Plant Physiology Preview. Published on July 9, 2015, as DOI:10.1104/pp.15.00626
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Running Head: Functional Regions of the ETR1 Receiver Domain
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Corresponding Author:
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Brad M. Binder
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Department of Biochemistry, Cellular, and Molecular Biology
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University of Tennessee - Knoxville
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M407 Walters Life Sciences
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Knoxville, Tennessee 37996-0849
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phone: (865) 974-7994
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email: [email protected]
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Journal Research Area: Signal and Response
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Title: Identification of Regions in the Receiver Domain of the ETHYLENE RESPONSE1 Ethylene
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Receptor of Arabidopsis Important for Functional Divergence1
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Authors: Arkadipta Bakshi2, Rebecca L. Wilson2, Randy F. Lacey, Heejung Kim3, Sai Keerthana
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Wuppalapati, and Brad M. Binder4
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Department of Biochemistry, Cellular, and Molecular Biology (R.L.W., R.F.L., H.K., S.K.W., B.M.B.)
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and the Genome Science and Technology Program (A.B., B.M.B.), University of Tennessee, Knoxville,
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Tennessee, USA.
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One sentence summary: The receiver domain of the ETHYLENE RESPONSE1 ethylene receptor has
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regions that control specific traits leading to receptor sub-functionalization.
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Footnotes:
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1
This project was supported by NSF grant MCB-0918430 to BMB and a NSF fellowship to RLW.
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2
These authors contributed equally to the article.
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3
Current address: Department of Chemistry & Molecular Biology, University of Gothenburg,
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Gothenburg, Sweden.
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Corresponding Author; email: [email protected]
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The author responsible for distribution of materials integral to the findings presented in this article in
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accordance with the Journal policy described in the Instructions for Authors
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(http://www.plantphysiol.org) is: Brad M. Binder ([email protected])
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ABSTRACT
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Ethylene influences the growth and development of Arabidopsis thaliana via five receptor isoforms.
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However, the ETHYLENE RESPONSE1 (ETR1) ethylene receptor has unique, and sometimes
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contrasting roles from the other receptor isoforms. Prior research indicates that the receiver domain of
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ETR1 is important for some of these non-canonical roles. We determined that the ETR1 receiver domain
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is not needed for ETR1’s predominant role in mediating responses to the ethylene antagonist, silver. To
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understand the structure-function relationship underlying the unique roles of the ETR1 receiver domain in
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the control of specific traits, we performed Ala scanning mutagenesis. We chose amino acids that are
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poorly conserved and are in regions predicted to have altered tertiary structure compared to the receiver
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domains of the other two receptors that contain a receiver domain, ETR2 and ETHYLENE
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INSENSITIVE4. The effects of these mutants on various phenotypes were examined in transgenic,
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receptor-deficient Arabidopsis plants. Some traits, such as growth in air and growth recovery after the
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removal of ethylene, were unaffected by these mutations. By contrast, three mutations on one surface of
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the receiver domain rendered the transgene unable to rescue ethylene-stimulated nutations. Additionally,
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several mutations on another surface altered germination on salt. Some of these mutations conferred
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hyper-functionality to ETR1 in the context of seed germination on salt, but not for other traits, that
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correlated with increased responsiveness to abscisic acid. Thus, the ETR1 receiver domain has multiple
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functions where different surfaces are involved in the control of different traits. Models are discussed for
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these observations.
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Introduction
Ethylene is a phytohormone that affects the growth and development of plants and mediates plant
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stress responses (Mattoo and Suttle, 1991; Abeles et al., 1992). Mutational, molecular, and biochemical
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analyses have identified components in the ethylene signal transduction pathway. The current model
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speculates that ethylene binding to the receptors reduces the activity of the receptors, leading to reduced
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activity of the CONSTITUTIVE TRIPLE RESPONSE1 (CTR1) protein kinase (Kieber et al., 1993).
