The Arabidopsis eer1 mutant has enhanced ethylene responses in the hypocotyl and stem - PubMed (original) (raw)

The Arabidopsis eer1 mutant has enhanced ethylene responses in the hypocotyl and stem

P B Larsen et al. Plant Physiol. 2001 Feb.

Abstract

By screening for enhanced ethylene-response (eer) mutants in Arabidopsis, we isolated a novel recessive mutant, eer1, which displays increased ethylene sensitivity in the hypocotyl and stem. Dark-grown eer1 seedlings have short and thick hypocotyls even in the absence of added ethylene. This phenotype is suppressed, however, by the ethylene biosynthesis inhibitor 1-aminoethoxyvinyl-glycine. Following ethylene treatment, the dark-grown eer1 hypocotyl response is greatly exaggerated in comparison with the wild type, indicating that the eer1 phenotype is not simply due to ethylene overproduction. eer1 seedlings have significantly elevated levels of basic-chitinase expression, suggesting that eer1 may be highly sensitive to low levels of endogenous ethylene. Adult eer1 plants display exaggerated ethylene-dependent stem thickening, which is an ethylene response previously unreported in Arabidopsis. eer1 also has enhanced responsiveness to the ethylene agonists propylene and 2,5-norbornadiene. The eer1 phenotype is completely suppressed by the ethylene-insensitive mutation etr1-1, and is additive with the constitutive ethylene-response mutation ctr1-3. Our findings suggest that the wild-type EER1 product acts to oppose ethylene responses in the hypocotyl and stem.

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Figures

Figure 1

Figure 1

Dark-grown eer1 seedlings have an enhanced ethylene response when exposed to the ethylene precursor ACC. A, Dark-grown wild-type and eer1 seedlings treated with either 10 μ

m

AVG, 0 μ

m

ACC (nt, no treatment), or 10 μ

m

ACC for 3.5 d. B, ACC dose response curves for hypocotyl length of 3.5-d-old dark-grown wild-type and_eer1_ seedlings. Control treatments included no ACC and 10 μ

m

AVG. Mean ±

se

values were determined from 25 to 30 seedlings. C, ACC dose response curves for hypocotyl diameter of 3.5-d-old dark-grown wild-type and eer1 seedlings as in B. D, ACC dose response curves for root length of 3.5-d-old dark-grown wild-type and_eer1_ seedlings as in B.

Figure 2

Figure 2

Ethylene treatment causes an exaggerated response in dark-grown eer1 seedlings. A, Dark-grown wild-type and_eer1_ 3.5-d-old seedlings grown in air or 10 μL L−1 ethylene in the presence of 10 μ

m

AVG. B, Ethylene dose response curves for hypocotyl length of 3.5-d-old dark-grown wild-type and eer1 seedlings grown in the presence of 10 μ

m

AVG. Left, Actual hypocotyl length. Center, Relative inhibition of hypocotyl length (length/length at 0 μL L−1 ethylene) with the concentration of ethylene that gives 50% inhibition denoted by (- - -). Right, The ratio of eer1 hypocotyl length over wild-type hypocotyl length for each ethylene concentration with (- - -) denoting the predicted ratio if the eer1 mutant were not ethylene responsive. Mean ±

se

values were determined from 25 to 30 seedlings. C, Ethylene dose response curves for hypocotyl diameter of dark-grown wild-type and eer1 seedlings treated as in B. Left, Actual hypocotyl diameter. Middle, The relative increase in hypocotyl diameter (diameter/diameter at 0 μL L−1 ethylene). Right, The ratio of_eer1_ hypocotyl diameter over wild-type hypocotyl diameter for each ethylene concentration, with (- - -) denoting the predicted ratio if the eer1 mutant were not ethylene-responsive.

Figure 3

Figure 3

Dark-grown ctr1 seedlings respond to ethylene treatment. A, Dark-grown ctr1-3 seedlings grown in air or 10 μL L−1 ethylene for 3.5 d. B, Hypocotyl length of the wild type (Col-0) and three different_ctr1_ mutant alleles grown in the dark for 3.5 d either in the presence or absence of 10 μL L−1 ethylene gas. The ctr1-1 missense mutation results in an amino acid substitution of a highly conserved residue within the kinase domain. ctr1-3 carries a stop codon in the N-terminal regulatory domain. The ctr1-15 mutation has not yet been identified. Mean ±

se

values were determined from 25 to 30 seedlings. C, Hypocotyl diameter of the wild type (Col-0) and the three ctr1 mutant alleles treated as in B.

Figure 4

Figure 4

The enhanced ethylene response of eer1 is restricted to the basal portion of the hypocotyl. Confocal microscopy of dark-grown wild-type and eer1 seedlings treated with either 10 μ

m

AVG or 10 μL L−1 ethylene for 3.5 d. Top two sets, The cotyledons, apical hook, and apical portion of the hypocotyl (10× magnification). Bottom, The transition zone between the apical hook region and basal portion of the hypocotyl (20× magnification).

Figure 5

Figure 5

eer1 hypocotyls have an enhanced response to ethylene agonists. A, Propylene dose response curves for hypocotyl length of 3.5-d-old dark-grown wild-type and eer1 seedlings grown in the presence of 10 μ

m

AVG. Left, Actual hypocotyl length. Middle, Relative inhibition of hypocotyl length (length/length at 0 μL L−1 ethylene) with the concentration of propylene causing 50% inhibition denoted by (- - -). Right, The ratio of eer1 hypocotyl length over wild-type hypocotyl length for each propylene concentration with (- - -) denoting the predicted ratio if the eer1 mutant were not propylene responsive. Mean ±

se

values were determined from 25 to 30 seedlings. B, Hypocotyl length was measured for dark-grown wild-type and eer1 seedlings exposed to either 229 μL L−1 NBD, air, or 1 μL L−1 ethylene for 3.5 d. Mean ±

se

values were determined from 25 to 30 seedlings.

Figure 6

Figure 6

Ethylene-dependent regulation of_basic-chitinase_ expression in dark-grown seedlings. Top, Autoradiograms of northern blots hybridized with a_basic-chitinase_ gene probe. Dark-grown seedlings of the wild type and eer1 were treated with either 0, 0.1, or 1 μL L−1 ethylene, either in the presence or absence of 10 μ

m

AVG for 3.5 d. Bottom, Ethidium bromide-stained rRNA bands in the corresponding gels prior to blotting.

Figure 7

Figure 7

Ethylene treatment causes radial swelling of Arabidopsis stems. A, Stems of 3-week-old wild-type and eer1 plants treated with either 229 μL L−1 NBD, air, or 1 μL L−1 ethylene for 8 d. B, Measurements of stem thickness immediately following the treatments described in A. Mean ±

se

values were determined from 10 plants.

Figure 8

Figure 8

The eer1 ctr1-3 double mutant phenotype is additive. Dark-grown 3.5-d-old seedlings of the wild type (Ws-2 and Col-0), eer1, ctr1-3, and the double mutant_eer1 ctr1-3_ without ethylene treatment. Hypocotyl inhibition of the eer1 ctr1-3 double mutant was greater than for either_eer1_ or ctr1-3 alone, indicating that the two mutations are additive.

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