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First published online October 1, 2004; 10.1104/pp.104.050369 Plant Physiology 136:2913-2920 (2004) © 2004 American Society of Plant Biologists Arabidopsis Seedling Growth Response and Recovery to Ethylene. A Kinetic Analysis1Department of Botany, University of Wisconsin, Madison, Wisconsin 53706
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.
Ethylene regulates a number of developmental processes in higher plants, including growth in etiolated seedlings. Inhibition of growth in etiolated seedlings by ethylene is a convenient and useful bioassay that has been used to quantify the dose-response characteristics of ethylene (Chen and Bleecker, 1995  ) and, in mutant screens, to identify components in the ethylene signal transduction pathway (Bleecker et al., 1988; Guzman and Ecker, 1989). Mutational analysis of the ethylene signaling pathway has led to an increasingly refined model for signaling (Guo and Ecker, 2004). According to this model, responses to ethylene are mediated by a family of five receptors in Arabidopsis that are related to bacterial two-component receptors (Chang et al., 1993; Hua et al., 1998; Hua and Meyerowitz, 1998; Sakai et al., 1998). The ethylene receptors are thought to transduce signal via Ser/Thr kinase activity in CTR1 (Kieber et al., 1993; Huang et al., 2003). CTR1 may negatively regulate the ethylene response pathway by inhibiting activity of an Nramp-related protein, EIN2, which is required for responses to ethylene (Alonso et al., 1999). Ethylene binding to the receptors reduces the activity of the receptors, leading to reduced activity of CTR1 protein and an increase in activity of EIN2 protein along with subsequent signaling associated with it. At least some responses to ethylene, including the seedling growth response, are mediated by activation of a transcriptional cascade (Solano et al., 1998), suggesting that ethylene responses are mediated by differential gene activation and inactivation. In support of this, a number of genes have been shown to be ethylene responsive (Schenk et al., 2000; Van Zhong and Burns, 2003), including the genes for the ERS1, ERS2, and ETR2 ethylene receptors (Hua et al., 1998).
The ethylene receptors can be divided into two subfamilies. Subfamily I consists of ETR1 and ERS1, which contain all amino acid residues thought to be needed for His-kinase activity (Chang et al., 1993
Despite our growing understanding of the ethylene signal transduction pathway, questions remain as to the role(s) of each receptor isoform in the control of etiolated seedling growth by ethylene. Previous kinetic studies indicate that ethylene rapidly inhibits the growth rate of etiolated pea (Pisum sativum) seedlings within 10 min and seedlings return to pretreatment growth within 20 min after ethylene is removed (Warner and Leopold, 1971
Growth Inhibition and Recovery Kinetics Short-term changes in growth rates of etiolated Arabidopsis seedlings were obtained using 2-d-old seedlings growing along the surface of a vertical agar plate. Under the conditions used in this study, we found that the growth rate of wild-type and mutant hypocotyls in air prior to ethylene treatment ranged from 0.17 to 0.42 mm h–1 (Table I). Control experiments carried out in air showed that hypocotyls had linear growth for at least 12 h under the conditions used in these experiments (data not shown).
Figure 1 shows typical growth responses of etiolated wild-type Arabidopsis (Columbia) hypocotyls to a treatment with 10 µL L–1 ethylene. Addition of exogenous ethylene caused the growth rate to decrease within 15 min, reaching a new steady-state growth rate approximately 75 min after ethylene was added. Closer examination of the response kinetics showed that there appears to be at least two phases to growth inhibition by ethylene. The first, rapid deceleration phase had a lag of approximately 15 min after ethylene was applied, lasted approximately 15 min, and resulted in a steady-state growth rate of 0.13 mm h–1. Following this, growth rate was stable for approximately 20 min before a second, slower phase of growth inhibition ensued. The rate of change for this slower phase was approximately one-sixth the rate of change observed with the onset of the first phase. This lasted another 20 min until the growth rate reached the new steady-state rate of 0.03 mm h–1. Growth rate was suppressed to this low level until ethylene was removed.
Following the withdrawal of ethylene from the treatment chamber, hypocotyls that had been growing in the presence of ethylene for 2 h began to recover, attaining pretreatment growth rates approximately 90 min later (Fig. 1). It is interesting to note that the time needed for growth recovery was much faster than the time required for dissociation of ethylene to occur from yeast-expressed ETR1 or ETR2 receptors (Schaller and Bleecker, 1995 ; F.I. Rodriguez, unpublished data).
