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. 2012 May 7;159(3):1263–1276. doi: 10.1104/pp.112.193979

Arabidopsis RTE1 Is Essential to Ethylene Receptor ETR1 Amino-Terminal Signaling Independent of CTR11,[C]

Liping Qiu 1,2, Fang Xie 1,2, Jing Yu 1, Chi-Kuang Wen 1,*
PMCID: PMC3387708  PMID: 22566492

Abstract

The Arabidopsis (Arabidopsis thaliana) ethylene receptor Ethylene Response1 (ETR1) can mediate the receptor signal output via its carboxyl terminus interacting with the amino (N) terminus of Constitutive Triple Response1 (CTR1) or via its N terminus (etr11-349 or the dominant ethylene-insensitive etr1-11-349) by an unknown mechanism. Given that CTR1 is essential to ethylene receptor signaling and that overexpression of Reversion To Ethylene Sensitivity1 (RTE1) promotes ETR1 N-terminal signaling, we evaluated the roles of CTR1 and RTE1 in ETR1 N-terminal signaling. The mutant phenotype of ctr1-1 and ctr1-2 was suppressed in part by the transgenes etr11-349 and etr1-11-349, with etr1-11-349 conferring ethylene insensitivity. Coexpression of 35S:RTE1 and etr11-349 conferred ethylene insensitivity in ctr1-1, whereas suppression of the ctr1-1 phenotype by etr11-349 was prevented by rte1-2. Thus, RTE1 was essential to ETR1 N-terminal signaling independent of the CTR1 pathway. An excess amount of the CTR1 N terminus CTR17-560 prevented ethylene receptor signaling, and the CTR17-560 overexpressor CTR1-Nox showed a constitutive ethylene response phenotype. Expression of the ETR1 N terminus suppressed the CTR1-Nox phenotype. etr11-349 restored the ethylene insensitivity conferred by dominant receptor mutant alleles in the ctr1-1 background. Therefore, ETR1 N-terminal signaling was not mediated by full-length ethylene receptors; rather, full-length ethylene receptors acted cooperatively with the ETR1 N terminus to mediate the receptor signal independent of CTR1. ETR1 N-terminal signaling may involve RTE1, receptor cooperation, and negative regulation by the ETR1 carboxyl terminus.

The gaseous plant hormone ethylene is perceived by a small family of ethylene receptors. Arabidopsis (Arabidopsis thaliana) has five ethylene receptors that are structurally similar to prokaryotic two-component histidine kinase (HK) proteins. Mutants defective in multiple ethylene receptor genes show a constitutive ethylene response phenotype, which indicates a negative regulation of ethylene responses by the receptor genes (Hua and Meyerowitz, 1998).

The receptor N terminus has three or four transmembrane domains that bind ethylene. The GAF (for cGMP-specific phosphodiesterases, adenylyl cyclases, and FhlA) domain, which follows the transmembrane helices, mediates noncovalent receptor heterodimerization and may have a role in receptor cooperation (Gamble et al., 2002; O’Malley et al., 2005; Xie et al., 2006; Gao et al., 2008). The subfamily I receptors Ethylene Response1 (ETR1) and Ethylene Response Sensor1 (ERS1) have a conserved HK domain following the GAF domain. For subfamily II members ETR2, Ethylene Insensitive4 (EIN4), and ERS2, the HK domain is less conserved, and they lack most signature motifs essential for HK activity (Chang et al., 1993; Gamble et al., 1998; Hua et al., 1998; Qu and Schaller, 2004; Xie et al., 2006). Among the five receptors, ETR1, ETR2, and EIN4 have a receiver domain following the HK domain. The ETR1 HK domain may have a role in mediating the receptor signal to downstream components, and the HK activity facilitates the ethylene signaling (Clark et al., 1998; Huang et al., 2003; Hall et al., 2012). The receiver domain can dimerize and could involve receptor cooperation (Müller-Dieckmann et al., 1999). However, differential receptor cooperation occurs between the receiver domain-lacking ERS1 and the other ethylene receptors, which does not support the hypothesis that the domains involve receptor cooperation (Liu and Wen, 2012).

Acting downstream of the ethylene receptors is Constitutive Triple Response1 (CTR1), a MEK kinase (mitogen-activated protein kinase kinase kinase) with Ser/Thr kinase activity, and the kinase domain locates at the C terminus. The CTR1 N terminus does not share sequence similarity to known domains and can physically interact with the ethylene-receptor HK domain (Clark et al., 1998; Huang et al., 2003). ctr1 mutants showing attenuated CTR1 kinase activity or the ETR1-CTR1 association exhibit various degrees of the constitutive ethylene-response phenotype. For example, the ctr1-1 and ctr1btk mutations result from the D694E and E626K substitutions, respectively, in the CTR1 kinase domain, and ctr1-1 shows a stronger ethylene-response phenotype than ctr1btk, with ctr1-1 having much weaker kinase activity than ctr1btk (Kieber et al., 1993; Huang et al., 2003; Ikeda et al., 2009). The ctr1-8 mutation results in the G354E substitution that prevents the ETR1-CTR1 association, and the mutant exhibits a constitutive ethylene-response phenotype. Overexpression of the CTR1 N terminus CTR17-560, which is responsible for interaction with ethylene receptors, leads to constitutive ethylene responses, possibly by titrating out available ethylene receptors (Kieber et al., 1993; Huang et al., 2003). These studies suggest that CTR1 kinase activity and the interaction of CTR1 with the receptor HK domain may be important to the ethylene receptor signal output in suppressing constitutive ethylene responses.

Although the ETR1-CTR1 interaction via the HK domain is essential to the ethylene receptor signal output, evidence suggests that the ETR1 receptor signal output can also be independent of the HK activity or domain. The etr1 ers1 loss-of-function mutant displays extreme growth defects. The etr1[HGG] mutation inactivates ETR1 HK activity, and expression of the getr1[HGG] transgene rescues the etr1 ers1 growth defects, which indicates a lack of association of ETR1 receptor signaling and its kinase activity (Wang et al., 2003). The dominant etr1-1 mutation results in the C65Y substitution and confers ethylene insensitivity (Chang et al., 1993), and the expression of the HK domain-lacking etr11-349 and ethylene-insensitive etr1-11-349 isoforms partially suppresses the growth defects of etr1 ers1-2. Loss-of-function mutations of subfamily II members do not affect etr1-11-349 functions. Therefore, etr1-11-349 predominantly cooperates with subfamily I receptors to mediate the ethylene receptor signal output (Xie et al., 2006). Biochemical and transformation studies showing that ethylene receptors can form heterodimers and that each receptor is a component of high-molecular-mass complexes explain how ethylene receptors may act cooperatively (Gao et al., 2008; Gao and Schaller, 2009; Chen et al., 2010).

