Five components of the ethylene-response pathway identified in a screen for weak ethylene-insensitive mutants in Arabidopsis

Abstract

Five ethylene-insensitive loci (wei1-wei5) were identified by using a low-dose screen for "weak" ethylene-insensitive mutants. wei1, wei2, and wei3 seedlings showed hormone insensitivity only in roots, whereas wei4 and wei5 displayed insensitivity in both roots and hypocotyls. The genes corresponding to wei1, wei4, and wei5 were isolated using a positional cloning approach. The wei1 mutant harbored a recessive mutation in TIR1, which encodes a component of the SCF protein ubiquitin ligase involved in the auxin response. wei4, a dominant mutant, resulted from a mutation in the ethylene receptor ERS, whereas wei5, a semidominant mutant, was caused by a mutation in the EIN3-related transcription factor gene EIL1. The simultaneous loss of functional WEI5EIL1 and EIN3 nearly completely abolished the ethylene response in etiolated seedlings, and adult plants were highly susceptible to infection by the necrotrophic fungal pathogen Botrytis cinerea. Moreover, wei5eil1 ein3 double mutants were able to fully suppress constitutive signaling caused by ctr1, suggesting a synergistic interaction among these gene products. Unlike previously known root ethylene-insensitive mutants, wei2 and wei3 were not affected in their response to auxin and showed a normal response to gravity. Genetic mapping studies indicate that wei2 and wei3 correspond to previously unidentified ethylene pathway genes that may control cell-elongation processes functioning at the intersection of the ethylene and auxin response pathways.

Figure 1

Mutant screen using nonsaturating levels of ethylene. (A) Schematic representation of the screening strategy. Mutagenized Arabidopsis plants were screened at a low concentration of the ethylene precursor ACC. Plants that showed hormone insensitivity were selected and retested in the next generation. Only putative mutants that showed weak hormone insensitivity were characterized further. (B) Comparison of the phenotypes of wei mutants in different concentrations of ACC. Seedlings were grown in the dark for 3 days in the presence of the indicated concentrations of ACC and photographed.

Figure 2

Quantification of the effects of ACC on hypocotyl (A) and root length (B) of the wei mutants. Wild-type and mutant seedlings (30–40 per treatment per genotype) were grown in the dark in the presence of the indicated concentrations of ACC for 3 days, photographed, and measured by using the program NIH IMAGE.

Figure 3

Epistasis analysis of the wei mutants. Double mutants were constructed between ctr1 and five wei mutants. Seedlings of the corresponding double mutants were grown in the dark for 3 days in the absence of exogenously applied ethylene or ACC and photographed.

Figure 4

Schematic representation of the ERS1 (A), EIL1 (B), and TIR1 (C) proteins. The approximate positions of the mutations found in wei4/ers1-10 (R to C), wei5-1/eil1-1 (transposon insertion), wei5-2/eil1-2 (frame shift), and wei1/tir1-101 (stop codon) are indicated.

Figure 5

Pathogen susceptibility of ethylene mutants. Rosettes of 4-week-old adult plants of Col, eil1-1, ein3-1, ein2-5, and eil1-1 ein3-1 were infected with the spore suspensions of B. cinerea. The percentage of dead plants was scored 9 days after inoculation. The data represent averages with standard deviations of three independent experiments performed with 12 or more plants per genotype.

Figure 6

Quantification of the effects of 2,4-dichlorophenoxyacetic acid on the hypocotyls (A) and roots (B) of the wei mutants. Wild-type and mutant seedlings (30–40 per treatment per genotype) were grown in the dark for 3 days in the presence of the indicated concentrations of 2,4-dichlorophenoxyacetic acid, photographed, and measured by using the program NIH IMAGE.