To study the role(s) of specific receptors in response to and recovery from ethylene, we examined growth-rate kinetics in various ethylene receptor null mutants. Of all the single null mutations tested, the etr1-7 null mutant showed the strongest phenotype. Figure 2 shows that this mutant took significantly longer to recover from ethylene-induced growth inhibition following a 2-h ethylene treatment. This longer recovery time was also noted for shorter (30-min) ethylene treatment (data not shown). Following ethylene removal, the etr1-7 mutant required more than 2.5 h to return to initial growth rates compared to 1.5 h for wild-type seedlings (Fig. 2A). Both the etr2-3 (Fig. 2B) and ein4-4 (Fig. 2C) mutants also displayed a slowed growth-rate recovery, each needing slightly over 2 h. Seedlings which contained only the ERS1 and ERS2 ethylene receptors (etr1-6;etr2-3;ein4-4 triple mutants) showed a very slow recovery of approximately 4 h (Fig. 2D). In general, the main effect of these receptor null mutations on the growth-rate response profile appeared to be a delay in the onset of measurable growth recovery. The exception to this was the etr2-3 mutant where the lag time and the rate of growth recovery contributed equally to the slower recovery to pretreatment growth rates. Growth-rate recovery following ethylene treatment was unaltered in ers1-2 and ers2-3 mutants, as well as the ers1-2;ers2-3 double null mutant (Fig. 2E). Thus, these two receptor isoforms that lack a receiver domain do not appear to contribute to the rapid recovery of seedlings from ethylene treatments.
We have previously found that receptor transcript levels correlate with ethylene binding (R.C. O'Malley and J. Esch, unpublished data). We used quantitative reverse transcription (RT)-PCR to measure the mRNA levels of each receptor isoform in etiolated seedlings treated with ethylene for various amounts of time to examine whether the altered growth-rate response profiles observed for the various receptor null mutants correlated with any altered expression of receptor isoform genes. Results from this survey are shown in Figure 3.
In air prior to ethylene treatment, the ETR1 transcript comprised approximately 50% and ERS1 approximately 37% of total receptor transcript levels. The other 3 receptor isoforms each comprised 6% or less of total receptor transcript levels. Upon treatment with 10 µL L–1 ethylene, transcript levels for each receptor isoform remained constant for the first 30 min, but after 2 h in the presence of ethylene the mRNA for ERS1, ERS2, and ETR2 showed large increases of approximately 3.8-, 6.6-, and 14.3-fold, respectively (Fig. 3). The mRNA levels of ETR1 and EIN4 remained constant in the presence of ethylene as reported previously (Chang et al., 1993 ; Hua et al., 1998). At this point, mRNA for ERS1 comprised approximately 54%, ETR1 approximately 20%, and ERS2 approximately 14% of total receptor transcript. The other 2 receptor isoforms each comprised 7% or less of total transcript levels (Fig. 3). The lack of contribution to recovery by ERS1, despite its high level of expression at the time of recovery in these experiments, further highlights a special role for receiver domain-containing receptor isoforms in the recovery process.
In a previous study, it was shown that a kinase-deficient mutant of ETR1 could rescue the etr1;ers1 double mutant for a number of ethylene responses, indicating that canonical His-kinase activity was not essential for signaling by the receptor (Wang et al., 2003 The double loss-of-function mutant, ers1-2;etr1-7, had slow growth recovery (Fig. 4). However, its recovery was faster than that observed in the etr1-7 mutant. This faster-than-expected recovery in the ers1-2;etr1-7 mutant plants is probably due to our observation that ETR2 transcript levels are elevated in the double mutant plants but not the etr1-7 mutants (data not shown). Transformation of the double mutant with wild-type genomic ETR1 (gETR1) rescued the slow growth recovery phenotype. However, when transformed with a kinase-inactivated ETR1 genomic clone (getr1-[HGG]), the slow growth recovery phenotype was not rescued, supporting a specific role for His-kinase activity in growth recovery. To determine whether phosphotransfer to the receiver domain is required for normal growth recovery, we used plants deficient in receptor isoforms containing receiver domains. When etr1-6;etr2-3;ein4-4 triple loss-of-function mutant seedlings were transformed with genomic ETR1 (gETR1), the slow recovery phenotype was rescued (Fig. 5). This was seen in two independent plant lines. As would be predicted, the time for recovery in these transformants was similar to recovery seen in either the etr2-3 or the ein4-4 single loss-of-function mutants. A mutant ETR1 lacking the conserved Asp-659, getr1-[D], transformed into the triple null seedlings showed only a partial rescue of the recovery phenotype (Fig. 5). This result was seen in three independent transgenic plant lines. This reduced rescue by the getr1-[D] transgene could be due to lower expression levels of getr1-[D] compared to gETR1. However, using end-point analysis, growth in air of the etr1-6;etr2-3;ein4-4 triple loss-of-function mutant seedlings was rescued equally well by either the gETR1 or getr1-[D] transgene (data not shown), suggesting that both transgenes are functioning equally well in this context.