Reversion To Ethylene Sensitivity1 (RTE1), a Golgi/endoplasmic reticulum protein, was isolated from a suppressor screen of the dominant ethylene-insensitive etr1-2 mutation. The cross-species complementation of the rte1-2 loss-of-function mutation by the rice (Oryza sativa) RTE Homolog1 (OsRTH1) suggests a conserved mechanism that modulates the ethylene receptor signaling across higher plant species (Zhang et al., 2012). RTE1 and OsRTH1 overexpression led to ethylene insensitivity in wild-type Arabidopsis but not the etr1-7 loss-of-function mutant, and expression of etr11-349 restored ethylene insensitivity with RTE1 overexpression in etr1-7 (Resnick et al., 2006; Zhou et al., 2007; Zhang et al., 2010). Coimmunoprecipitation of epitope-tagged ETR1 and RTE1 and Trp fluorescence spectroscopy revealed the physical interaction of RTE1 and ETR1 (Zhou et al., 2007; Dong et al., 2008, 2010). Therefore, RTE1 may directly promote ETR1 receptor signal output through the ETR1 N terminus, but whether RTE1 has an essential role in ETR1 N-terminal signaling remains to be addressed.

Currently, the biochemical nature of the ethylene receptor signal is unknown, and the underlying mechanisms of mediation of the ethylene receptor signal output remain uninvestigated. Genetic and biochemical studies suggest that activation of CTR1 by ethylene receptors may suppress constitutive ethylene responses; upon ethylene binding, the receptors are converted to an inactive state and fail to activate CTR1, and the suppression of ethylene responses by CTR1 is alleviated (Hua and Meyerowitz, 1998; Klee, 2004; Wang et al., 2006; Hall et al., 2007). However, this model does not address how the ETR1 N terminus, which does not have the CTR1-interacting site, mediates the receptor signal to suppress constitutive ethylene responses. The receptor signal of the truncated etr1 isoforms may be mediated by other full-length ethylene receptors and then activate CTR1; alternatively, the ETR1 N-terminal signal may be mediated by a pathway independent of CTR1 (Gamble et al., 2002; Qu and Schaller, 2004; Xie et al., 2006). Results showing that mutants defective in multiple ethylene receptor genes exhibit a more severe ethylene-response phenotype than ctr1 and that ctr1 mutants are responsive to ethylene support the presence of a CTR1-independent pathway (Hua and Meyerowitz, 1998; Cancel and Larsen, 2002; Huang et al., 2003; Liu et al., 2010).

In this study, we investigated whether mediation of ETR1 N-terminal signaling is independent of CTR1 and whether RTE1 is essential to the CTR1-independent ETR1 N-terminal signaling. The ETR1 N-terminal signaling was not mediated via other full-length ethylene receptors, but the signal of full-length ethylene receptors could be mediated by the ETR1 N terminus independent of CTR1. The ETR1 C terminus may inhibit ETR1 N-terminal signaling, whereby deletion of the C terminus facilitates N-terminal signaling. We propose a model for the possible modulation of ETR1 receptor signaling.

RESULTS

Expression of etr11-349 or etr1-11-349 Partly Suppresses the Constitutive Ethylene Response Phenotype of ctr1-1

To investigate whether the ETR1 N terminus can mediate the ethylene receptor signal to constitutively suppress ethylene responses in the absence of CTR1, we expressed transgenes encoding etr11-349 and the ethylene-insensitive etr1-11-349 in the ctr1-1 loss-of function mutant. If ETR1 N-terminal signaling was mediated independent of CTR1, expression of the transgenes might suppress the constitutive ethylene response in ctr1-1. The dominant nature of etr1-11-349 can facilitate the study of ETR1 N-terminal signaling in ctr1-1. Given that the etr1-1 mutation confers a gain of function, we examined the effects of etr11-349 expression on the ctr1-1 mutant phenotype to address ETR1 N-terminal signaling in the absence of CTR1.

The structures and domains of the etr1 and ctr1 isoforms are shown in Figure 1A. ctr1-1 lines that expressed the ETR1p:etr1-11-349 or ETR1p:etr11-349 transgene (designated etr1-11-349 and etr11-349, respectively) were obtained by transformation or genetic crossing. Here, Lx refers to the line that was obtained from the genetic cross of ctr1-1 and the previously obtained etr1-7 ers1-2 lines that carry etr1-11-349 or etr11-349 (Xie et al., 2006).

Figure 1.

Figure 1.

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Expression of the ETR1 N terminus partially suppresses constitutive ethylene responses mediated by ctr1-1. A, Structure and mutations of ETR1 and CTR1 isoforms described in this study. TM, Transmembrane domain. B, Seedling hypocotyl length of ctr1-1 and ctr1-1 expressing etr1-11-349 or etr11-349. C and D, Phenotype (C) and hypocotyl length (D) of etiolated seedlings of ctr1-1 and etr1-1 ctr1-1. E, Rosette phenotype of ctr1-1 and etr1-1 ctr1-1. F and G, Phenotype of light-grown seedlings (F) and rosettes (G) of ctr1-1 and ctr1-1 expressing etr11-349 or etr1-11-349. H, ERF1 expression in ctr1-1 and ctr1-1 expressing the ETR1 N terminus. I and J, Measurement of the relative transgene expression for etr1-11-349 (I) and etr11-349 (J) of corresponding ctr1-1 transformation lines. etr11-349 and etr1-11-349 denote the etr11-349 and etr1-11-349 transgenes, respectively, that are driven by the native ETR1 promoter. L indicates the transformation lines used in this assay. Data are means ± sd (n ≥ 30). ** P < 0.01 by Student’s t test. [See online article for color version of this figure.]

As expected, dark-grown ctr1-1 seedlings showed a constitutive ethylene-response phenotype, with a short hypocotyl and root and an exaggerated apical hook in the absence of ethylene treatment. ctr1-1 was marginally responsive to ethylene, and ethylene-treated ctr1-1 seedlings had a shorter hypocotyl than air-grown seedlings (Fig. 1, B and C; Student’s t test, P < 0.01). Interestingly, the expression of the ETR1 N terminus rescued in part the ctr1-1 seedling growth defects. Air-grown, etiolated etr1-11-349 ctr1-1 and etr11-349 ctr1-1 seedlings had a longer seedling hypocotyl than ctr1-1 seedlings. Ethylene treatment inhibited the etiolated-seedling hypocotyl growth of etr11-349 ctr1-1 but not etr1-11-349 ctr1-1 (Fig. 1B; Fisher’s lsd, α = 0.01).

We examined whether the dominant ethylene-insensitive etr1-1 allele has the same effect as etr1-11-349 on suppression of the ctr1-1 mutant phenotype. The etr1-1 ctr1-1 seedlings phenotypically resembled but were slightly longer than ctr1-1 seedlings regardless of ethylene treatment (Student’s t test, P < 10−6; Fig. 1, C and D). Consistently, at the adult stage, ctr1-1 growth defects were slightly suppressed by etr1-1 (Fig. 1E).