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Lower CTR1 activity results in reduced phosphorylation of ETHYLENE INSENSITIVE2 (EIN2) protein
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(Chen et al., 2011; Ju et al., 2012; Qiao et al., 2012). The reduction in EIN2 phosphorylation leads to a
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decrease in the ubiquitination of EIN2 resulting in a rise in EIN2 protein levels and proteolytic release of
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the C-terminal portion of the protein (Qiao et al., 2009; Ju et al., 2012; Qiao et al., 2012; Wen et al.,
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2012). The C-terminal portion of EIN2, through mechanisms not completely understood, modulates levels
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of the EIN3 and EIN3-LIKE1 transcription factors and leads to most ethylene responses (Chao et al.,
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1997; Solano et al., 1998; Alonso et al., 1999; Guo and Ecker, 2003; Yanagisawa et al., 2003; Binder et
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al., 2004; Gagne et al., 2004; Qiao et al., 2012). As predicted by this model, loss of multiple ethylene
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receptors results in a constitutive ethylene response (Hua and Meyerowitz, 1998; Hall and Bleecker,
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2003; Wang et al., 2003; Qu et al., 2007). Even though this signaling pathway was mostly worked out in
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the model plant Arabidopsis thaliana, similar genes have been described in other plants such as rice
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(Oryza sativa), strawberry (Fragaria vesca), and tomato (Solanum lycopersicum), as well as in plants
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from ancient divergent lineages such as Physcomitrella patens and Selaginella moellendorffii and the
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charaphyte Spirogyra (Rensing et al., 2008; Rzewuski and Sauter, 2008; Ma et al., 2010; Banks et al.,
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2011; Klee and Giovannoni, 2011; Shulaev et al., 2011; Ju et al., 2015). This suggests that a similar
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signaling pathway is found in all land plants and likely evolved prior to colonization of land.
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Responses to ethylene are mediated by a receptor family that is predominantly located in the
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membrane of the endoplasmic reticulum (Chen et al., 2002; Ma et al., 2006; Grefen et al., 2007; Bisson et
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al., 2009). In Arabidopsis there are five receptor isoforms called ETHYLENE RESPONSE1 (ETR1),
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ETR2, ETHYLENE RESPONSE SENSOR1 (ERS1), ERS2, and EIN4 (Chang et al., 1993; Hua and
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Meyerowitz, 1998; Hua et al., 1998; Sakai et al., 1998; Gao et al., 2003). The plant receptors form
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homodimers with each monomer predicted to have three membrane-spanning α helices at the amino-
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terminus containing the ethylene-binding site (Schaller and Bleecker, 1995; Schaller et al., 1995;
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Rodriguez et al., 1999). This is followed by the cytosolic portion of the receptor consisting of a GAF (for
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cGMP-specific phosphodiesterases, adenylyl cyclases, and FhlA) domain, kinase domain, and in a subset
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of the receptors (ETR1, ETR2, EIN4), a receiver domain (Fig. 1A). We know that all five ethylene
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receptor isoforms in Arabidopsis are involved with ethylene signaling because all five members bind
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ethylene with high affinity (Schaller and Bleecker, 1995; Hall et al., 2000; O'Malley et al., 2005;
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McDaniel and Binder, 2012) and specific missense mutations in the ethylene binding domain of any
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single isoform leads to dominant ethylene insensitivity that affects responses throughout the plant
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(Bleecker et al., 1988; Hua et al., 1995; Hua and Meyerowitz, 1998; Wang et al., 2006). The nature of
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signal transmission through the ethylene receptors is unknown. They have homology to bacterial two-
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component receptors (Chang et al., 1993; Hua and Meyerowitz, 1998; Hua et al., 1998; Sakai et al., 1998)
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which transduce signal via the autophosphorylation of a His residue in the kinase domain, followed by the
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transfer of phosphate to a conserved Asp residue in the receiver domain of a response regulator protein
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(West and Stock, 2001). Biochemical studies show that some of the receptor isoforms possess functional
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His kinases (Gamble et al., 1998; Moussatche and Klee, 2004) and ethylene binding might modulate His-
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kinase activity of ETR1 (Voet-van-Vormizeele and Groth, 2008). However, genetic studies suggest that
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His-kinase activity is not required for responses to ethylene (Wang et al., 2003; Binder et al., 2004; Qu
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and Schaller, 2004; Xie et al., 2006; Cho and Yoo, 2007; Hall et al., 2012). Rather, this activity modulates
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growth in air, sensitivity to ethylene, and growth recovery after ethylene removal (Gamble et al., 1998;
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Binder et al., 2004; Cho and Yoo, 2007; Hall et al., 2012). Thus, the plant ethylene receptors have
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diverged in functional output from other two-component receptors.
