Analysis of the ethylene response in the epinastic mutant of tomato

Abstract

Ethylene can alter plant morphology due to its effect on cell expansion. The most widely documented example of ethylene-mediated cell expansion is promotion of the "triple response" of seedlings grown in the dark in ethylene. Roots and hypocotyls become shorter and thickened compared with controls due to a reorientation of cell expansion, and curvature of the apical hook is more pronounced. The epinastic (epi) mutant of tomato (Lycopersicon esculentum) has a dark-grown seedling phenotype similar to the triple response even in the absence of ethylene. In addition, in adult plants both the leaves and the petioles display epinastic curvature and there is constitutive expression of an ethylene-inducible chitinase gene. However, petal senescence and abscission and fruit ripening are all normal in epi. A double mutant (epi/epi;Nr/Nr) homozygous for both the recessive epi and dominant ethylene-insensitive Never-ripe loci has the same dark-grown seedling and vegetative phenotypes as epi but possesses the senescence and ripening characteristics of Never-ripe. These data suggest that a subset of ethylene responses controlling vegetative growth and development may be constitutively activated in epi. In addition, the epi locus has been placed on the tomato RFLP map on the long arm of chromosome 4 and does not demonstrate linkage to reported tomato CTR1 homologs.

Figure 1

Phenotypes of the epiepi;Nr/Nr double mutant. A, Vegetative growth; note the epinastic growth of double-mutant plants (genotypes from left to right are as follows: AC [nr/nr], Nr/Nr, VFN8 [Epi/Epi], epi/epi, and epi/epi;Nr/Nr). B, Delayed petal senescence and abscission in epi/epi;Nr/Nr plants; note epinastic curvature of leaves. C, Fruit ripening is impaired in the double mutant. Genotypes are indicated as follows: a, AC (nr/nr); b, Nr/Nr; c, VFN8 (Epi/Epi); d, (epi/epi); and e, epi/epi;Nr/Nr. All fruit were of equivalent age and were harvested when normal ripening lines reached the red ripe stage. The difference in fruit locule number is due to cultivar differences. Fruit of AC typically have two locules, whereas those of the VFN8 cultivar are multilocular.

Figure 2

Effect of ACC and 1-methylcyclopropene on the dark-grown seedling phenotype of epi. Seedlings were grown as described in “Materials and Methods” and were photographed 2 weeks after sowing. Genotypes are indicated as follows: a, AC (nr/nr); b, Nr/Nr; c, VFN8 (Epi/Epi); d, (epi/epi); and e, epi/epi;Nr/Nr. A, Seedlings grown in absence of ACC (top); note shortening and thickening of epi/epi and double-mutant hypocotyls. Bottom, Seedlings grown in 20 μm ACC; note further reduction in epi/epi elongation but reduced inhibition in Nr and double-mutant seedlings. B, Growth in 20 μm ACC (top) and 2 μL L⁻¹ of 1-MCP and 20 μm ACC (bottom). Note that 1-MCP reverses the effect of ACC but does not restore epi/epi or double mutants to normal.

Figure 3

Ethylene-inducible gene expression in epi. Total RNA was isolated from leaves of greenhouse-grown plants either from control leaves (− ethylene) or from plants that had been treated with 20 μL L⁻¹ ethylene for 8 h (+ ethylene). Genotypes are indicated as follows: a, AC (nr/nr); b, Nr/Nr; c, VFN8 (Epi/Epi); d, epi/epi; and e, epi/epi;Nr/Nr. Twenty μg of RNA was loaded per lane and the filter was hybridized to probes for CHITINASE9 (CHI9), E4, and ACTIN.

Figure 4

Morphology of adaxial epidermal cells. Genotypes are indicated as follows: a, AC (nr/nr); b, Nr/Nr; c, VFN8 (Epi/Epi); d, epi/epi, and e, epi/epi Nr/Nr. Note swollen morphology of the epi/epi mutant (d) compared with the isogenic control VFN8 (c). Magnification is ×700; bar = 10 μm.

Figure 5

Placement of the epi locus on the tomato RFLP map. An F₂ population segregating for epi and RFLP loci was generated from a cross between L. esculentum (epi/epi) × L. pennellii (Epi/Epi). A total of 139 individuals were scored on the basis of the epi phenotype, and linkage to markers was determined as described in “Materials and Methods.” The tomato RFLP linkage map of Tanksley et al. (1992) is shown to demonstrate conservation of marker order.

Figure 6

Models proposing the action of the EPI protein in tomato. A, EPI lies within the ethylene signal transduction pathway to regulate a subset of ethylene responses that lead to normal cell expansion. Mutation of EPI results in abnormal cell expansion and leads to elevated ethylene synthesis (+ve). B, EPI acts to control normal cell expansion and growth in a separate pathway to the ethylene response pathway. Mutation of EPI leads to abnormal cell expansion that in turn activates the ethylene response pathway, causing elevated ethylene synthesis.