Grown under light, etr1-11-349 had a greater effect than etr11-349 on suppressing the ctr1-1 seedling growth defects. Ethylene treatment inhibited the seedling growth of the wild type (ecotype Columbia [Col-0]), ctr1-1, and ctr1-1 lines expressing the etr11-349 transgene. As expected, etr1-11-349 ctr1-1 lines showed minor growth inhibition with ethylene treatment because etr1-11-349 expression conferred ethylene insensitivity (Fig. 1F). At the adult stage, ctr1-1 growth defects were substantially suppressed by etr1-11-349 and etr11-349 (Fig. 1G). These results suggested that the receptor signal output was mediated by etr11-349 in the absence of CTR1 in suppressing constitutive ethylene responses and was prevented by ethylene treatment. The etr1-11-349 signaling was independent of CTR1 and was not affected by ethylene because ethylene binding was prevented by the C65Y mutation (Wang et al., 2006).

Ethylene Response Factor1 (ERF1) is a primary target of the ethylene signal with expression induced by ethylene (Solano et al., 1998). ERF1 is thus ideal for measuring the degree of ethylene responsiveness. Quantitative reverse transcription (qRT)-PCR revealed ERF1 expression attenuated by about 60% in ctr1-1 lines that expressed etr1-11-349 or etr11-349 (Fig. 1H). These results suggest an association of growth recovery and suppression of the constitutive ethylene response in air-grown ctr1-1 plants by the transgenes.

The etr11-349 and etr1-11-349 transgenes were each introduced into ctr1-1 by genetic crossing or transformation, and their expression levels were measured to determine their effect on the suppression of the ctr1-1 mutant phenotype. qRT-PCR revealed the etr1-11-349 transgene to be expressed at a lower level in the ctr1-1 transformation line Lx than in the other lines (Fig. 1I). The expression of etr11-349 in the three transformation lines varied, but the difference was minor (±20% relative to that of line L10; Fig. 1J), which may explain why the etr1-11-349 ctr1-1 line Lx exhibited stronger growth inhibition than the other etr1-11-349 ctr1-1 lines at the seedling stage.

Because ctr1-1 did not completely suppress the effect of the etr1-1 mutation and ctr1-1 was slightly ethylene responsive, ctr1-1 might not be a null mutation; alternatively, the receptor signal might be mediated in part via an alternative pathway independent of CTR1. Our data show that the growth defects and the constitutive ethylene response caused by the ctr1-1 mutation were suppressed in part by expression of the ETR1 N terminus and that expression of etr1-11-349 but not etr11-349 conferred ethylene insensitivity in ctr1-1. These results support a model in which ETR1 N-terminal signaling is mediated independently of CTR1.

The Constitutive Ethylene Response of ctr1-2 Is Partly Suppressed by Transgenes Encoding the ETR1 N Terminus

The ctr1-1 mutation substantially weakens the Ser/Thr kinase activity and does not prevent ctr1-1 protein expression (Huang et al., 2003). ctr1-1 may not be a null mutation, so it may mediate a small amount of ethylene receptor signal output. The ctr1-2 mutation results in a 17-bp deletion in the CTR1 coding region, and ctr1-2 may encode a truncated protein of 462 residues that lacks the kinase domain (Huang et al., 2003; Fig. 1A). We investigated whether the constitutive ethylene response caused by the ctr1-2 mutation can be suppressed by expression of the ETR1 N terminus to determine whether ETR1 N-terminal signaling requires CTR1.

We introduced etr1-11-349 and etr11-349 transgenes into ctr1-2 by genetic crossing from line Lx so that the corresponding transgenes were expressed at the same locus in ctr1-1 and ctr1-2. Etiolated ctr1-2 seedlings had a short hypocotyl when grown in air and with ethylene treatment; notably, ethylene treatment slightly inhibited ctr1-2 seedling growth (Student’s t test, P < 0.01). Expression of etr11-349 or etr1-11-349 in part suppressed the ctr1-2 growth defects in air, and ctr1-2 seedlings with each transgene had longer hypocotyls than ctr1-2 seedlings alone (Fisher’s lsd, α = 0.01). As expected, ethylene treatment inhibited the seedling growth of etr11-349 ctr1-2 but not etr1-11-349 ctr1-2 (Fig. 2, A and B). Consistently, both etr1 isoforms suppressed the growth inhibition of light-grown ctr1-2 seedlings. etr11-349 ctr1-2 and etr1-11-349 ctr1-2 seedlings showed larger cotyledons and longer hypocotyls and roots than ctr1-2 when grown in air. As expected, ethylene treatment inhibited the seedling growth of ctr1-2 and etr11-349 ctr1-2 but not etr1-11-349 ctr1-2 (Fig. 2C). At the adult stage, ctr1-2 showed strong growth inhibition and the rosette was extremely small; rosettes were larger for both etr11-349 ctr1-2 and etr1-11-349 ctr1-2 than for ctr1-2 (Fig. 2D). ERF1 expression was attenuated by about 40% and 50% in ctr1-2 plants with etr11-349 and etr1-11-349, respectively (Fig. 2E).

Figure 2.

Figure 2.

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Expression of the ETR1 N terminus partially rescues the growth defects of ctr1-2. A and B, Phenotype (A) and hypocotyl measurement (B) of etiolated seedlings of ctr1-2 and ctr1-2 expressing etr11-349 or etr1-11-349. C and D, Phenotype of light-grown seedlings (C) and rosettes (D) of ctr1-2 and ctr1-2 expressing etr11-349 or etr1-11-349. E, Relative ERF1 expression level of ctr1-2 and ctr1-2 expressing the truncated etr1 isoforms. F, Relative transgene expression in the transgene donor (ctr1-1, Lx) and recipient (ctr1-2, Lx). Lx indicates the transformation lines. Data are means ± sd. ** P < 0.01 by Student’s t test (B and F) and Fisher’s lsd (E). [See online article for color version of this figure.]

The transgenes were introduced by genetic crossing into ctr1-2. qRT-PCR to determine whether the expression of the transgenes would vary in transgene donors (ctr1-1 lines) and recipients (ctr1-2 lines) revealed the expression of the transgenes to be slightly lower in ctr1-2 than in ctr1-1 (Fig. 2F; Student’s t test, P < 0.01). However, the reduction was minor (about 20%) and did not contradict our conclusion that expression of the truncated etr1 isoforms in part suppressed ctr1-2 growth inhibition and attenuated the constitutive ethylene response.

Our results were in agreement with the mediation of the ETR1 N-terminal signal output to suppress the constitutive ethylene response being independent of the wild-type CTR1 and ethylene binding to the ETR1 N terminus preventing the N-terminal receptor signal output. In addition, similar to ctr1-1, ctr1-2 was slightly ethylene responsive, which implied an alternative signal transduction pathway.

The Suppression of the ctr1 Phenotype by the ETR1 N Terminus Occurs Independently of Wild-Type ETR1

We showed that expression of etr11-349 or etr1-11-349 suppressed the constitutive ethylene-response phenotype of ctr1-1 and ctr1-2, both with the wild-type ETR1. Given that the full-length ETR1 and the truncated isoforms physically interact and may cooperate (Xie et al., 2006; Gao et al., 2008), we evaluated whether suppression of the ctr1 mutant phenotype resulted from coexpression of the wild-type and truncated ETR1 isoforms.