In this study, we examined the process of Arabidopsis hypocotyl growth inhibition and recovery in response to ethylene treatment and removal, respectively. This kinetic analysis uncovered details of these processes that would have been unavailable through the use of long-term ethylene treatments and end-point growth analysis. Kinetic analysis of the ethylene response has found that growth inhibition occurs in two distinct phases. An initial rapid phase occurs shortly after the onset of ethylene treatment and is then supplanted by a more prolonged phase of stronger growth inhibition. None of the receptor null mutations used in this study measurably altered the kinetics of this biphasic ethylene response. Previous studies showed that the ethylene receptors have redundant function in the continuing, long-term presence of air or ethylene (Hua et al., 1995 , 1998; Hua and Meyerowitz, 1998; Hall and Bleecker, 2003) but may also have unique functions or properties (Hall and Bleecker, 2003; Wang et al., 2003). Results from this study showed that receptors have redundant function during the initial responses to ethylene since no single, receptor null mutation altered responses to ethylene. Differences in the ethylene growth response that occurred as a consequence of deficiencies in specific receptor isoforms were clearly exposed during the recovery phase after ethylene was purged from the treatment chamber. Here, there appears to be a transition where ETR1, ETR2, and EIN4 are more important for rapid recovery of growth than ERS1 or ERS2. The most prominent structural difference between these two groups of receptors is that ETR1, ETR2, and EIN4 each contain a receiver domain. While loss-of-function mutations in these three receptors all caused a delay in growth recovery, the largest delay was noted for etr1-7. This correlated with the observation that of these three isoforms, ETR1 mRNA accounted for approximately 85% of the transcript in etiolated seedlings in air and approximately 65% of the transcript after 2 h in ethylene, when ethylene withdrawal was initiated. The delay in growth recovery in some receptor-deficient lines does not appear to simply reflect diminished growth rates in air. For instance, the largest delay in recovery for single loss-of-function mutants was observed in the etr1-7 mutants, yet this mutation had no measurable effect on growth in air. Also, while plants with the etr1-6;etr2-3;ein4-4 triple loss-of-function mutation had slower growth in air and took longer to recover than any of the single mutants, the etr1-6;etr2-3;ein4-4 gETR-1 plants also had slow growth in air, yet had recovery comparable to etr2-3 or ein4-4 mutant plants.
The relatively rapid rate of recovery from ethylene treatment exhibited by etiolated seedlings must be reconciled with binding studies of the ethylene receptors indicating that the ETR family of receptors show very slow release kinetics for ethylene (half-life 10–12 h; Schaller and Bleecker, 1995
While both of these processes presumably contribute to a shift in the proportions of total receptors in the signaling and nonsignaling states, it should require a very rapid ethylene dissociation rate and/or an extremely rapid receptor turnover rate to significantly shift the equilibrium of total receptors from the bound inactive state to the unbound active state within 90 min of exogenous ethylene removal. However, existing evidence suggests that ethylene release occurs on a much longer time scale for the ETR family of receptors (Schaller and Bleecker, 1995
Positing a model in which receptors act cooperatively offers one way to reconcile the relatively slow changes in receptor occupancy with the more rapid changes in response output found in this study. Receptor-clustering models are currently being utilized to describe the behavior of the two-component bacterial chemoreceptors that are evolutionarily related to the ethylene receptors (Bray et al., 1998
The mechanisms by which His-kinase activity and phosphotransfer to the receiver domain might accelerate recovery are unknown. One possibility consistent with the proposed receptor cooperativity model is that transphosphorylation of receptors between receptor dimers in a cluster could favor the active (signaling) conformational state of the receptors. Imagine that receptors are normally in equilibrium between the active (unbound) and inactive (bound) conformational states with regard to downstream signaling. Unbound receptors could exist primarily in the active (signaling) state, while ethylene binding shifts the equilibrium to the inactive (nonsignaling) state. If receptor phosphorylation shifts the equilibrium of ethylene-bound receptors back to the active signaling state, the cooperative signaling proposed in the model would occur.
An alternative role for receiver domains in growth recovery could involve the rate at which newly synthesized receptors are incorporated into active signaling complexes. All five receptor isoforms form homodimers (Schaller et al., 1995
L-  -(2-Ammino ethoxyvinyl)-Gly was kindly supplied by Dr. Tarlochan S. Dhadialla at Rohm Haas (Philadelphia). Mutants were in the Columbia background except for ers1-2 and ers2-3, which were in the Wassilewskija (WS) background.
