The relationship between ethylene binding and dominant insensitivity conferred by mutant forms of the ETR1 ethylene receptor - PubMed (original) (raw)

The relationship between ethylene binding and dominant insensitivity conferred by mutant forms of the ETR1 ethylene receptor

A E Hall et al. Plant Physiol. 1999 Sep.

Abstract

Ethylene responses in Arabidopsis are mediated by a small family of receptors, including the ETR1 gene product. Specific mutations in the N-terminal ethylene-binding domain of any family member lead to dominant ethylene insensitivity. To investigate the mechanism of ethylene insensitivity, we examined the effects of mutations on the ethylene-binding activity of the ETR1 protein expressed in yeast. The etr1-1 and etr1-4 mutations completely eliminated ethylene binding, while the etr1-3 mutation severely reduced binding. Additional site-directed mutations that disrupted ethylene binding in yeast also conferred dominant ethylene insensitivity when the mutated genes were transferred into wild-type Arabidopsis plants. By contrast, the etr1-2 mutation did not disrupt ethylene binding in yeast. These results indicate that dominant ethylene insensitivity may be conferred by mutations that disrupt ethylene binding or that uncouple ethylene binding from signal output by the receptor. Increased dosage of wild-type alleles in triploid lines led to the partial recovery of ethylene sensitivity, indicating that dominant ethylene insensitivity may involve either interactions between wild-type and mutant receptors or competition between mutant and wild-type receptors for downstream effectors.

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Figure 4

Figure 4

Analysis of ethylene binding and ethylene responses in C65S and H69A mutants. A, Ethylene binding by C65S and H69A proteins expressed in yeast. Dark bars represent the amount of [14C]ethylene bound (disintegrations per minute per gram of yeast) by transgenic yeast incubated with 0.07 μL L−1 [14C]ethylene. Samples were run in triplicate and

sd

bars are shown. White bars represent the amount of [14C]ethylene bound (disintegrations per minute per gram of yeast) by single samples of transgenic yeast incubated with 0.07 μL L−1 [14C]ethylene and 1,000 μL L−1 [12C]ethylene (background binding). WT, Wild type. B, Analysis of the triple response in dark-grown seedlings. Transgenic lines are shown for plants transformed with the wild-type ETR1 gene (WT), the ETR1 gene containing a C65S mutation (C65S), and the ETR1 gene containing an H69A mutation (H69A). For comparison, wild-type (ethylene-sensitive) and _etr1_-1 mutant (ethylene-insensitive) seedlings were grown under the same conditions. Seeds were grown for 4 d in 35 μL L−1 ethylene, and two independent transgenic lines are shown for each construct used. C, Expression level of ETR1 protein. Expression levels of ETR1 protein were determined for the transgenic lines from B. Membranes were isolated from etiolated seedlings grown in air for 4 d. Membrane protein (15 μg) was subjected to SDS-PAGE, and ETR1 was visualized by western blot.

Figure 1

Figure 1

Ethylene dose-response curves of seedling growth for the etr1 alleles. A schematic of ETR1 indicates the protein's predicted domains. The three predicted transmembrane subdomains within the N-terminal hydrophobic domain are shaded light gray. The GAF domain (Aravind and Ponting, 1997) is shaded dark gray. The His kinase domain is indicated by diagonal lines; black boxes within this area represent the five consensus motifs (H, N, G1, F, and G2) found in bacterial His kinases. The response regulator domain is indicated by diagonal bars. The point mutations that constitute the four dominant etr1 alleles are indicated. Single-letter abbreviations for amino acids are designated. Ethylene dose-response curves of seedling growth for the etr1 alleles are shown for hypocotyl (A–D) and root (E–H). Dose-response curves are shown for wild type (•, WT) and mutants (▴), including _etr1_-1 (C and G), _etr1_-2 (D and H), _etr1_-3 (A and E), and _etr1_-4 (B and F). Mutant and wild-type dark-grown seedlings were incubated for 3 d in air or a range of ethylene concentrations (0–1,000 μL L−1) (log values shown on the x axis). Hypocotyl and root length values are reported as a percentage of the wild-type maximum (wt max) and represent the means ±

se

of 50 measurements. Error bars are not shown if smaller than the smallest displayed. ND, No detectable ethylene.

Figure 2

Figure 2

Analysis of ethylene binding by mutant ETR1 proteins. A schematic of the amino acids 1 to 128 of the ETR1 protein is shown, and relative positions of amino acid changes (•) in the three hydrophobic domains are included. For site-directed mutations, single-letter abbreviations are noted for amino acids. Dark bars represent the amount of [14C]ethylene bound (disintegrations per minute per gram of yeast) by transgenic yeast incubated with 0.07 μL L−1 [14C]ethylene. Samples were run in triplicate and

sd

bars are shown. White bars represent the amount of [14C]ethylene bound (disintegrations per minute per gram of yeast) by single samples of transgenic yeast incubated with 0.07 μL L−1 [14C]ethylene and 1,000 μL L−1 [12C]ethylene (background binding). Total yeast proteins were isolated and analyzed by western-blot analysis. The expression level of each of the mutants was determined by densitometric quantification of western blots, and is reported as expression level relative to wild-type ETR1 protein.

Figure 3

Figure 3

Western-blot analysis indicates mutant ETR1 proteins form disulfide-linked dimers. Membranes isolated from yeast expressing each of the mutant ETR1 proteins were incubated in the presence (+) or absence (−) of 100 m

m

DTT for 1 h at 37°C, and separated by SDS-PAGE. Western-blot analysis comparing wild-type ETR1 protein to the mutant proteins was carried out using an anti-ETR1 antibody (HRR). In the presence of reducing agent (A), the proteins migrate as a 79-kD monomer, while in the absence of reducing agent (B), a 147-kD dimer is also detected.

Figure 5

Figure 5

Effects of gene dosage on the etr1, etr2, and ein4 dominant mutant phenotypes. Dose-response curves for seedling-growth response in hypocotyl tissues are shown for wild-type (•) and mutants (▴) including _etr1_-1 (A–C), _etr1_-2 (D–F), _etr2_-1(G–I), and _ein4_-3 (J–L). Genotypes of mutants are indicated as mt/mt for homozygous diploid mutants, mt/wt for heterozygous diploid mutants, and mt/wt/wt for heterozygous triploid mutants. Values represent the means ±

se

of 20 measurements. Error bars are not shown if smaller than the symbol. ND, No detectable ethylene.

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