The etr1-7 mutation results in the W74stop substitution and is believed to be amorphic (Hua and Meyerowitz, 1998; Xie et al., 2006). The etr1-11-349 and etr11-349 transgenes were introduced into etr1-7 ctr1-1 and etr1-7 ctr1-2, respectively, by genetic crossing from the transgene donors as shown in Figures 1 and 2, so that each transgene was expressed at the same locus in the corresponding transgene donor and recipient. Transformation lines labeled with the same number carry the transgene from a common transgene donor. qRT-PCR revealed similar expression of the transgene from the same donor in different genotypes. One exception was that etr1-11-349 expression was slightly reduced (by 20%) in etr1-7 ctr1-1 etr1-11-349 line L6 (Fig. 3, A and B).

Figure 3.

Figure 3.

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Growth rescue of etr1 ctr1 mutants by the ETR1 N terminus. A and B, etr11-349 (A) and etr1-11-349 (B) expression in transgene donors (ctr1-1 and ctr1-2 lines) and recipients (etr1-7 ctr1-1 and etr1-7 ctr1-2 lines). C and D, Phenotype (C) and hypocotyl length (D) of etiolated etr1-7 ctr1 seedlings expressing the etr1-11-349 or etr11-349 transgene. E and F, Phenotype of light-grown seedlings (E) and rosettes (F) of mutants expressing the etr1-11-349 or etr11-349 transgene. G, Relative ERF1 expression level of transformation mutants. L indicates the transformation lines. Data are means ± sd. ** P < 0.01 by Student’s t test. [See online article for color version of this figure.]

The hypocotyl was shorter for ethylene-treated, etiolated etr1-7 ctr1-1 than air-grown seedlings (Fig. 3, C and D). The constitutive ethylene-response phenotype was stronger for dark-grown etr1-7 ctr1-1 than ctr1-1 seedlings, regardless of ethylene treatment (Figs. 1B and 3D). On the basis of the seedling triple-response phenotype and hypocotyl length, our data show that expression of etr1-11-349 or etr11-349 suppressed the growth inhibition of etr1-7 ctr1-1 and etr1-7 ctr1-2 to a large extent. As expected, ethylene treatment inhibited the hypocotyl elongation of etr1-7 ctr1-1 and etr1-7 ctr1-2 seedlings that expressed etr11-349 (Student’s t test, P < 0.01) but not those that expressed etr1-11-349 (Fig. 3, C and D).

Grown under light, seedlings of ctr1-1, ctr1-2, etr1-7 ctr1-1, and etr1-7 ctr1-2 showed severe growth inhibition, small and compact cotyledons, and short hypocotyls and primary roots. As expected, expression of etr1-11-349 or etr11-349 substantially suppressed the mutant growth defects, and the transformation mutants showed larger and more expanded cotyledons and longer seedling hypocotyls and roots (Fig. 3E). At the adult stage, etr1-7 ctr1-1 and etr1-7 ctr1-2 plants showed severe growth inhibition and small rosettes. Expression of etr1-11-349 or etr11-349 substantially suppressed the growth defects of the two mutants. Lack of the wild-type ETR1 seemed to have a minor effect on suppression of the ctr1 mutant phenotype by the transgenes, and ctr1-1 and ctr1-2 lines that expressed the ETR1 N terminus were slightly larger than corresponding ctr1 etr1-7 transformation lines (Fig. 3F).

We evaluated the degree of constitutive ethylene response in transformation mutants by measuring ERF1 expression. ERF1 levels were higher (greater than 5-fold) in air-grown ctr1-1, ctr1-2, etr1-7 ctr1-1, and etr1-7 ctr1-2 plants than in wild-type (Col-0) plants. ERF1 levels were slightly higher in ctr1 transformation lines (L10 and Lx) with etr11-349 than in corresponding etr1-7 ctr1 etr11-349 lines (Student’s t test, P < 0.01). In contrast, ERF1 expression was identical in ctr1 etr1-11-349 transformation lines (L6 and Lx) and corresponding etr1-7 ctr1 etr1-11-349 transformation lines (Student’s t test, P > 0.05; Fig. 3G).

The constitutive ethylene response of ctr1-1 and ctr1-2 suppressed by the ETR1 N terminus was not affected in the absence of ETR1. Given that the suppression of the constitutive ethylene response by etr1-11-349 predominantly depends on ETR1 and ERS1 and that the ethylene receptors cooperate differentially (Xie et al., 2006; Gao et al., 2008; Liu and Wen, 2012), the ETR1 N terminus may cooperate differentially with other ethylene receptors to mediate the receptor signal independent of CTR1.

RTE1 Is Essential for Suppression of the ctr1-1 Mutant Phenotype by etr11-349

RTE1 overexpression leads to ethylene insensitivity in wild-type (Col-0) plants but not in the etr1-7 loss-of-function mutant, and expression of the etr11-349 transgene restores the ethylene insensitivity conferred by RTE1 overexpression in etr1-7 (Resnick et al., 2006; Zhou et al., 2007). The physical association of ETR1 and RTE1 plays a role in regulating ETR1 signaling (Dong et al., 2010). These studies indicate that excess RTE1 promotes ETR1 N-terminal signaling but do not conclude whether RTE1 is essential for ETR1 N-terminal signaling. We wondered whether the RTE1-promoted ETR1 N-terminal signaling could also be independent of CTR1. We addressed these questions by examining whether expression of the ETR1 N terminus still confers ethylene insensitivity both in the ctr1-1 background when RTE1 is overexpressed and in the rte1-2 ctr1-1 loss-of-function mutant.

Air-grown etiolated ctr1-1 seedlings showed the constitutive ethylene-response phenotype and produced a short seedling hypocotyl. RTE1 overexpression (designated RTE1ox) did not alleviate the seedling growth inhibition of ctr1-1. In contrast, when 35S:RTE1 and etr11-349 were coexpressed, the constitutive ethylene-response phenotype was substantially suppressed in air-grown ctr1-1 seedlings and ethylene treatment did not inhibit seedling growth (Fig. 4A). Seedling hypocotyl length was the same as in the seedling triple-response assay, with the hypocotyl elongation of RTE1ox etr11-349 ctr1-1 seedlings unaffected by ethylene treatment (Student’s t test, P > 0.14; Fig. 4B).

Figure 4.

Figure 4.

