Mutants at the Slender1 locus of barley cv Himalaya. Molecular and physiological characterization

Abstract

A dominant dwarf mutant of barley (Hordeum vulgare) that resembles dominant gibberellin (GA) "-insensitive" or "-nonresponsive" mutants in other species is described. alpha-Amylase production by endosperm half-grains of the mutant required GA3 at concentrations about 100 times that of the WT. The mutant showed only a slight growth response to GA3, even at very high concentrations. However, when additionally dwarfed, growth rate responded to GA3 over the normal concentration range, although only back to the original (dwarf) elongation rate. Genetic studies indicated that the dominant dwarf locus was either closely linked or identical to the Sln1 (Slender1) locus. A barley sequence related to Arabidopsis GAI/RGA was isolated, and shown to represent the Sln1 locus by the analysis of sln1 mutants. The dominant dwarf mutant was also altered in this sequence, indicating that it too is an allele at Sln1. Thus, mutations at Sln1 generate plants of radically different phenotypes; either dwarfs that are largely dominant and GA "-insensitive/-nonresponsive," or the recessive slender types in which GA responses appear to be constitutive. Immunoblotting studies showed that in growing leaves, SLN1 protein localized almost exclusively to the leaf elongation zone. In mutants at the Sln1 locus, there were differences in both the abundance and distribution of SLN1 protein, and large changes in the amounts of bioactive GAs, and of their metabolic precursors and catabolites. These results suggest that there are dynamic interactions between SLN1 protein and GA content in determining leaf elongation rate.

Figure 1

LERs of M640 segregants that are heterozygous (Sln1d,Grd3) or homozygous (Sln1d,grd3) for a GA deficiency allele, and of grd3, growing in different concentrations of GA₃. Grains were germinated and seedlings grown in the presence of GA₃ at the indicated concentrations. Maximal leaf elongation rate (LER_max; mean ± se) was determined for the grd3 mutant, and for Sln1d,Grd3 and Sln1d,grd3 seedlings as previously described (Chandler and Robertson, 1999). At GA₃ concentrations higher than 1 μm, it was no longer possible to reliably identify Sln1d grd3 seedlings in the segregating population, so LER data are for the whole population. Where not visible, error bars are within the symbol.

Figure 2

α-Amylase production by endosperm half-grains. A, α-Amylase production by half-grains of barley cv Himalaya and M640 in response to GA₃. Half-grains were incubated in GA₃ solutions of the indicated concentrations (m), and samples were frozen at the indicated time of incubation before homogenization, extraction, and assay of α-amylase activity. The legend in the right applies to both graphs, and the data represent means ± se of triplicate samples. Where not visible, error bars are within the symbol. B, α-Amylase production by half-grains of barley cv Himalaya and slender segregants of M770. Half-grains corresponding to sln1c homozygous segregants in the M770 stock were identified by scoring growth (slender or normal) of the corresponding embryo half-grain. Slender half-grains were incubated without addition of GA₃, whereas barley cv Himalaya half-grains were incubated with or without GA₃ at 10⁻⁶ m. Samples were frozen at the indicated time of incubation before homogenization, extraction, and assay of α-amylase activity. The data represent means ± se of triplicate samples. Where not visible, error bars are within the symbol.

Figure 3

Representation of mutants in the SLN1 sequence. The barley cv Himalaya (WT) open reading frame (ORF) is 618 amino acid residues in length. Slender mutants: sln1b has a frameshift mutation in amino acid residue 93 (Thr, ACC to A-C), resulting in an early termination codon at residue 252, and sln1c has a G to A substitution in amino acid residue 602 (Trp, TGG to TGA), resulting in an early termination codon. Dominant dwarf: Sln1d has a G to A substitution in amino acid residue 46 (Gly, GGG to GAG), causing a Gly to Glu change in the DELLA region, namely ³⁹DELLAALG⁴⁶ → ³⁹DELLAALE⁴⁶

Figure 4

Above-ground parts of seedlings of sln1c, WT, and Sln1d 2 weeks after sowing.

Figure 5

Distribution of SLN1 mRNA along the growing blade of L2 of barley cv Himalaya, sln1c, and Sln1d. Blades were harvested at approximately 50% final length, cut into five segments of equal length, frozen and RNA extracted, electrophoresed, blotted, and the filter hybridized with a SLN1 probe. The upper panel shows the hybridization profile, and the lower panel the ethidium bromide-stained gel before transfer. Lanes from left to right: Sln1d, base (B) to tip (T), five segments; Himalaya, base to tip (five segments); and sln1c, base to tip (five segments). The basal segment in each case is contained within the EZ.

Figure 6

Distribution of SLN1 protein in growing leaf blades. A, Distribution of SLN1 protein along the growing L3 blade of barley cv Himalaya. Blades of L3 were harvested when 90 mm in length (approximately 50% final length), and cut into six 10-mm segments from the base, and then a single 30-mm segment remaining at the tip. Protein was extracted from each segment, electrophoresed, blotted, and the filter developed with antibodies prepared against SLN1. Lanes from left to right represent protein from the six 10-mm segments, and then the single 30-mm segment (base of blade to tip of blade). The EZ is 30 mm, represented by the first three lanes. B, Contents of SLN1 protein in EZ and next segment of growing L3 blades of barley cv Himalaya and mutants at Sln1. Blades of L3 were harvested at approximately 50% final length. One segment equal in length to the EZ was cut from the base, and then another segment of equal length adjoining the first (“next” segment). Segment lengths were 30, 50, and 14 mm for barley cv Himalaya, slender, and dominant dwarf types, respectively. Protein was extracted from each segment, electrophoresed, blotted, and the filter incubated with antibodies prepared against SLN1. Lanes from left to right represent protein from the basal (B) and next (N) segments of Himalaya, sln1b, sln1c, and Sln1d.