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Even though the receptors have largely overlapping roles for many phenotypes, there are traits where
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the receptors are not redundant (Binder et al., 2004; Binder et al., 2006; Plett et al., 2009; Plett et al.,
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2009; Liu et al., 2010; Kim et al., 2011; McDaniel and Binder, 2012; Wilson et al., 2014; Wilson et al.,
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2014). For instance, ETR1, ETR2, and EIN4 have a role in normal growth recovery after ethylene
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removal, whereas ERS1 and ERS2 do not (Binder et al., 2004). Another example of non-redundancy is
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that ETR1 has the predominant role in mediating the inhibitory effects of silver ions (McDaniel and
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Binder, 2012). Silver ions inhibit ethylene perception in plants (Beyer, 1976), but etr1 loss-of-function
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mutants have little response to silver ions (McDaniel and Binder, 2012). Surprisingly, there are also some
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traits where ethylene receptor isoforms have contrasting roles. For example, ethylene stimulates
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nutational bending of the hypocotyls (Binder et al., 2006). Mutant seedlings lacking ETR1 fail to nutate
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when treated with ethylene, whereas, mutants lacking the other four isoforms have constitutive nutations
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in air (Binder et al., 2006; Kim et al., 2011). Additionally, we recently reported that ETR1 and ETR2 have
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contrasting roles in the control of Arabidopsis seed germination in darkness or during salt stress in the
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light where ETR1 inhibits germination and ETR2 stimulates germination (Wilson et al., 2014; Wilson et
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al., 2014). Correlating with these seed germination results are the observations that etr1 loss-of-function
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mutants have reduced sensitivity to abscisic acid (ABA), whereas, etr2 loss-of-function mutants have
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increased sensitivity to ABA (Wilson et al., 2014). These observations are not explained by current
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models of ethylene signaling and point to unique roles for the ETR1 receptor. It has been suggested that
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the receptors may have ethylene signaling-independent roles (Gamble et al., 1998; Beaudoin et al., 2000;
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Desikan et al., 2005; Binder et al., 2006; Wilson et al., 2014; Wilson et al., 2014) which may underlie
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these observations. Determining the basis for non-canonical receptor signaling is of wide interest since
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non-canonical signal transduction has been observed in other signal transduction pathways including
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Hedgehog and Wnt (for Wingless/int-1) signaling in animal cells (Jenkins, 2009; Miller and McCrea,
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2010) and quorum sensing in bacteria (Federle and Bassler, 2003).
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The receiver domain of ETR1 is required for both ethylene-stimulated nutations and the inhibitory
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role of ETR1 on seed germination during salt stress (Kim et al., 2011; Wilson et al., 2014). Interestingly,
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this domain is not required for the inhibitory role of ETR1 on seed germination in the dark (Wilson et al.,
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2014). Additionally, a common feature of the three receptors that have a role in growth recovery is that
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they contain a receiver domain and genetic evidence indicates that ETR1 His autophosphorylation
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followed by phosphotransfer through these receiver domains is important for normal growth recovery
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(Binder et al., 2004; Kim et al., 2011). In order to understand the structure-function relationship leading to
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the unique role of the ETR1 receiver domain in the control of various traits, we performed Ala scanning
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mutagenesis. For this we chose amino acids that are poorly conserved and are in regions predicted to have
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altered tertiary structure when compared to the receiver domains of ETR2 and EIN4. The effects of these
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mutants on various phenotypes were examined in transgenic, receptor-deficient Arabidopsis plants. From
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these experiments we identified regions of the receiver domain important for the control of specific
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phenotypes.