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RTE1 is required for the rescue of ctr1-1 growth by etr11-349. A and B, Phenotype (A) and hypocotyl length (B) of etiolated ctr1-1 seedlings with or without the etr11-349 and 35S:RTE1 transgenes. C and D, Phenotype of light-grown ctr1-1 seedlings (C) and adult plants (D) with or without the etr11-349 and 35S:RTE1 transgenes. E and F, Phenotype (E) and seedling hypocotyl length (F) of ctr1-1, ctr1-1 rte1-2, and mutants with the etr11-349 transgene. G and H, Phenotype of light-grown seedlings (G) and rosettes (H) of ctr1-1, ctr1-1 rte1-2, and mutants with the etr11-349 transgene. I to K, Relative expression of ERF1 (I), CHIB (J), and PDF1.2 (K) in ctr1-1 with or without etr11-349 and 35S:RTE1 transgenes. L, Relative expression of the etr11-349 transgene in etr11-349 ctr1-1 and RTE1ox etr11-349 ctr1-1. L10, The etr11-349 transgene was from the transformation line etr11-349ctr1-1 L10 by genetic cross. Data are means ± sd. ** P < 0.01 by Student’s t test (B) and Fisher’s lsd (G–I). [See online article for color version of this figure.]

Light-grown ctr1-1 seedlings showed a constitutive ethylene-response phenotype in air: the cotyledons were small, and the seedling hypocotyls and roots were short. The ctr1-1 seedling growth defects were substantially suppressed by the etr11-349 transgene: etr11-349 ctr1-1 (L10) showed larger and more expanded cotyledons, with longer seedling hypocotyls and roots than ctr1-1. As expected, RTE1 overexpression had little effect on ctr1-1 seedling growth but substantially promoted the growth of etr11-349 ctr1-1 (L10) seedlings, which had much larger cotyledons and a longer hypocotyl and root than etr11-349 ctr1-1 (L10) seedlings (Fig. 4C).

Grown to the adult stage, the ctr1-1 mutant phenotype was in part suppressed by the etr11-349 transgene: rosettes were larger for etr11-349 ctr1-1 (L10) than for ctr1-1 plants. As expected, RTE1 overexpression did not affect ctr1-1 plant growth but substantially promoted the growth of etr11-349 ctr1-1 (L10) plants, and rosettes were larger for RTE1ox etr11-349 ctr1-1 (L10) than for etr11-349 ctr1-1 (L10) plants (Fig. 4D).

Our data suggested that RTE1 overexpression promoted ETR1 N-terminal signaling in the absence of CTR1. We examined whether RTE1 is essential to the CTR1-independent ETR1 N-terminal signaling by examining whether the rte1-2 allele reverses the etr11-349 ctr1-1 phenotype. In the seedling triple-response assay, we showed that in the presence of the etr11-349 transgene, seedling hypocotyls were longer for ctr1-1 than for rte1-2 ctr1-1, and ethylene treatment inhibited in part the hypocotyl elongation (Fig. 4, E and F). Hypocotyls were slightly longer for ctr1-1 than for rte1-2 ctr1-1 seedlings in air and ethylene (Student’s t test, P < 0.01). Light-grown rte1-2 ctr1-1 seedlings showed severe growth inhibition and had much smaller cotyledons and shorter hypocotyls than ctr1-1 seedlings. Interestingly, the ctr1-1 growth defects suppressed by etr11-349 were prevented by the rte1-2 allele (Fig. 4G). Grown to the adult stage, rosettes were smaller for rte1-2 etr11-349 ctr1-1 than for ctr1-1 plants. As expected, rosettes were smaller for rte1-2 ctr1-1 than for ctr1-1 (Fig. 4H).

We quantitatively evaluated the degree of the constitutive ethylene response of ctr1-1 altered by the expression of 35S:RTE1 and etr11-349. With the ERF1 expression of ethylene-treated ctr1-1 plants set to 1, the level of ERF1 was not altered in RTE1ox ctr1-1, whereas that of etr11-349 RTE1ox ctr1-1 was substantially attenuated (Fisher’s lsd, α = 0.01; Fig. 4I). We next examined the expression of CHIB and PDF1.2, whose expression is elevated by ethylene treatment (Hua and Meyerowitz, 1998). As expected, the levels of both CHIB and PDF1.2 in ctr1-1 were similar to that in RTE1ox ctr1-1 and were highly attenuated in etr11-349 RTE1ox ctr1-1 (Fisher’s lsd, α = 0.01; Fig. 4, J and K). qRT-PCR revealed the expression of the etr11-349 transgene to be identical in the transgene donor (etr11-349 ctr1-1, L10) and the recipient (RTE1ox etr11-349 ctr1-1; Student’s t test, P > 0.25; Fig. 4L).

Our data here indicate that RTE1 was essential to the CTR1-independent ETR1 N-terminal signaling, which suppresses constitutive ethylene responses. The stronger growth defects of etr1-7 ctr1-1 and rte1-2 ctr1-1 than of ctr1-1 agree with the suggestion that RTE1 promotes a portion of ETR1 signaling that is independent of CTR1, so that the lack of ETR1 or RTE1 will strengthen the ctr1-1 mutant phenotype.

The Constitutive Ethylene-Response Phenotype of CTR1-Nox Is Suppressed by etr11-349 and etr1-11-349

Our results suggested that mediation of ETR1 N-terminal signaling is independent of CTR1. Given that the ethylene receptors act cooperatively and that the ETR1 N-terminal signaling primarily depends on ETR1 and ERS1, ETR1 N-terminal signaling to full-length ethylene receptors may be mediated by a pathway independent of CTR1. Alternatively, the signal output of full-length ethylene receptors may be cooperatively mediated to the ETR1 N terminus via a CTR1-indpendent pathway (Fig. 5A; Gamble et al., 2002; Xie et al., 2006; Gao et al., 2008; Gao and Schaller, 2009; Chen et al., 2010; Liu and Wen, 2012). Given that excess CTR17-560 prevents ethylene receptor signaling (Huang et al., 2003), if the constitutive ethylene-response phenotype of CTR1-Nox is suppressed by expression of the ETR1 N terminus, ETR1 N-terminal signaling may not be mediated via full-length ethylene receptors.

Figure 5.

Figure 5.

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Expression of the ETR1 N terminus suppresses the CTR1-Nox constitutive ethylene response. A, Scenarios of the ETR1 N-terminal signal mediated by full-length ethylene receptors (black arrows) or full-length ethylene receptor signals mediated by the ETR1 N terminus (red arrows) by an alternative pathway when excess CTR17-560 prevents ethylene receptor signaling. B to D, Phenotype (B), hypocotyl length (C), and seedling growth (D) of wild type (Col-0) plants expressing 35S:CTR17-560 (designated CTR1-Nox). E, Immunoassay of expression of the CTR17-560 fragment in CTR1-Nox lines. F to H, Hypocotyl length (F), seedling growth (G), and rosette phenotype (H) of CTR1-Nox L50 expressing etr1-11-349 or etr11-349. I, Relative ERF1 expression in CTR1-Nox and CTR1-Nox expressing the ETR1 N terminus. J, Relative transgene expression in the transgene donor (Col-0) and recipient (CTR1-Nox). K, Immunoassay of CTR17-560 expression. L6 and L10 indicate the origins of the etr1-11-349 and etr11-349 transformation lines, respectively. CHEM, The pseudocolor bar indicates relative chemiluminescence strength from weak (dark) to strong (bright); CTR1-Ab, chemiluminescence detected by the monoclonal CTR1 antibody; Immunoblot, staining with Coomassie blue to show relative protein amount; ND, not determined. Data are means ± sd. ** P < 0.01 by Student’s t test. [See online article for color version of this figure.]

