Short-term growth responses to ethylene in Arabidopsis seedlings are EIN3/EIL1 independent

Abstract

Kinetic studies indicate there are two phases to growth inhibition by ethylene for the hypocotyls of etiolated Arabidopsis seedlings. Phase I is transient, while phase II results in sustained growth inhibition. The EIN2 membrane protein is required for both the first and second phases of growth inhibition by ethylene, while the transcription factors EIN3 and EIL1 are required for the second phase but not the first phase. The first phase lasts no more than 2 h. It is less sensitive to the ethylene response inhibitor 1-methylcyclopropene and more sensitive to ethylene than the second phase. The first phase shows adaptation at low concentrations of ethylene (< or =0.01 microL L(-1)) with a relative refractory period of 5 h after ethylene is added. A modified signal transduction model is proposed that accounts for the two phases of growth inhibition.

Figure 1.

Rapid kinetic analysis of growth in etiolated Arabidopsis seedlings. Measurements were made in air for 1 h prior to introducing 10 μL L⁻¹ ethylene (↓). Seedling growth was measured for another 5 h. The rate of growth was determined throughout. Columbia (wild-type) seedlings (▴) were compared with ein3-1;eil1-1 (♦) and ein2-1 (○) seedlings.

Figure 2.

1-MCP prevents both phases of inhibition by ethylene. Seedlings were pretreated with 1-MCP for 17 h prior to growth measurements. At 45 min (↓), 10 μL L⁻¹ ethylene was introduced. Concentrations of 1-MCP used were 0 (×), 10 (▪), 50 (▴), and 1,000 (•) nL L⁻¹.

Figure 3.

Growth inhibition by various concentrations of ethylene. A, Growth response kinetics at various concentrations of ethylene. Ethylene was introduced 1 h after measurements were initiated (↓). Concentrations used were: 1 μL L⁻¹ (×), 100 nL L⁻¹ (▪), 10 nL L⁻¹ (▴), 1.5 nL L⁻¹ (•), and 0.2 nL L⁻¹ (♦). B, Dose-response relationship for phase I (▴) and phase II (▪) growth inhibition responses. Growth inhibition was normalized to inhibition obtained at 10 μL L⁻¹ ethylene. For most doses of ethylene, the amount of growth inhibition was averaged from 20 to 35 min after ethylene was added to determine the percent inhibition for phase I. The exceptions to this were the two lowest doses of ethylene, which started to recover 30 min after ethylene was added. For these data points, the amount of inhibition between 20 and 25 min was averaged. For phase II responses, the amount of growth inhibition between 5 and 6 h was averaged and used to determine the percent inhibition.

Figure 4.

Etiolated seedlings adapt to low concentrations of ethylene. Seedlings were treated with 10 nL L⁻¹ ethylene at 1 h. Growth rate returned to pretreatment rates approximately 2 h after ethylene was introduced. A second, equal dose of ethylene was introduced 2 h 10 min (A) or 5 h (B) after the first dose was initiated. The response profile of seedlings only given the initial dose of ethylene (dashed line) is shown in each section for comparison.

Figure 5.

Alternative models of ethylene signal transduction for the two phases of seedling growth inhibition. Feedback is invoked in both models to explain the observation that phase I shows adaptation. In model I, EIN2 mediates the two phases of seedling growth response through two independent mechanisms. Phase I is EIN3/EIL1 independent and subject to feedback inhibition, while phase II is EIN3/EIL1 dependent and not subject to feedback inhibition. In model II, both phases of growth response occur via a single EIN2-dependent mechanism. At very low ethylene concentrations, a feedback loop reverses the growth inhibition by either stimulating components upstream of EIN2 or inhibiting components at or downstream of EIN2, resulting in a transient growth inhibition. At higher ethylene concentrations, a secondary feedback loop, mediated through EIN3/EIL1, inhibits the primary feedback loop, resulting in sustained growth inhibition.