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Results
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Responses To Silver Ions Do Not Require The ETR1 Receiver Domain
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ETR1 has been shown to have the major role in mediating the effects of the ethylene antagonist,
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AgNO3, and etr1-6;etr2-3;ein4-4 triple mutants have no response to silver ions (McDaniel and Binder,
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2012). We previously showed silver responses were rescued when the triple mutants were transformed
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with a full-length genomic ETR1 transgene, a transgene deficient in His-kinase activity, or a transgene
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lacking the conserved Asp659 required for phosphorelay (McDaniel and Binder, 2012). However, it is
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unknown whether or not the ETR1 receiver domain is required for this trait. To address this, we examined
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the growth response kinetics to the application of 1 µL L-1 ethylene in the presence of 100 µM AgNO3.
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Our prior studies determined that there are two phases of ethylene-induced growth inhibition that are
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genetically distinct (Binder et al., 2004; Binder et al., 2004). The first phase starts approximately 10 min
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after the addition of ethylene and reaches a plateau approximately 10 min later. This plateau lasts
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approximately 30 min and is followed by a second phase of growth inhibition that lasts for as long as
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ethylene is present (Binder et al., 2004; Binder et al., 2004). The application of 100 µM AgNO3 severely
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attenuates or eliminates the first phase and entirely blocks the second phase of growth inhibition
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(McDaniel and Binder, 2012). Consistent with our prior study (McDaniel and Binder, 2012), in the
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presence of AgNO3 etr1-6;etr2-3;ein4-4 triple mutants showed two phases of growth inhibition when
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ethylene was added and wild-type seedlings showed no response to ethylene (Fig. 1B). Transformation of
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the etr1-6;etr2-3;ein4-4 triple mutants with a cDNA encoding for either a full-length ETR1 transgene
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(cETR1) or a truncated transgene lacking the receiver domain (cetr1-ΔR) resulted in seedlings that had a
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small, transient growth inhibition response when ethylene was applied (Fig. 1B). Neither transformant
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had a long-term response to ethylene in the presence of AgNO3 indicating that the receiver domain of
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ETR1 has little or no role in mediating ETR1 output important for responses to silver ions.
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Identification Of Amino Acid Residues In The ETR1 Receiver Domain For Mutagenesis
To better define unique regions within the ETR1 receiver domain that may be important for ETR1
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sub-functionalization for other traits, we carried out site-directed mutagenesis on the receiver domain of
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full-length ETR1 genomic DNA. To define which amino acids residues to target, we first looked for
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amino acids not conserved in the ETR1 receiver domain compared to the receiver domains of ETR2 and
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EIN4 as well as regions where the structure of the ETR1 receiver domain is predicted to diverge from the
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other two receptors. As shown in figure 2, a comparison of the predicted amino acid sequences of the
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receiver domains shows that there are over sixty residues not conserved between ETR1 and the other two
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receptors that could potentially underlie the unique functions of ETR1 (Fig. 2A). One region that shows
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high divergence in amino acid sequence is the γ loop which consists of six amino acids just beyond the
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conserved Asp required for phosphorelay. The γ loop of ETR1 is in a different orientation from other
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structurally characterized γ loops and may underlie functional differences (Müller-Dieckmann et al.,
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1999). We therefore mutagenized several amino acids in this loop. We also targeted amino acids in the C-
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terminal tail which has been identified as a region that might be important in ETR1-protein interactions
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(Müller-Dieckmann et al., 1999) and is another region where there is high amino acid divergence. Finally,
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autodephosphorylation activity has been observed in receiver domains and amino acids corresponding to
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Asn618, Cys661, and Asn694 in ETR1 have been implicated as critical for this activity in other receiver
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domains (Pazy et al., 2009). We also homology modeled the receiver domains of ETR2 and EIN4 and
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compared these models to the crystal structure of the ETR1 receiver domain (Fig. 2B). This modeling
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revealed that there are several loops where the tertiary structures may be diverged (marked with asterisks
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in Fig 2B). Several amino acids in these regions were targeted for mutagenesis. Based on these criteria,
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eleven amino acids were changed to Ala (marked with open circles in Fig. 2A). These mutant transgenes
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and wild-type genomic ETR1 (gETR1) were transformed into etr1-6;etr2-3;ein4-4 triple loss-of-function
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mutants. Two to three transgenic lines were created for each mutant and transcript levels measured with
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RT-PCR (Fig. 2C). For each transgene construct, we chose the line with the highest expression level for
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physiological analyses.