We transformed 35S:CTR17-560 into the wild type (Col-0) and selected three transformation lines (designated CTR1-Nox) representing three categories of constitutive ethylene response. Seedling triple-response phenotype and hypocotyl length data showed CTR1-Nox (L50) with the strongest constitutive ethylene-response phenotype and L63 with the weakest (Fig. 5, B and C). Consistently, growth was inhibited in light-grown seedlings of the three lines, which showed small cotyledons and short hypocotyls and roots, with L50 showing the greatest growth inhibition and L63 the least (Fig. 5D). Immunoassay revealed an association of the expression of the CTR17-560 fragment and the degree of the constitutive ethylene-response phenotype, with L50 showing the highest CTR17-560 expression and L63 the least (Fig. 5E).

We introduced the etr1-11-349 and etr11-349 transgenes from transgene donors L6 and L10, respectively, by genetic crossing (Fig. 1), into CTR1-Nox (L50). As expected, the seedling hypocotyl length of CTR1-Nox (L50) was longer with than without the transgenes, which indicates that the growth inhibition of CTR1-Nox (L50) was prevented in part by each transgene, with etr1-11-349 having a greater effect than etr11-349. Ethylene treatment did not affect the growth of etr1-11-349 CTR1-Nox, and the hypocotyl lengths of air- and ethylene-grown etr1-11-349 CTR1-Nox (L50) seedlings were identical (Student’s t test, P > 0.29). As expected, the elongation of etr11-349 CTR1-Nox (L50) seedlings was inhibited by ethylene treatment (Student’s t test, P < 0.01; Fig. 5F). Grown under light, CTR1-Nox (L50) seedlings showed severe growth inhibition and produced small cotyledons and short hypocotyls and roots. The expression of etr1-11-349 and etr11-349 substantially suppressed the growth defects: the CTR1-Nox seedlings carrying the transgene showed expanded cotyledons and produced long hypocotyls and roots (Fig. 5G). At the adult stage, the strong rosette growth inhibition of CTR1-Nox was substantially prevented by the expression of each transgene (Fig. 5H).

We evaluated the degree of CTR1-Nox (L50) ethylene response altered by the etr1 isoforms by measuring ERF1 expression. qRT-PCR revealed that the expression of etr1-11-349 and etr11-349 substantially attenuated ERF1 expression in CTR1-Nox (L50) by about 70% (Fig. 5I). As well, levels of transgenes were slightly lower in CTR1-Nox than in corresponding donors (Fig. 5J). Transgene attenuation of ERF1 expression and prevention of the constitutive ethylene-response phenotype of CTR1-Nox were unlikely to be affected by minor alterations in transgene expression. We used an immunoassay to examine whether the truncated etr1 isoform recovery of growth and attenuation of ERF1 expression in CTR1-Nox resulted from impaired CTR17-560 expression. CTR17-560 expression in the two transformation lines was not reduced as compared with that of the 35S:CTR17-560 donor [CTR1-Nox (L50); Fig. 5K].

The constitutive ethylene-response phenotype of CTR1-Nox was prevented in part by expression of the ETR1 N terminus, so the ETR1 N-terminal signaling was not mediated by full-length ethylene receptors. Rather, full-length ethylene receptor signaling likely occurs cooperatively via the ETR1 N terminus through a pathway independent of CTR1.

Expression of etr11-349 Suppresses the Constitutive Ethylene-Response Phenotype and Confers Ethylene Insensitivity in ctr1-1 Harboring an Ethylene-Insensitive Ethylene Receptor Gene

The signal output of full-length ethylene receptors via the ETR1 N terminus may be mediated by a pathway independent of CTR1 (Fig. 5A). Double mutants that carry ctr1-1 and a dominant ethylene-insensitive receptor gene show the constitutive ethylene-response phenotype. If expression of the ethylene-responsive etr11-349 restores the ethylene insensitivity conferred by the ethylene-insensitive receptor gene, the ETR1 N terminus should mediate the signal output of full-length ethylene receptors by a pathway independent of CTR1.

etr1-1, ers1-1, etr2-1, ein4-1, and ers2-1 are dominant ethylene receptor gene mutations, and each confers ethylene insensitivity. Ethylene treatment had little effect on their seedling growth, as seen by seedling hypocotyl length (Fig. 6A). Consistent with previous genetic analyses, the ctr1-1 mutation suppressed each of the dominant, ethylene-insensitive receptor gene mutations, and the corresponding double mutants showed inhibited seedling growth regardless of ethylene treatment (Fig. 6B). In each of these double mutants, ectopic expression of etr11-349 suppressed the growth inhibition to various degrees, with or without ethylene treatment (Student’s t test, P < 0.01; Fig. 6C). Among the double mutants, etr11-349 had the greatest effect on promoting the growth of ers1-1 ctr1-1 and ers2-1 ctr1-1. Light-grown ctr1-1 seedlings showed small and compact cotyledons and short hypocotyls and roots. Seedlings of these ethylene-insensitive receptor mutants showed large, expanded cotyledons and long hypocotyls and roots (Fig. 6D). As expected, growth was inhibited in these light-grown double-mutant seedlings, and double mutants carrying etr11-349 showed normal seedling growth, with large, extended cotyledons and long hypocotyls and roots (Fig. 6E). At the adult stage, the double mutants produced small rosettes; etr11-349 expression promoted the growth of these double mutants to a large extent. Among these mutants, etr11-349 ctr1-1 carrying ers-1 or ers2-1 had the largest rosettes, and that carrying ein4-1 had the smallest (Fig. 6F).

Figure 6.

Figure 6.

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Expression of etr11-349 restores ethylene insensitivity conferred by dominant receptor mutant alleles in the ctr1-1 background. A to C, Seedling hypocotyl length of ctr1-1 and ethylene-insensitive receptor mutants (A), double mutants of ctr1-1 with an ethylene-insensitive receptor mutation (B), and double mutants expressing etr11-349 (C). D, Phenotype of light-grown seedlings of ctr1-1 and ethylene-insensitive receptor mutants. E and F, Phenotype of double mutants of ctr1-1 with an ethylene-insensitive receptor mutation, with and without the etr11-349 transgene, at seedling (E) and rosette (F) stages. G to I, Relative ERF1 expression in ctr1-1 and each ethylene-insensitive receptor mutant (G), ctr1-1 with an ethylene-insensitive receptor mutation (H), and double mutants expressing etr11-349(I). J, Relative etr11-349 transgene expression in the transgene donor (ctr1-1 L10) and recipients (the double mutants). K, Difference in expression (by Fisher’s lsd) of the etr11-349 transgene for double mutants and transgene donor. Data are means ± sd. ** P < 0.01 by Student’s t test. [See online article for color version of this figure.]

