Arabidopsis seedling growth response and recovery to ethylene. A kinetic analysis

Abstract

Responses to the plant hormone ethylene are mediated by a family of five receptors in Arabidopsis that act in the absence of ethylene as negative regulators of response pathways. In this study, we examined the rapid kinetics of growth inhibition by ethylene and growth recovery after ethylene withdrawal in hypocotyls of etiolated seedlings of wild-type and ethylene receptor-deficient Arabidopsis lines. This analysis revealed that there are two phases to growth inhibition by ethylene in wild type: a rapid phase followed by a prolonged, slower phase. Full recovery of growth occurs approximately 90 min after ethylene removal. None of the receptor null mutations tested had a measurable effect on the two phases of growth inhibition. However, loss-of-function mutations in ETR1, ETR2, and EIN4 significantly prolonged the time for recovery of growth rate after ethylene was removed. Plants with an etr1-6;etr2-3;ein4-4 triple loss-of-function mutation took longer to recover than any of the single mutants, while the ers1;ers2 double mutant had no effect on recovery rate, suggesting that receiver domains play a role in recovery. Transformation of the ers1-2;etr1-7 double mutant with wild-type genomic ETR1 rescued the slow recovery phenotype, while a His kinase-inactivated ETR1 construct did not. To account for the rapid recovery from growth inhibition, a model in which clustered receptors act cooperatively is proposed.

Figure 1.

Rapid kinetic analysis of growth in etiolated Arabidopsis hypocotyls. Etiolated hypocotyls respond to ethylene within 15 min of applying 10 μL L⁻¹ ethylene. There is a rapid decrease in growth rate, followed by a plateau in the change in growth rate. This is followed by a slow decrease in growth rate until a new steady-state growth rate is reached approximately 75 min after applying ethylene. When ethylene is removed, there is a lag of approximately 30 min before a measurable increase in growth rate is observed. Hypocotyls return to pretreatment growth rates approximately 90 min after ethylene is removed. In this and Figures 2, 4, and 5, measurements were made in air for 1 h prior to introducing 10 μL L⁻¹ ethylene (↓). Ethylene was removed 2 h later (↑).

Figure 2.

Comparisons of growth response kinetics in wild-type and loss-of-function receptors mutants. The responses of wild-type seedlings (▴) are shown in each section compared to etr1-7 mutants (A); etr2-3 mutants (B); ein 4-4 mutants (C); etr1-6;etr2-3;ein4-4 triple mutants (D); and ers1-2 (▪), ers2-3 (•), and ers1-2;ers2-3 (▵) mutants (E). Columbia (wild-type) seedlings were used for comparison with etr1-7, etr2-3, ein4-4, and etr1-6;etr2-3;ein4-4 (A–D), while WS (wild-type) seedlings were used for comparison with the ers1-2, ers2-3, and ers1-2;ers2-3 mutants (E). Ethylene was introduced at 1 h (↓) and removed 2 h later (↑).

Figure 3.

RT-PCR analysis from Columbia (wild-type) seedlings treated with ethylene. The mRNA from individual ethylene receptor isoforms was analyzed from etiolated seedlings before and during treatment with ethylene for 30 min or 2 h. All measurements were made in triplicate.

Figure 4.

The slow growth recovery phenotype of plants deficient in His-kinase receptor isoforms was rescued by a wild-type ETR1 transgene but not a kinase-inactivated ETR1 transgene. A, The ers1-2;etr1-7 double loss-of-function receptor mutant (▪) had slower recovery than wild-type plants (▴). B, Transformation of this double mutant with genomic ETR1 (gETR1; •) rescued this slow recovery while a kinase-inactivated ETR1 transgene (getr1-[HGG]; ▪) did not. Ethylene was introduced at 1 h (↓) and removed 2 h later (↑).

Figure 5.

The slow growth recovery phenotype of plants deficient in receiver domain receptor isoforms was rescued by a wild-type ETR1 transgene but rescued poorly by a phosphotransfer-inactivated ETR1 mutant. Transformation of the etr1-6;etr2-3;ein4-4 triple mutant with genomic ETR1 (gETR1; •) results in rescue of the slow recovery phenotype. However, transformation with an ETR1 transgene mutated at Asp-659 (getr1-[D]; ▪) rescued the slow recovery phenotype poorly. The responses of wild type (dashed line) and the triple mutant (gray line) are shown for comparison. Ethylene was introduced at 1 h (↓) and removed 2 h later (↑).

Figure 6.

Receptor-clustering model for recovery of growth after ethylene is removed. Based on the negative regulator model for ethylene signaling, ethylene treatment should result in receptors almost exclusively in the ethylene-bound, inactive state, leading to an inactive CTR1. This releases EIN2 from inhibition and leads to growth inhibition in etiolated seedlings. When ethylene is removed, recovery from growth inhibition should involve an increase in receptors in the active signaling state. Accumulation of active receptors can result directly from dissociation of ethylene from existing ethylene-bound receptors or synthesis of new unbound receptors. To account for the rapidity of growth recovery after ethylene withdrawal, a receptor interaction model is proposed in which unbound active receptors can alter the signaling status of neighboring ethylene-bound receptors as denoted by the arrows (→), resulting in an amplification of overall receptor signal output. Inactive proteins are dark gray, active proteins light gray, bound receptors shown by the presence of ethylene on the receptor, and unbound receptors shown by the absence of ethylene on the receptor.