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Effect Of ETR1 Receiver Domain Point Mutations On ETR1 Signaling
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To delineate amino acids in the ETR1 receiver domain important for sub-functionalization, we
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studied the ability of these eleven point mutants to rescue several traits when transformed into the etr1-
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6;etr2-3;ein4-4 triple mutants. This triple mutant was chosen because it has reduced growth in air (Hua
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and Meyerowitz, 1998; Binder et al., 2004; Qu and Schaller, 2004; Liu et al., 2010), has very slow growth
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recovery after ethylene removal (Binder et al., 2004), fails to nutate when ethylene is applied (Binder et
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al., 2006), and has an accelerated germination time-course under salt stress (Wilson et al., 2014). Thus,
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we could compare the rescue of these various traits by the mutant transgenes to determine residues
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important for one or more functions.
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Using high-resolution, time-lapse imaging of growing seedlings in the dark, we analyzed growth rate
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in air, ethylene-stimulated nutations, and growth response kinetics when ethylene was applied and
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removed (Fig. 3). Transformation of the etr1-6;etr2-3;ein4-4 triple null mutant with a wild-type gETR1
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transgene reversed the effects of the triple mutant resulting in faster growth in air (Fig. 3A), larger
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ethylene-stimulated nutational amplitude (Fig. 3B), and accelerated growth recovery after removal of
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ethylene (Fig. 3C). Different patterns of rescue were obtained with the point mutants, but all were
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functional for at least one trait (summarized in Table 1). All were at least partially functional in the rescue
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of growth in air and growth recovery after ethylene removal. For this later trait, several transgenes
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rescued slightly better than wild-type gETR1 (Fig. 3C), some equally well (Fig. 3D), and some slightly
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worse than gETR1 (Fig. 3 E). In addition, all of the transformants had growth inhibition kinetics similar to
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wild-type seedlings. In general, the mutant transgenes were less effective at rescuing nutations than
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gETR1 (Fig. 3B, Table 1). This is consistent with our previous observations that indicate this trait is easily
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disrupted (Kim et al., 2011). Nonetheless, eight mutant transgenes partially rescued ethylene-stimulated
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nutations. Three of the mutant transgenes failed to rescue ethylene-stimulated nutations; this was
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confirmed by examining multiple transgenic lines for each mutant (Fig. 3B). One of the mutations
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(Q684A) is in a loop region and two (E730A, L734A) are in the C-terminal tail of ETR1.
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We also examined the effect of the point mutants on seed germination in the presence of 150 mM
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NaCl. Previously, we generated and characterized an Asp659Ala mutant (D659A) transformed into the
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etr1-6;etr2-3;ein4-4 triple mutant background to assess the role of phosphotransfer in the control of
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growth in air, growth recovery after ethylene removal, and ethylene-stimulated nutations (Binder et al.,
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2004; Kim et al., 2011). In addition to the eleven point mutants described above, we used this mutant in
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these seed germination experiments to determine the role of phosphotransfer in this phenotype. In the
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absence of NaCl, most seed lines germinated with a time-course indistinguishable from wild-type seeds
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(Fig. 4). However, several transformants (C661A, V665A, E666A, Q681A) germinated slightly slower
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than wild-type seeds with the E666A mutant receptor giving the slowest germination time-course with a
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time for 50% seed germination of approximately 1.8d compared to 1.3d for the wild-type seeds
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(Supplemental Figure S1).