In addition to measuring growth phenotype alterations, we examined ERF1 expression by qRT-PCR to evaluate quantitatively the degree of ethylene responsiveness attenuated by the expression of etr11-349 in each double mutant. ERF1 levels in ethylene-insensitive receptor mutants were only 2% to 5% that of ctr1-1 levels (Fig. 6G). In ctr1-1, the ERF1 level was attenuated to different levels with each of the ethylene-insensitive receptor gene mutations, except for ers1-1 (Fig. 6H), which is in line with the ctr1-1 mutation not completely suppressing each of the ethylene-insensitive receptor mutations (Fig. 6, B and E). As expected, ERF1 levels were highly attenuated with etr11-349 expression in the double mutants (3.6%–12% that of ctr1-1 levels; Fig. 6I). qRT-PCR analysis of etr11-349 transgene expression showed etr11-349 expression attenuated by about 20% to 45% that of the transgene donor (etr11-349 ctr1-1 L10). Therefore, the prevention of ethylene responses by etr11-349 expression in the double mutants was not due to elevated etr11-349 expression (Fig. 6, J and K).

Coexpression of etr11-349 and an ethylene-insensitive receptor gene substantially suppressed the ctr1-1 mutant phenotype and conferred ethylene insensitivity. Our results support that the signal of each of the five ethylene receptors can be cooperatively mediated via the ETR1 N terminus to suppress the constitutive ethylene response via a pathway independent of CTR1.

DISCUSSION

Previous studies suggested that CTR1 acts downstream of ethylene receptors and that the ETR1-CTR1 association mediates the ethylene receptor signal output. Notably, ctr1 mutants show a constitutive ethylene-response phenotype but are somewhat ethylene responsive. The transcription factors EIN3 and EIN3-Like1 (EIL1) up-regulate the expression of ethylene-inducible genes. The constitutive ethylene-response phenotype is stronger in mutants defective in multiple ethylene receptors or the F-box proteins EBF1 and EBF2, which mediate EIN3/EIL1 degradation, than in ctr1 mutants (Kieber et al., 1993; Chao et al., 1997; Clark et al., 1998; Hua and Meyerowitz, 1998; Cancel and Larsen, 2002; Gao et al., 2003; Guo and Ecker, 2003; Huang et al., 2003; Potuschak et al., 2003). Therefore, a portion of the ethylene receptor signal may be mediated independently of CTR1, whereby lack of CTR1 substantially weakens but does not abolish the ethylene receptor signal output. Interestingly, we found that without CTR1, the ETR1 N terminus could produce the ethylene receptor signal output to suppress the constitutive ethylene response, which supports the hypothesis that the ethylene receptor signal can be mediated by a pathway independent of CTR1.

The ETR1 N terminus consists of three transmembrane helices and the GAF domain, of which the transmembrane helices are the ethylene-binding site and the GAF domain is involved in noncovalent receptor dimerization. ETR1 N-terminal signaling primarily depends on ETR1 and ERS1, which suggests cooperative receptor signaling of full-length ethylene receptors with the ETR1 N-terminal fragment (Rodríguez et al., 1999; Wang et al., 2006; Xie et al., 2006; Gao et al., 2008). The cooperative signaling could occur via mediation of the ETR1 N-terminal signal to full-length ethylene receptors or the opposite way, to a pathway independent of CTR1 (Fig. 5A; Gamble et al., 2002; Xie et al., 2006). We found the constitutive ethylene-response phenotype of CTR1-Nox, with the receptor signaling prevented by excess CTR17-560, suppressed by expression of the ETR1 N terminus. These results do not favor the scenario of the ETR1 N-terminal signal output mediated by full-length ethylene receptors. Rather, the favored scenario is that the full-length ethylene receptor signal is mediated cooperatively via the ETR1 N terminus by a pathway independent of CTR1. This hypothesis is also supported by results showing that expression of etr11-349 suppressed the constitutive ethylene-response phenotype and conferred ethylene insensitivity in double mutants harboring ctr1-1 and an ethylene-insensitive receptor allele. Given that the ETR1 N-terminal GAF domain can form heterodimers with other ethylene receptors (Xie et al., 2006; Gao et al., 2008), the GAF domain may have a role in interreceptor signaling and may be where the N-terminal signal output occurs.

The components that mediate or modulate the ETR1 N-terminal signal have yet to be identified. RTE1 overexpression promotes ETR1 N-terminal signaling in etr1-7 (Zhou et al., 2007) and ETR1 and RTE1 physically associate (Dong et al., 2010), so RTE1 may be involved in the CTR1-independent ETR1 N-terminal signaling. This hypothesis was supported by our results showing that expression of etr11-349 did not suppress the ctr1-1 mutant phenotype in the presence of the rte1-2 loss-of-function mutation and that RTE1 overexpression conferred ethylene insensitivity and suppressed the constitutive ethylene-response phenotype in the ctr1-1 mutant expressing the etr11-349 transgene. These results also reveal an essential role of RTE1 in ETR1 N-terminal signaling.

We observed CTR1-independent ETR1 N-terminal signaling only in the presence of the etr11-349 or etr1-11-349 transgene, which encodes the truncated ETR1 protein lacking the C terminus. Therefore, ETR1 N-terminal signaling mediated by the native, full-length ETR1 was largely prevented in ctr1 mutants. The ETR1 C terminus may inhibit N-terminal signaling so that the N-terminal signaling mediated by the full-length ETR1 did not occur in ctr1 mutants or ctr1-1 carrying 35S:RTE1 or an ethylene-insensitive receptor allele. In contrast, deletion of the ETR1 C terminus alleviated the signaling inhibition, thereby activating ETR1 N-terminal signaling. The C terminus could directly prevent N-terminal signaling or disrupt the ethylene receptor cooperativity, which is essential to N-terminal signaling. Given that the full-length ETR1 ethylene receptor has the C-terminal portion, if the ETR1 C terminus could disrupt receptor cooperativity, it would also disrupt the cooperativity of the full-length ETR1 and the ETR1 N terminus and disrupt signaling. Thus, we do not favor the scenario that the ETR1 C terminus disrupts ethylene receptor cooperativity with the ETR1 N terminus.

