Gibberellin signaling in barley aleurone cells. Control of SLN1 and GAMYB expression

Abstract

We have previously identified GAMYB, a gibberellin (GA)-regulated transcriptional activator of alpha-amylase gene expression, in aleurone cells of barley (Hordeum vulgare). To examine the regulation of GAMYB expression, we describe the use of nuclear run-on experiments to show that GA causes a 2-fold increase in the rate of GAMYB transcription and that the effect of GA can be blocked by abscisic acid (ABA). To identify GA-signaling components that regulate GAMYB expression, we examined the role of SLN1, a negative regulator of GA signaling in barley. SLN1, which is the product of the Sln1 (Slender1) locus, is necessary for repression of GAMYB in barley aleurone cells. The activity of SLN1 in aleurone cells is regulated posttranslationally. SLN1 protein levels decline rapidly in response to GA before any increase in GAMYB levels. Green fluorescent protein-SLN1 fusion protein was targeted to the nucleus of aleurone protoplasts and disappeared in response to GA. Evidence from a dominant dwarf mutant at Sln1, and from the gse1 mutant (that affects GA "sensitivity"), indicates that GA acts by regulating SLN1 degradation and not translation. Mutation of the DELLA region of SLN1 results in increased protein stability in GA-treated layers, indicating that the DELLA region plays an important role in GA-induced degradation of SLN1. Unlike GA, ABA had no effect on SLN1 stability, confirming that ABA acts downstream of SLN1 to block GA signaling.

Figure 1

Effect of GA₃ and ABA on GAMYB transcription and amount of GAMYB mRNA in aleurone cells. A, Run-on transcription analyses of nuclei isolated from isolated aleurone layers treated without hormone (C), with 10⁻⁶ m GA₃ (GA) and 10⁻⁶ m GA3 and 5 × 10⁻⁵ m ABA (GA + ABA) for 2 h. Newly synthesized transcripts were hybridized to a 3′-specific fragment of the GAMYB cDNA and an rRNA cDNA (clone pTa250–10; Gerlach and Bedbrook, 1979). B, Quantitation of transcript amount using a phosphorimager. The values represent the mean of three independent experiments and se values. C, RNA-blot analysis of isolated barley aleurone layers that were incubated without hormone (C), with 10⁻⁶ m GA₃ (GA), 5 × 10⁻⁵ m ABA (ABA), and 10⁻⁶ m GA₃ and 5 × 10⁻⁵ m ABA (GA + ABA) for 12 h. The blot was probed with a 3′-specific GAMYB cDNA probe and an α-amylase cDNA (AMY).

Figure 2

The dominant dwarf allele (Sln1d) of the SLN1 gene affects responsiveness of GAMYB gene expression to GA₃ in aleurone cells. RNA-blot analysis of wild-type (wt) and mutant Sln1d aleurone layers after incubation without hormone, and 10⁻⁹ and 10⁻⁶ m GA₃ for 12 h. The blots were hybridized with a 3′-specific GAMYB cDNA probe. RNA loading was monitored by ethidium bromide staining.

Figure 3

SLN1 mRNA accumulation is not regulated by GA₃ in aleurone cells. RNA-blot analysis of SLN1 expression in isolated aleurone layers incubated for up to 12 h without hormone (Control) and 10⁻⁶ m GA₃ (GA). The blots were hybridized with the SLN1 cDNA and an α-amylase cDNA. RNA loading was monitored by ethidium bromide staining.

Figure 4

Anti-SLN1 antibodies specifically recognize the product of the SLN1 gene in aleurone cells. Total protein was extracted from wild-type (wt) and sln1b mutant aleurone layers and run on an SDS-PAGE gel. SLN1 protein was detected on protein blots by using anti-SLN1 antibodies.

Figure 5

GA₃ reduces the amount of SLN1 protein in aleurone cells. A, Time course analyses of SLN1 and GAMYB proteins in aleurone layers incubated for up to 12 h without hormone (Control) and 10⁻⁶ m GA₃ (GA). Total protein was extracted from aleurone layers and run on SDS-PAGE gels. SLN1 protein and GAMYB protein were detected on blots using antibodies to SLN1 and GAMYB. B, Short time course analysis of SLN1 protein expression in aleurone layers treated without hormone (Control) and 10⁻⁶ m GA₃ (GA) for up to 30 min. SLN1 protein was detected as described above. C, Effect of GA₃ and ABA on the amount of SLN1 protein in aleurone layers. Aleurone layers were incubated without hormone (Control), and with 10⁻⁶ m GA₃ (GA), 5 × 10⁻⁵ m ABA (ABA), and 10⁻⁶ m GA₃ and 5 × 10⁻⁵ m ABA (GA + ABA) for 30 min. SLN1 protein was detected as described above.

Figure 6

GFP-SLN1 fusion protein responds to GA₃ in transfected aleurone protoplasts. A, Scanning laser confocal microscope images of aleurone protoplasts transfected with GFP-SLN1 construct. Left, GFP fluorescence; right, autofluorescence. B, GFP-positive nuclei were expressed as a percentage of total numbers of cells after treatment with 10⁻⁶ m GA₃ (gray histogram) or without GA₃ (control; black histogram) for 5 h. Error bars represent the se values of the means.

Figure 7

Effects of GA on SLN1 protein levels in Sln1d and sln1c aleurone layers. A, Wild-type (wt) and mutant Sln1d aleurone layers were treated with and without GA₃ for 10 min. Protein blots of total aleurone protein were probed with anti-SLN1 antibodies. B, Wild-type (wt) and mutant sln1c aleurone layers were incubated with and without 10⁻⁶ m GA₃ for 30 min. Protein blots of total aleurone protein were probed with anti-SLN1 antibodies.

Figure 8

Effects of GA on SLN1 protein levels in aleurone of wild-type and gse1 grains. A, Wild-type (wt) and mutant gse1 aleurone layers were incubated with GA₃ or without GA₃ for 30 min. Protein blots of total aleurone protein were probed with anti-SLN1 antibodies. B, Total protein was extracted from wild-type and gse1 aleurone layers that were incubated without (Control) and with the protein biosynthesis inhibitor, cycloheximide (CHX). SLN1 protein levels were detected on protein blots using anti-SLN1 antibodies.