Gibberellin dose-response curves and the characterization of dwarf mutants of barley

Abstract

Dose-response curves relating gibberellin (GA) concentration to the maximal leaf-elongation rate (LERmax) defined three classes of recessive dwarf mutants in the barley (Hordeum vulgare L.) 'Himalaya. ' The first class responded to low (10(-8)-10(-6) M) [GA3] (as did the wild type). These grd (GA-responsive dwarf) mutants are likely to be GA-biosynthesis mutants. The second class of mutant, gse (GA sensitivity), differed principally in GA sensitivity, requiring approximately 100-fold higher [GA3] for both leaf elongation and alpha-amylase production by aleurone. This novel class may have impaired recognition between the components that are involved in GA signaling. The third class of mutant showed no effect of GA3 on the LERmax. When further dwarfed by treatment with a GA-biosynthesis inhibitor, mutants in this class did respond to GA3, although the LERmax never exceeded that of the untreated dwarf. These mutants, called elo (elongation), appeared to be defective in the specific processes that are required for elongation rather than in GA signaling. When sln1 (slender1) was introduced into these different genetic backgrounds, sln was epistatic to grd and gse but hypostatic to elo. Because the rapid leaf elongation typical of sln was observed in the grd and gse backgrounds, we inferred that rapid leaf elongation is the default state and suggest that GA action is mediated through the activity of the product of the Sln gene.

Figure 1

Growth of L₁ of wild-type barley. Grains were surface-sterilized, placed in moist paper envelopes, stratified, and incubated as described in Methods. At the indicated times the mean L₁ lengths of 10 seedlings were determined, as well as the mean lengths of the blade and sheath. Where not visible, error (se) bars lie within the symbols. Inset, Mean elongation rate of the blade of L₁ in the previous 24 h was plotted as a function of days of growth. The LER_max value for this set of data is indicated.

Figure 2

Dose-response curves relating LER_max of L₁ to [GA₃]. Grains of the indicated lines were surface-sterilized, placed in paper envelopes moistened with the appropriate [GA₃], stratified, incubated in low light, and LER_max (mean ± se) of seedling L₁ was determined as described in Methods. Curves in the top six panels were fitted using PEST software (Weyers et al., 1987). Note differences in the range of GA₃ concentrations in different panels.

Figure 6

LER_max of L₁ of elo mutants and a grd mutant growing with or without tetcyclacis and GA₃. Grains of M21 (elo1), M626 (elo2), and M117 (grd1) were surface-sterilized, placed in moist paper envelopes containing, where appropriate, 2 μm tetcyclacis or 2 μm tetcyclacis plus 10 μm GA₃, stratified, and incubated under low light; and the mean LER_max of seedling L₁ was determined as described in Methods. A replicate consisted of 10 seedlings, and there were three replicates for each genotype and treatment. Within a genotype, letters (a, b, or c) indicate significance (P < 0.05) for the differences between the means of each treatment.

Figure 7

LER_max of L₁ of Sln1− and sln1sln1 segregants in different genetic backgrounds. Grains from stocks in which the sln1 allele was segregating in different genetic backgrounds (WT, wild type; grd1, M117; gse1, M121; and elo1; M21) were surface-sterilized, placed in moist paper envelopes, stratified, and incubated in low light, and the LER_max (means ± se) of seedling L₁ was determined as described in Methods. In an elo1 background, slender (sln1sln1) seedlings cannot be distinguished at the first leaf stage from Sln1− seedlings, but after transplanting and further growth, the early stem elongation characteristic of sln1sln1 plants, which still occurs in an elo1 genetic background, allowed the genotype to be determined.

Figure 3

Dose-response curves comparing the effects of GA₃ on either LER_max or final blade length. Grains of the indicated lines were surface-sterilized, placed in paper envelopes moistened with the appropriate [GA₃], stratified, and incubated in low light, and LER_max (mean ± se) or final blade length of seedling L₁ was determined as described in Methods. To allow direct comparison, response ratios are plotted in which the LER_max or L₁ blade length in the absence of GA₃ is assigned a value of unity. Curves are the same (minus data points) as shown in Figure 2. Individual data points (▾) are for final blade length.

Figure 4

LER_max (means ± se) of different grd mutants in the presence of GA-biosynthetic intermediates. Grains of M117 (grd1), M359 (grd2), and M411 (grd3) were surface-sterilized, placed in paper envelopes moistened with the indicated GA at 2 μm, stratified, and incubated in low light, and LER_max (means ± se) of seedling L₁ was determined as described in Methods. GA₄₄, GA₁₉, and GA₂₀ are successive biosynthetic intermediates in the early 13-hydroxylation pathway leading to the formation of the bioactive GA₁.

Figure 5

α-Amylase activity of de-axised grains of the wild type and a gse1 mutant incubated with different [GA₃]. The embryonic axes of wild type and M488 (gse1) grains were removed, and the resulting de-axised grains were surface-sterilized, placed in paper envelopes moistened with the indicated [GA₃], stratified, and incubated in low light as described in Methods. At the indicated times, duplicate samples (five grains each) were harvested and α-amylase was extracted and assayed. Each data point is the mean of duplicate samples.