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. 2003 Sep;15(9):2032-41.
doi: 10.1105/tpc.013060.

Analysis of combinatorial loss-of-function mutants in the Arabidopsis ethylene receptors reveals that the ers1 etr1 double mutant has severe developmental defects that are EIN2 dependent

Anne E Hall  1 , Anthony B Bleecker
Affiliations

Analysis of combinatorial loss-of-function mutants in the Arabidopsis ethylene receptors reveals that the ers1 etr1 double mutant has severe developmental defects that are EIN2 dependent

Anne E Hall et al. Plant Cell. 2003 Sep.

Abstract

Ethylene responses in Arabidopsis are controlled by the ETR receptor family. The receptors function as negative regulators of downstream signal transduction components and fall into two distinct subfamilies based on sequence similarity. To clarify the levels of functional redundancy between receptor isoforms, combinatorial mutant lines were generated that included the newly isolated ers1-2 allele. Based on the etiolated seedling growth response, all mutant combinations tested exhibited some constitutive ethylene responsiveness but also remained responsive to exogenous ethylene, indicating that all five receptor isoforms can contribute to signaling and no one receptor subtype is essential. On the other hand, light-grown seedlings and adult ers1 etr1 double mutants exhibited severe phenotypes such as miniature rosette size, delayed flowering, and sterility, revealing a distinct role for subfamily I receptors in light-grown plants. Introduction of an ein2 loss-of-function mutation into the ers1 etr1 double mutant line resulted in plants that phenocopy ein2 single mutants, indicating that all phenotypes observed in the ers1 etr1 double mutant are EIN2 dependent.

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Figures

Figure 1.
Figure 1.
Light-Grown ers1 etr1 Mutants Exhibit a Severe Constitutive Ethylene-Response Phenotype, Whereas Dark-Grown Seedlings Exhibit a Partial Ethylene-Response Phenotype. (A) ers1 etr1 mutants are capable of responding to ethylene. Hypocotyl lengths (in mm) of etiolated seedlings grown for 4 days in either air (dark gray) or 35 ppm of ethylene (light gray) are shown. At least 15 seedlings were measured for each treatment. ers1 etr1 mutants were identified phenotypically once the plates were removed from the dark and left in the light for 7 days. Col, Columbia wild type. (B) Seedlings grown in the dark on agar plates for 4 days in air. Compared with other constitutive response mutants, ers1 etr1 seedlings exhibited only a partial inhibition of hypocotyl growth but a more complete inhibition of root growth relative to wild-type (Ws) seedlings. (C) Seedlings grown in the light for 3 days in air. Ws seedlings appear wild type, whereas the ctr1 and etr1 etr2 ein4 mutants exhibit shorter hypocotyls, short roots with prolific root hairs, and smaller, less expanded cotyledons than wild-type seedlings. The ers1 etr1 mutants phenocopied the severe ran1 mutants, exhibiting shorter hypocotyls and roots and small, dark, unexpanded cotyledons.
Figure 2.
Figure 2.
ers1 etr1 Mutants Exhibit an Extremely Compact Rosette, Delayed Flowering, and Stunted Inflorescence Stems. (A) Rosette-stage phenotype of a 3-week-old ers1-2 etr1-7 homozygous mutant is shown next to a wild-type Columbia plant (at right) for comparison. (B) ers1-2 etr1-6 homozygous mutants (far right) photographed next to an etr1 etr2 ein4 triple mutant and a ctr1-2 mutant for comparison. All plants were 6 weeks old. (C) Close-up of a 12-week-old ers1-2 etr1-7 mutant that has not bolted. Bar = 1 cm. (D) Close-up of a 10-week-old ers1-2 etr1-6 mutant that has bolted and produced flowers (plant size, ∼4 cm).
Figure 3.
Figure 3.
Defects in Reproductive Development in the ers1 etr1 Mutant. (A) First bud off the apex of the inflorescence from an ers1-2 etr1-6 mutant exhibiting a severe phenotype (approximately floral stage 10 to 11). Petals are level with the short stamens, and stigmatic papillae are beginning to appear. (B) Older bud taken off the same inflorescence stem as the bud in (A), which also appears arrested in approximately stage 11. All buds are green, and sepals still enclose the floral organs. (C) ers1-2 etr1-6 flower exhibiting a weaker phenotype, approximately stage 13. Petals have elongated past the sepals, and mature stigmatic papillae are present. Anthers are dehiscent but are not in the proximity of the stigma. (D) Wild-type (Ws) flower in approximately stage 13 for comparison. (E) Floral bud of an ers1-2 etr1-6 individual. An abnormal stamen can be seen at bottom right. (F) Close-up of the defective stamen structure in (E). (G) Close-up of an anther from an ers1 etr1 mutant. There is breakage along the stomium, although it is not nearly as pronounced as that in wild-type flowers. (H) Dehiscent anther of a wild-type (Ws) individual. (I) Floral bud from a severe ers1-2 etr1-7 mutant. Sepals were dissected off. (J) An anther from the bud in (I) was dissected open, revealing aberrant pollen with a flattened appearance. (K) Wild-type (Ws) bud in approximately stage 10 to 11 for comparison. (L) An anther from the bud in (K) was dissected open, revealing normal pollen grains. Thin bars = 240 μm; thick bars in (G), (J), and (L) = 24 μm.
Figure 4.
Figure 4.
Chitinase B mRNA Expression Is Highly Increased in ers1 etr1 Mutants. Total RNA was isolated from rosette tissue of 3.5-week-old plants. Ten micrograms of total RNA was probed with a Chitinase B–specific probe. A ubiquitin-specific probe (UBQ10) was used as a loading control. Lanes with asterisks at top represent wild-type-looking siblings of the double mutants that were heterozygous for the ers1-2 allele and homozygous for the etr1-6 or etr1-7 allele. Col, Columbia wild type.
Figure 5.
Figure 5.
ers1 etr1 Developmental Defects Are Rescued Completely by the ein2-1 Mutation (A) Phenotypes of etiolated seedlings. Seedlings were grown on agar plates in the dark for 4 days in either air or 35 ppm of ethylene (Eth). ein2 ers1 etr1 triple mutants exhibit complete ethylene insensitivity. (B) Phenotypes of light-grown seedlings. Seedlings were grown in the light for 3 days. ein2 ers1 etr1 mutants no longer exhibit cupped cotyledons, shortened roots, and prolific root hairs. Col, Columbia wild type. (C) Floral phenotypes of adult plants. Flowers are of equivalent ages starting with developmental stage 12. ein2 ers1 etr1 mutants no longer exhibit defects in floral development. (D) Quantification of the hypocotyl response of mutant and wild-type seedlings. Seedlings were grown on agar plates in the dark for 3 days in either air (dark bars) or 35 ppm of ethylene (light bars). At least 20 seedlings were measured for each treatment.
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