Regulation of abscisic acid signaling by the ethylene response pathway in Arabidopsis

Abstract

Although abscisic acid (ABA) is involved in a variety of plant growth and developmental processes, few genes that actually regulate the transduction of the ABA signal into a cellular response have been identified. In an attempt to determine negative regulators of ABA signaling, we identified mutants, designated enhanced response to ABA3 (era3), that increased the sensitivity of the seed to ABA. Biochemical and molecular analyses demonstrated that era3 mutants overaccumulate ABA, suggesting that era3 is a negative regulator of ABA synthesis. Subsequent genetic analysis of era3 alleles, however, showed that these are new alleles at the ETHYLENE INSENSITIVE2 locus. Other mutants defective in their response to ethylene also showed altered ABA sensitivity; from these results, we conclude that ethylene appears to be a negative regulator of ABA action during germination. In contrast, the ethylene response pathway positively regulates some aspects of ABA action that involve root growth in the absence of ethylene. We discuss the response of plants to ethylene and ABA in the context of how these two hormones could influence the same growth responses.

Figure 1.

Germination of Wild-Type and era3 Seed. (A) Germination in the presence of exogenous ABA. Open squares, wild type; filled diamonds, era3-1; filled circles, era3-3; open triangles, era1-2. (B) Seed dormancy in response to increasing periods of chilling. Open squares, wild type; filled squares, era3-1 at 0 days of chilling at 4°C; filled triangles, era3-1 at 2 days of chilling at 4°C; filled circles, era3-1 at 4 days of chilling at 4°C. Each point represents a germination test of 20 to 40 seed. Tests in (A) were performed in duplicate, and similar results were obtained in all cases. Tests in (B) were performed in triplicate. Vertical bars represent the standard errors. The percentage of germination was determined by dividing the number of seeds that germinated after 5 days of imbibition by the total number of seed.

Figure 2.

ABA Accumulation in Wild-Type and era3 Plants. (A) Endogenous ABA concentrations in wild-type (WT), era3-1, and era1-2 plants. Wild-type and era3-1 values are the average of three independent measurements. era1-2 ABA content was determined only once. Averages and standard deviation are shown for WT and era3-1 mutants. FW, fresh weight. (B) RNA transcript accumulation of zeaxanthin epoxidase (ABA1). After hybridization with the ABA1 probe, blots were stripped for β-tubulin (β-TUB) hybridization, which meant that absolute amounts could not be compared between hybridization experiments. Five micrograms of total RNA was loaded per lane.

Figure 3.

Root Growth of Wild-Type and Ethylene Response Mutants Grown on Media Containing ABA. (A) Root elongation of wild type (filled squares), ein2-44 (open triangles), and etr1-4 (open circles). (B) Root elongation of wild type (WT), abi1-1, and several ethylene-insensitive mutants when grown on media containing 10 μM ABA. Seven- to 10-day-old seedlings were placed on minimal media supplemented with increasing concentrations of ABA. Root elongation was measured after 4 days. Root growth on ABA was relative to the mean root elongation of the same genotype on minimal media. Each value represents the mean measurement for five to 15 seedlings. Experiments were conducted twice with wild-type and ein2 mutants and gave similar results, whereas experiments involving various etr1 alleles and ein3 were performed once.

Figure 4.

ABA-Inducible Gene Expression in RAB18–GUS and ein2-44 RAB18–GUS Roots. RAB18–GUS (WT) and ein2-44 RAB18–GUS (ein2-44) seedlings were treated with 0 (−ABA) or 10 μM (+ABA) ABA over a 24-hr period, and lines were stained to determine GUS activity for 12 hr at 37°C. Each point represents results for roots from three separate plants.

Figure 5.

Root Growth of Wild-Type Seedlings in the Presence of Ethylene Biosynthetic (AVG) and Action Inhibitors (AgNO₃). (A) Seven-day-old wild-type seedlings were placed on minimal medium supplemented with no inhibitor (filled squares), 10 μM AgNO₃ (open circles), or 2 μM AVG (open triangles). Root elongation was measured after 4 days. Relative growth is indicated in relationship to the mean root elongation of wild-type seedlings transferred to minimal medium without inhibitors. Each value represents the mean of measurements from at least 10 seedlings. Experiments were done in duplicate, and similar results were obtained in all cases. (B) Seven-day-old wild-type seedlings were placed on medium free of ABA (open bars) or medium containing 1 μM ABA (striped bars). Treatment 1, no ACC or AVG supplement; treatment 2, added 1 μM AVG; treatment 3, added 2 μM ABG and 2 μM ACC. Root growth shown is the mean of the absolute root length of at least 10 seedlings. The experiment was performed in triplicate. Error bars represent sd.

Figure 6.

Effects of Ethylene on Root Growth and Germination in the Presence of ABA. (A) Relative root growth of wild-type (filled squares) and eto1-1 (open circles) seedlings in the presence of ABA. Experiments were performed as described earlier, and each point represents the mean value for five to 15 seedlings. (B) Germination of wild-type seeds on minimal media in the presence (open squares) and absence (filled squares) of ACC and on media containing 3 μM ABA in the presence (open circles) and absence (filled circles) of ACC. Each point represents a germination test with ⩾20 seed. Each experiment was performed in duplicate, and similar results were obtained in all cases. The percentage of germination was determined by dividing the number of seeds that germinated after 5 days of imbibition by the total number of seed.

Figure 7.

Hypothetical Model for the Role of ABA and Ethylene in Regulating Root Growth in Arabidopsis. (A) In the absence of ethylene, one way for ABA to inhibit root growth is by signaling through the ETR1 response pathway. Because this pathway is also regulated by ethylene, mutations in the pathway interfere with both ABA and ethylene signaling. (B) In the presence of ethylene, ABA is unable to use this pathway; thus, mutations that increase ethylene synthesis confer an ABA-insensitive root phenotype.