Interactions between abscisic acid and ethylene signaling cascades

Abstract

We screened for mutations that either enhanced or suppressed the abscisic acid (ABA)-resistant seed germination phenotype of the Arabidopsis abi1-1 mutant. Alleles of the constitutive ethylene response mutant ctr1 and ethylene-insensitive mutant ein2 were recovered as enhancer and suppressor mutations, respectively. Using these and other ethylene response mutants, we showed that the ethylene signaling cascade defined by the ETR1, CTR1, and EIN2 genes inhibits ABA signaling in seeds. Furthermore, epistasis analysis between ethylene- and ABA-insensitive mutations indicated that endogenous ethylene promotes seed germination by decreasing sensitivity to endogenous ABA. In marked contrast to the situation in seeds, ein2 and etr1-1 roots were resistant to both ABA and ethylene. Our data indicate that ABA inhibition of root growth requires a functional ethylene signaling cascade, although this inhibition is apparently not mediated by an increase in ethylene biosynthesis. These results are discussed in the context of the other hormonal regulations controlling seed germination and root growth.

Figure 1.

Morphological Phenotypes of Mutants. (A) Eight-day-old seedling of mutant line H3 (subsequently renamed abi1-1 ctr1-10) grown with a 16-hr-light photoperiod. (B) Eight-day-old abi1-1 seedling grown with a 16-hr-light photoperiod. (C) Eight-day-old seedling of mutant line 84B (subsequently renamed abi1-1 ein2-45) grown with a 16-hr-light photoperiod. The root is out of focus but was similar to that of abi1-1. (D) Three-day-old abi1-1 seedling grown in the dark. (E) Three-day-old seedling of mutant line H3 (subsequently renamed abi1-1 ctr1-10) grown in the dark. (F) Three-day-old Landsberg erecta (Ler) seedling grown in the dark on medium supplemented with 10 μM 1-aminocylcopropane-1-carboxylic acid (ACC). (G) Three-day-old seedling of mutant line 84B/1 (subsequently renamed ein2-45) grown in the dark on medium supplemented with 10 μM ACC. .

Figure 2.

ABA Dose Response for Germination Inhibition. (A) Seeds of Ler wild type (open squares), abi1-1 (open circles), and H3 (subsequently renamed abi1-1 ctr1-10; filled diamonds) were plated on medium supplemented with the indicated concentrations of ABA, chilled for 4 days at 4°C in darkness, and incubated for 5 days at 21°C with a 16-hr-light photoperiod. The number of germinated seeds (with green cotyledons, elongated root with hairs, or both) was expressed as the percentage of the total number of seeds plated (100 to 200). (B) Seeds of Ler wild type (open squares), abi1-1 (open circles), and 84B (subsequently renamed abi1-1 ein2-45; filled circles) were plated on medium supplemented with the indicated concentrations of ABA, chilled for 4 days at 4°C in darkness, and incubated for 3 days at 21°C with a 16-hr-light photoperiod. The number of germinated seeds (with green cotyledons) was expressed as the percentage of the total number of seeds plated (100 to 200).

Figure 3.

ABA Dose Response for Germination Inhibition. Seeds were plated on medium supplemented with the indicated concentrations of ABA, chilled for 4 days at 4°C in darkness, and incubated at 21°C with a 16-hr-light photoperiod. The number of germinated seeds (with fully emerged radicle tip) was expressed as the percentage of the total number of seeds plated (100 to 200). (A) Seeds of Ler wild type (open squares), ctr1-10 (filled diamonds), and ein2-45 (filled circles) were incubated for 2 days at 21°C. (B) Seeds of the Col wild type (open squares), ctr1-1 (filled diamonds), ein2-1 (filled circles), and etr1-1 (filled triangles) were incubated for 3 days at 21°C.

Figure 4.

Seed Dormancy. Freshly harvested seeds were plated on ABA-free medium and incubated directly (without cold pretreatment) at 21°C with a 16-hr-light photoperiod. The number of germinated seeds (with fully emerged radicle tip) at a given time was expressed as the percentage of the total number of seeds plated (100 to 200). In simultaneous experiments in which seeds from the same batches were first chilled for 4 days at 4°C in darkness to break dormancy, all genotypes displayed 100% germination after 5 days at 21°C (data not shown). (A) Seeds of the Ler wild type (open squares), ctr1-10 (filled diamonds), and ein2-45 (filled circles). (B) Seeds of the Col wild type (open squares), ctr1-1 (filled diamonds), ein2-1 (filled circles), and etr1-1 (filled triangles).

Figure 5.