The CTR1 N terminus associates physically with the ETR1 C-terminal HK domain, which mediates the ethylene receptor signal. CTR1 isoforms with mutations in the C-terminal kinase domain, such as ctr1-1 and ctr1-4, may associate with ethylene receptors on the endoplasmic reticulum (Gao et al., 2003), so CTR1 kinase activity is essential to ethylene receptor signaling. This argument is in agreement with the result showing that an excess amount of CTR17-560, which does not have a CTR1 kinase domain, prevents ethylene receptor signaling, possibly by titrating out available ethylene receptors (Huang et al., 2003). The suppression of the CTR1-Nox phenotype by expression of etr11-349 and etr1-11-349, the latter of which also conferred ethylene insensitivity, indicated that CTR17-560 did not physically affect ETR1 N-terminal functions in cooperation with full-length ethylene receptors. Another implication is that CTR1 kinase activity may modulate ETR1 N-terminal signaling, in addition to its role in mediating ethylene receptor signaling. CTR1 kinase activity may inhibit the ETR1 C-terminal inhibition with the ETR1-CTR1 association, thereby facilitating ETR1 N-terminal signaling. The association of a kinase-defective CTR1 isoform with ETR1 fails to alleviate the ETR1 C-terminal inhibition, and the ETR1 N-terminal signaling is not facilitated. This argument may explain the lack of ETR1 N-terminal signaling in ctr1 mutants with defective kinase activity.

In summary, we hypothesize that modulation of the ethylene receptor ETR1 signal output involves CTR1, RTE1, and possibly ethylene receptor cooperation and negative regulation by the ETR1 C terminus. The receptor signal of full-length ETR1 can be mediated via CTR1 and a CTR1-independent pathway. The ethylene receptor signaling mediated via CTR1 has been well addressed in previous studies. Results from our study suggest that the ETR1 N terminus plays important roles in ethylene receptor signaling via a CTR1-independent pathway. The ETR1 C terminus may inhibit the ETR1 N-terminal signaling. Deletion of the C terminus or the association of CTR1 with the ETR1 C terminus alleviates the ETR1 C-terminal negative regulation. CTR1 kinase activity may have a role in eliminating the C-terminal inhibition. Without the ETR1 C-terminal inhibition, RTE1 promotes the ETR1 N-terminal signaling. The GAF domain may involve receptor interaction, by which receptor cooperation occurs and the signal of full-length ethylene receptors is mediated via CTR1 and the ETR1 N terminus, with the latter independent of CTR1, to repress ethylene responses.

MATERIALS AND METHODS

Plant Materials and Transgenes

Arabidopsis (Arabidopsis thaliana) etr1-1, ers1-1, etr2-1, ein4-1, and ers2-1 were each genetically crossed with ctr1-1 to obtain double mutants. etr1-7 was crossed with ctr1-1 or ctr1-2 to obtain double mutants. Receptor-defective mutants were described previously (Xie et al., 2006; Zhou et al., 2007). Corresponding transgenes and transformation lines carrying 35S:RTE1, ETR1p:etr11-349, and ETR1p:etr1-11-349 were described previously (Xie et al., 2006; Zhou et al., 2007). ctr1-2 was obtained from J. Kieber (Huang et al., 2003). To clone the CTR17-560 -encoding fragment, a full-length genomic CTR1 fragment, restricted with EcoRV and SmaI, was cloned from the bacterial artificial chromosome clone F17C15 to pBluescript SK+. The primer set CTR1 N1-F (5′-CGCTCGAGATGAGATCTAATTACACTTTGC-3′) and CTR1 N1-R (5′-AGGTCGATGCATAAGGT-3′) amplified a 5′ fragment carrying the start codon (ATG). The primer set CTR1 N2-F (5′-CCGGCTTAATGAATTCTAGTGC-3′) and CTR1 N2-R (5′-CAACTAGTTTAACCTGAAAGAGCAGTCGCTCAA-3′) amplified a 3′ fragment carrying a stop codon (TAA). The 5′ ATG-containing fragment was swapped with the XhoI/NsiI fragment of the genomic CTR1 clone; the 3′ TAA-containing fragment was swapped with the EcoRI/SpeI fragment of the genomic CTR1 clone, of which the SpeI site was from the cloning vector. The resulting genomic fragment, encoding CTR17-560, was released with KpnI and XbaI and cloned to the cauliflower mosaic virus 35S promoter-containing binary vector pCAMBIA. Phenotypic analyses of these transformation lines were carried out in the F4, or more advanced, generations.

Ethylene Treatment and Seedling Hypocotyl Measurement

Ethylene treatment and measurement were as described (Zhang and Wen, 2010). Ethylene (10 μL L−1) was used in the seedling triple-response assay and gene expression analyses. For each hypocotyl measurement, at least 20 seedlings (n ≥ 20) were measured (Xie et al., 2006). For gene expression analyses, adult plants (4 weeks old) were treated with ethylene for 4 h and then subjected to qRT-PCR analysis.

Immunoassay

An IgG monoclonal antibody purified from mice ascites against the CTR11-452 fragment was prepared by an antibody service (Abmart). Total protein from 35S:CTR17-560 transformation lines was isolated and fractionated by SDS-PAGE (Wen et al., 1999; Liu et al., 2010). The protein was transferred to a polyvinylidene difluoride membrane and probed with the monoclonal antibody. The monoclonal IgG was next probed with a horseradish peroxidase-conjugated goat anti-mouse IgG and detected by enhanced chemiluminescence captured with the use of a cold (−110°C) CCD system (VersArray System; Princeton Instruments; Liu et al., 2010) and computed by the use of MetaMorph version 7.0 (Molecular Devices). The same immunoblot was stained with Coomassie blue to visualize relative protein amount on membranes.

Fluorescence qRT-PCR

qRT-PCR and primer sets were described previously (Liu et al., 2010; Zhang and Wen, 2010). qRT-PCR involved the use of the StepOnePlus system (ABI). Primers used were PDF1.2 F (5′-CTTTGCTGCTTTCGACGCACC-3′) and PDF1.2 R (5′-CATGGGACGTAACAGATACA-3′); etr1-1F (5′-GCTTTTATCGTTCTTTA-3′) and etr1-1R (5′-GCTTTATTTTTCAAGAAA-3′); and ETR1N-F (5′-CGCTGATCAGGTGGCTGTAG-3′) and ETR1N-R (5′-TCTAGAGGATCCTAAACCGC-3′). Each measurement was repeated three times with three independent biological replicates.

Statistical Analysis

Data were compared by Student’s t test or, for paired comparison of multiple means and estimation of the mean difference, Fisher’s lsd, with P < 0.05 or P < 0.01 indicating statistical significance.

Sequence data from this article can be found in the GenBank/EMBL data libraries under accession numbers NC_003070, NC_003071, NC_003074, NC_003074, NC_003070, NC_003071, and NC_003076.

Acknowledgments

We thank Dr. J. Kieber for providing the ctr1-2 mutant, the Arabidopsis Biological Resource Center for the bacterial artificial chromosome clone F17C15, and Dr. C. Chang for critical comments on the manuscript.

Glossary

HK

histidine kinase

Col-0

ecotype Columbia

qRT

quantitative reverse transcription

GAF

to be defined

Footnotes

1

This work was supported by the National Natural Science Foundation of China (grant nos. 31123006, 31070249, and 30770199), the Chinese Ministry of Science and Technology (grant nos. 2011CB100700 and 2012AA10A302–2), and the Chinese Academy of Sciences (grant no. KSCX2–EW–J–12).

[C]

Some figures in this article are displayed in color online but in black and white in the print edition.

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