription and α-amylase production. Inhibition of the proteasome pathway (C) prevents the destruction of SLN1, which therefore persists and continues to repress GAmyb transcription and α-amylase production. Because protein kinase and protein phosphatase inhibitors also block the destruction of SLN1, it is possible that protein phosphorylation or dephosphorylation is required for the modification of SLN1 or before the destruction of the modified form can occur.
Tables
- Table 1.
Effects of the Different Inhibitors on α-Amylase Production in Barley Aleurone Layers
α-Amylase Produced (milliunits/g) SLN1 sln1-1 Inhibitors Water GA3 Water GA3 Control 0.08 ± 0.01 9.60 ± 0.03 6.24 ± 0.07 7.21 ± 0.05 MG115 0.07 ± 0.02 0.29 ± 0.04 5.87 ± 0.06 6.37 ± 0.07 MG132 0.07 ± 0.03 0.34 ± 0.09 5.99 ± 0.08 6.29 ± 0.10 Pefabloc SC 0.09 ± 0.02 6.48 ± 0.11 5.47 ± 0.12 6.91 ± 0.08 SV 0.08 ± 0.03 0.45 ± 0.03 5.93 ± 0.11 6.72 ± 0.09 AG555 0.54 ± 0.09 0.81 ± 0.03 7.18 ± 0.05 7.89 ± 0.09 PP2 0.09 ± 0.02 8.36 ± 0.02 6.69 ± 0.10 7.03 ± 0.08 Staurosporine 0.10 ± 0.03 9.24 ± 0.09 6.03 ± 0.09 7.11 ± 0.07 Each value shown is the mean ± se from 12 half-grains.