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Consistent with our previous results (Wilson et al., 2014), 150 mM NaCl delayed germination of all
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seed lines (Fig. 4, Supplemental Figure S1). The triple mutant seeds were the least affected by 150 mM
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NaCl having a slight increase in the time for 50% seed germination from approximately 1.3d in the
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absence of salt to approximately 1.8d in the presence of salt (Supplemental Figure S1). Wild-type and
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triple mutants transformed with gETR1 reached approximately 78% germination after 7d, whereas, the
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triple mutants reached 100% germination by 3.5d. There were three patterns of rescue for germination on
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150 mM NaCl (Fig. 4, Table 1, Supplemental Figure S1). Some transgenes (V665A, E666A, Q681A)
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caused germination to be slower than germination of triple mutants transformed with gETR1 and resulted
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in a lower percent of seeds that germinated (Fig. 4B). These results were confirmed in multiple transgenic
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lines for each mutant (data not shown). This indicates that these mutant receptors are more functional than
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the wild-type receptor for this phenotype. Interestingly, these mutants are not more functional for the
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other traits examined (Table 1). The two mutations causing the slowest germination time-course (V665A,
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E666A) are both in the γ loop and these seed lines failed to reach 50% germination within the time-span
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of these experiments (Supplemental Figure S1). Three mutant transgenes (N617A, Q618A, R682A)
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rescued germination on salt to the same extent as gETR1 (Fig. 4C) and six mutant transgenes were less
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functional resulting in a faster germination time-course and better overall germination compared to
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gETR1 (Fig. 4D). Four of these less functional transgenes (N667A, Q684A, E730A, L734A) were only
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slightly less functional than gETR1 (Fig. 4D, Table 1). By contrast, two mutant transgenes (D659A,
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C661A) resulted in a faster germination time-course on salt with a time for 50% seed germination over
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30% faster than the gETR1 transformants (Table 1, Supplemental Figure S1). The D659A transgene
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affects the Asp that is believed to be involved in phosphotransfer based on homology to other receiver
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domains, and C661A affects a Cys that hydrogen bonds with Asp659 (Müller-Dieckmann et al., 1999).
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Both amino acid residues are located just before the γ loop. Thus, the γ loop and phosphotransfer through
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the receiver domain have important roles in mediating germination on salt.
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Effect Of The EIN4 Receiver Domain On Seed Germination On Salt
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We have previously generated cDNAs encoding chimeric ETR1-EIN4 receptors to examine the role
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of each domain in the regulation of various traits (Kim et al., 2011). These chimeric receptor transgenes
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were transformed into etr1-6;etr2-3;ein4-4 triple mutants and the rescue of various traits compared to
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rescue obtained with a cDNA encoding a full-length ETR1 (cETR1). From this we found that
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transformation with a chimeric ETR1-EIN4 receptor transgene containing the receiver domain of EIN4
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(1114) can rescue growth in air and growth recovery after ethylene removal, but not ethylene-stimulated
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nutations (Kim et al., 2011). This suggests that the ETR1 receiver domain is unique and needed for the
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control of some, but not all, phenotypes. Therefore, we were curious to know whether or not the EIN4
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receiver domain can substitute for the ETR1 receiver domain in the control of seed germination on salt.
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To determine this, we examined the germination time-course of triple mutants transformed with the 1114
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transgene. The germination time-course of this transformant was compared to wild-type, etr1-6;etr2-
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3;ein4-4 triple mutants, and triple mutants transformed with either cETR1 or cetr1-ΔR. All seed lines had
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similar germination time-courses in the absence of salt (Fig. 5; Supplemental Figure S2). Consistent with
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our prior results (Wilson et al., 2014), on salt the etr1-6;etr2-3;ein4-4 triple mutants transformed with
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cETR1 had slow germination comparable to wild-type seeds, whereas the cetr1-ΔR transgene was non-
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functional for this trait resulting in fast germination similar to the germination time-course of the triple
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mutant seeds (Fig. 5; Supplemental Figure S2). Similarly, the 1114 transgene did not have an effect on
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seed germination on salt indicating that the EIN4 receiver domain cannot substitute for the ETR1 receiver
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domain for this trait. Both cetr1-ΔR and 1114 are expressed and functional for other traits (Kim et al.,
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2011), providing evidence that the ETR1 receiver domain is specifically required to mediate germination
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on salt.