Seed Dormancy. Freshly harvested seeds of the Ler wild type (open squares), abi1-1 (open circles), ein2-45 (open triangles), and abi1-1 ein2-45 (filled circles) were plated on ABA-free medium and incubated directly (without cold pretreatment) at 21°C with a 16-hr-light photoperiod. The number of germinated seeds (with fully emerged radicle tip) at a given time was expressed as the percentage of the total number of seeds plated (100 to 200). In simultaneous experiments in which seeds from the same batches were first chilled for 4 days at 4°C in darkness to break dormancy, all genotypes displayed 100% germination after 5 days at 21°C (data not shown).

Figure 6.

Effect of the ein2-45 Mutation on the Seed Phenotypes of Severely ABA-Insensitive Mutants. (A) and (C) ABA dose response for germination inhibition. Seeds were plated on medium supplemented with the indicated concentrations of ABA, chilled for 3 days at 4°C in darkness, and incubated for 3 days at 21°C with a 16-hr-light photoperiod. The number of germinated seeds (with fully emerged radicle tip) was expressed as the percentage of the total number of seeds plated (70 to 100). Data from seeds of the Ler wild type (open squares), abi3-4 (open circles), ein2-45 (open triangles), and abi3-4 ein2-45 (filled circles) are shown in (A). Data from seeds of the Ler wild type (open squares), abi1-1 abi3-1 (open circles), ein2-45 (open triangles), and abi1-1 abi3-1 ein2-45 (filled circles) are shown in (C). (B) and (D) Seed dormancy. Freshly harvested seeds were plated on ABA-free medium and incubated directly (without cold pretreatment) at 21°C with a 16-hr-light photoperiod. The number of germinated seeds (with fully emerged radicle tip) at a given time was expressed as the percentage of the total number of seeds plated (100 to 200). In simultaneous experiments in which seeds from the same batches were first chilled for 4 days at 4°C in darkness to break dormancy, all genotypes displayed 100% germination after 5 days at 21°C (data not shown). Data from seeds of the Ler wild type (open squares), abi3-4 (open circles), ein2-45 (open triangles), and abi3-4 ein2-45 (filled circles) are shown in (B). Data from seeds of the Ler wild type (open squares), abi1-1 abi3-1 (open circles), ein2-45 (open triangles), and abi1-1 abi3-1 ein2-45 (filled circles) are shown in (D).

Figure 7.

ABA Dose Response for Root Growth Inhibition. Seeds of the Ler wild type (open squares), abi1-1 (open circles), and ein2-45 (filled circles) were germinated and grown for 8 days on ABA-free medium. These seedlings were then incubated vertically on medium supplemented with the indicated concentrations of ABA, and their root length was scored after 5 days. Root growth of ABA-treated seedlings was expressed as a percentage relative to controls incubated on ABA-free medium. Values shown are mean ±sd from samples consisting of 15 to 20 seedlings each.

Figure 8.

Sensitivity of Root Growth to Applied ABA and ACC. (A) and (B) Seeds of the indicated genotypes were germinated and grown for 6 days on medium containing no additional supplements. These seedlings were then incubated vertically on medium supplemented with 100 μM ABA (A) or 0.2 μM ACC (B), and their root length was scored after 4 days. Root growth of ABA- or ACC-treated seedlings was expressed as a percentage relative to controls incubated on normal medium. Values shown are mean ±sd from samples consisting of 15 to 20 seedlings each.

Figure 9.

ABA Dose Response for Germination Inhibition. Seeds of the Col wild type (open squares), ein2-1 (open triangles), and axr2-1 (filled diamonds) were plated on medium supplemented with the indicated concentrations of ABA, chilled for 4 days at 4°C in darkness, and incubated for 5 days at 21°C with a 16-hr-light photoperiod. The number of germinated seeds was expressed as the percentage of the total number of seeds plated (70 to 100).

Figure 10.

Schematic Representations of the Interactions between the ABA and Ethylene Signaling Cascades. (A) Regulation of seed germination. Ethylene and GA signaling cascades promote seed germination by decreasing sensitivity to endogenous ABA in imbibed seeds. The abi1-1 and abi3 mutants are ABA insensitive, the sly1 mutant is GA insensitive, and the etr1-1 and ein2 mutants are ethylene insensitive. The positions of the intersections between the ABA, ethylene, and GA pathways are speculative. (B) Inhibition of seedling root growth. Inhibition of root growth in response to increased amounts of ethylene is largely mediated through internal accumulation of auxin. Cytokinin inhibits root growth by stimulating ethylene biosynthesis. ABA inhibition of root growth is apparently not mediated by an increase in ethylene biosynthesis but requires an active ethylene signaling cascade. Either the basal activity of the ethylene pathway is required to synergize ABA action or ABA stimulates the ethylene signaling cascade.