Arabidopsis seeds were surface sterilized by treatment with 70% alcohol for 30 s, placed on sterile filter paper to dry, and then placed on agar plates containing half-strength Murashige and Skoog basal salt mixture, pH 5.7 (Murashige and Skoog, 1962
Seedlings were allowed to grow in darkness to a height of 3 to 4 mm (42–46 h) before the beginning of growth-rate measurements. The agar plates were fitted with a lid that allowed for continuous gas flow and placed vertically in a holder mounted on a micromanipulator. These manipulations were done in the dark or under dim, green light. Following 1 h of treatment with air to establish a basal growth rate, ethylene was introduced at a flow of 10 mL min–1. Overall gas flow was maintained at 100 mL min–1 throughout the experiment using Side-Trak mass flow meters and controller (Sierra Instruments, Bolsuen, The Netherlands). Gas chromatography using a Carboxen 1000, 45/60-mesh size column (Supelco, Bellefonte, PA) was used to measure the ethylene concentration in the effluent from the chamber. Under these conditions it took 4 min for the chamber to equilibrate to a steady-state concentration of 10.6 ± 0.6 µL L–1, and it took approximately 6 min for ethylene to dissipate to nondetectable levels after the ethylene flow was turned off.
Hypocotyl growth rates were measured in darkness using infrared radiation, an electronic camera, and custom software as previously described (Parks and Spalding, 1999
Genomic DNA was purified from 1 g of 5-d-old seedlings using the 10 columns of the DNeasy kit (Qiagen, Valencia, CA) and recombining all the samples. DNA concentration was determined by gel electrophoresis with ethidium bromide and confirmed by fluorometry using Hoechst reagent (Sigma-Aldrich, St. Louis). Size determination by gel electrophoresis indicated that the size of the genomic DNA was typically 15 to 10 kb.
Seedlings were grown in the absence or presence of ethylene under the conditions used for growth-rate measurements. To facilitate rapid harvest of seedlings, a piece of sterile, Whatman Number 2 filter paper (Clifton, NJ) was placed on the agar plate, and seeds were plated on top of this paper. At the end of air or ethylene treatment, seedlings were rapidly harvested by scraping the paper with a razor blade and placing the seedlings into liquid N2. Total RNA was extracted from 50 to 100 mg of frozen Arabidopsis seedlings with the Qiagen RNeasy kit. The quantity of total RNA was determined by UV spectrometry. Subsequent denaturing PAGE analysis confirmed the concentration and provided a qualitative check of RNA integrity. A total of 1.0 µg of Arabidopsis RNA from seedlings was digested with 1 unit of µg–1 DNase (Invitrogen, Carlsbad, CA) and used to prepare cDNA by RT (Superscript II; Invitrogen) with 500 nM oligo(dT)18 + oligo(N)3 in a 20-µL reaction with a 50-min RT step. The cDNA samples from treated and untreated seedlings were diluted 5-fold to 100 µL final volume and split into three equal 33.3-µL aliquots. A genomic DNA 4-fold dilution series of 40 x 103, 10 x 103, and 2.5 x 103 molecules/µL was prepared, and 33.3 µL of each genomic DNA dilution was added to one of the cDNA aliquots.
For each receptor isoform, the gene-specific primers spanned the last intron and were designed to generate cDNA and genomic PCR products that differed in size between 12% and 16%. The sense primers were designed to include approximately 700 bp of the 3'end of the mRNA, which ensures that all the primers are measuring cDNA that are greater than 700 bp in length.
The cDNA and genomic DNA mixtures were amplified for 26 cycles using PCR (45 s at 94°C, 60 s at 55°C, 70 s at 72°C, and a final 7 min at 72°C for extension) with specific primers for each of the 5 ethylene receptors. The products were separated on a 1% agarose gel (100 V, 45 min) containing ethidium bromide. Products were visualized with UV illumination, photographed with a digital camera (Kodak DC120; Eastman-Kodak, Rochester, NY), and quantified with ImageQuant software (Molecular Dynamics, Sunnyvale, CA). Analysis of the data was performed as described previously (Pfaffl et al., 1998
The genomic ETR1 was previously cloned into pBluescript II SK– (Wang et al., 2003
We thank Matthew Touton for technical assistance. Received July 21, 2004; returned for revision August 30, 2004; accepted August 31, 2004.
1 This work was supported by the National Science Foundation (grant no. MCB–0131564 to A.B.B.).  Article, publication date, and citation information can be found at www.plantphysiol.org/cgi/doi/10.1104/pp.104.050369. * Corresponding author; e-mail bleecker{at}wisc.edu; fax 608–262–7509.
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ABSTRACT
RESULTS
). Ethylene was removed 2 h later (
).
) 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 (
), 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.
-loop, which is located next to the conserved Asp-659. The 