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Responses To ABA Are Altered to Affect Seed Germination
The hormone ABA is an inhibitor of germination that accumulates in plants in response to salt stress
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(Garciarrubio et al., 1997; Jakab et al., 2005). In a previous study, we demonstrated that the faster
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germination of etr1-6 loss-of-function mutants on salt correlated with a decrease in responsiveness to
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ABA, rather than changes in levels or responses to ethylene, gibberellic acid, or cytokinin (Wilson et al.,
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2014). We therefore wished to determine whether or not ABA was involved in the alterations we observe
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in seed germination on salt in the current study. We first compared the germination time-courses of wild-
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type to etr1-6;etr2-3;ein4-4 triple mutant seeds (Fig. 6). In the absence of ABA, both seed lines had
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similar germination time-courses. As we previously reported (Wilson et al., 2014), 1 µM ABA inhibited
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the germination of wild-type seeds (Fig. 6B) causing the time for 50% germination to increase by
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approximately 3d (Supplemental Figure S3). ABA had a smaller effect on the etr1-6;etr2-3;ein4-4 triple
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mutants showing that they are less responsive to this hormone. We also wished to determine if the altered
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germination of certain point mutants on salt involved ABA. For this, we chose the D659A transformant
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which germinates better than gETR1 on salt and the V665A and E666A transformants which germinate
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slower than gETR1 on salt. In solvent-control conditions (Fig. 6), germination time-courses were similar
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to what was observed in the absence of solvent (Fig. 4) with the E666A transgene causing a slight
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increase in the germination time-course (Fig. 6, Supplemental Figure S3). Transformation of the triple
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mutant with gETR1 caused the seeds to have a larger response to ABA and have slower germination that
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was comparable to wild-type seeds. Correlating with our germination results on salt, the D659A
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transformants germinated faster on ABA than gETR1 and had a germination time-course that was very
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similar to the triple mutants (Fig. 6, Supplemental Figure S3). By contrast, the V665A and E666A
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transgenes caused slower seed germination in response to ABA than what was observed with gETR1
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seeds (Fig. 6). These seed lines never reached 50% germination within the time-frame of these
313
experiments (Supplemental Figure S3). These data demonstrate that application of ABA phenocopies the
314
effects of NaCl on the seed lines tested suggesting that the difference in germination caused by these
315
mutant transgenes is due to differences in responsiveness to ABA.
316
To further explore the link between ABA and germination, we used quantitative real-time reverse
317
transcriptase (qRT)-PCR to examine the transcript abundance of two ABA-responsive genes in response
318
to ABA treatment. For this we chose CRUCIFERIN1 (CRA1) which encodes for a major seed storage
319
protein and RESPONSIVE TO ABA18 (RAB18) which encodes for a dehydrin (Lång and Palva, 1992;
320
Gliwicka et al., 2012). As shown in figure 7, treatment with 1 µM ABA for 2d increased the transcript
321
abundance of both genes in wild-type seeds (P < 0.05). Interestingly, the transcript levels of both genes
322
were lower in the triple mutant than the wild-type seeds after treatment with ABA (P < 0.05). ABA had
323
no effect CRA1 transcript levels in the etr1-6;etr2-3;ein4-4 triple mutants and had a smaller effect on
324
RAB18 transcript levels than seen with wild-type seeds. These data support the idea that the triple mutant
325
seeds are less responsive to ABA. Similar changes in CRA1 and RAB18 transcript abundance are seen in
326
response to 150 mM NaCl (Supplemental Figure S4A). We also examined the changes in CRA1 and
327
RAB18 transcript levels in triple mutants transformed with gETR1, D659A, or E666A. The patterns of
328
changes in these transcripts correlated with germination with the gETR1 transformants having responses
329
similar to wild-type seeds, the D659A transformants having little or no response to ABA, and the E666A
330
transformants having larger responses to ABA (Fig. 7).
331
To further examine the involvement of ABA, we treated seedlings with the ABA biosynthesis
332
inhibitor, norflurazon, in the presence of 150 mM NaCl. Solvent-control seedlings had germination time-
333
courses in response to 150 mM NaCl (Fig. 8) that were similar to what we observe in the absence of
334
solvent (Fig. 4). Application of 10 µM norflurazon caused germination time-courses to be faster (Fig. 8)
335
and significantly (P <0.05) reduced the time for 50% seed germination for all lines tested (Supplemental
336
Figure S5). Application of 100 µM norflurazon almost entirely eliminated the germination differences
337
between the seed lines tested (Fig. 8, Supplemental Figure S5). Application of 100 µM norflurazon also
338
almost entirely reversed the effects of 150 mM NaCl on these germinating seeds suggesting that the
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effects of the mutant transgenes on seed germination during salt stress are largely mediated by ABA.
340
Correlating with these changes, norfluorzon reversed the effects that salt treatment had on the transcript
341
levels of CRA1 and RAB18 (Supplemental Figure S4B).
342
343
344
Discussion
The ethylene receptors have become sub-functionalized and have both overlapping and non-
345
overlapping roles (Reviewed by Shakeel et al., 2013). Prior studies showed that ETR1 has a prominent
346
role in the control of several phenotypes including ethylene-stimulated nutations, growth recovery after
347
removal of ethylene, inhibition of ethylene responses by silver ions, seed germination during salt stress in
348
the light, and seed germination in darkness (Binder et al., 2004; Binder et al., 2006; Kim et al., 2011;
349
McDaniel and Binder, 2012; Wilson et al., 2014; Wilson et al., 2014). Interestingly, the ETR1 receiver
350
domain was previously shown to be important for normal growth recovery, ethylene-stimulated nutations,
351
and germination during salt stress in the light (Binder et al., 2004; Binder et al., 2006; Kim et al., 2011;
352
Wilson et al., 2014), but not for the control of seed germination in darkness (Wilson et al., 2014). In the
353
current study, we demonstrated that the ETR1 receiver domain also has little or no role in mediating the
354
inhibitory effects of silver ions. These results in combination with other studies demonstrate that the
355
control of various traits have different ETR1 domain requirements. Three traits (growth recovery after
356
ethylene removal, ethylene-stimulated nutations, germination during salt stress in the light) require the
357
full-length ETR1 receptor (Binder et al., 2004; Binder et al., 2006; Kim et al., 2011; Wilson et al., 2014).
358
By contrast, growth in air, growth inhibition by ethylene, germination in darkness, and response to silver
359
ions do not require the receiver domain (Gamble et al., 2002; Binder et al., 2004; Hall et al., 2012; Wilson
360
et al., 2014; this study). There is also evidence that ETR1 signaling occurs from the ethylene-binding and
361
GAF domains (Gamble et al., 2002; Qiu et al., 2012). This signaling appears to occur via REVERSION
362
TO ETHYLENE1, which modulates ETR1 function by interactions with the ethylene-binding and GAF
363
domains, and may signal via a CTR1-independent pathway (Resnick et al., 2006; Dong et al., 2008; Dong
364
et al., 2010; Qiu et al., 2012).
365
Within those traits that require the receiver domain there are differences. Genetic studies indicate that
366
phosphotransfer through the receiver domain is important for both normal growth recovery after the
367
removal ethylene (Binder et al., 2004) and germination under salt stress (this study). However, using
368
ETR1-EIN4 chimeric receptors containing the receiver domain of EIN4 (1114) we show that the receiver
369
domain of EIN4 can substitute for the receiver domain of ETR1 in the control of growth recovery (Kim et
370
al., 2011), but not germination on salt (this study). This suggests a model where there are two patterns of
371
phosphotransfer from ETR1 His kinase, where phosphotransfer to ETR1, ETR2, and EIN4 controls
372
growth recovery and phosphotransfer solely to ETR1 controls seed germination during salt stress (Fig. 9).
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The phosphotransfer solely to ETR1 appears to inhibit germination under salt stress. But, we cannot rule
374
out that subtle changes in tertiary structure have occurred in the chimeric receptor leading to these results.
375
Since ETR2 has the opposite effect on seed germination from ETR1, it will be interesting to determine
376
whether a chimeric ETR1-ETR2 receptor containing the receiver domain of ETR2 has the opposite effect
377
on seed germination from the ETR1 receptor. Interestingly, the changes in seed germination on salt
378
involve ABA since there are differences in ABA responsiveness. However, the mechanism for this is
379
unknown. In contrast to these two traits, genetic studies show that ETR1 His-kinase activity and
380
phosphotransfer are not required for ethylene-stimulated nutations, even though the ETR1 receiver
381
domain is specifically needed for this trait (Kim et al., 2011). Thus, the ETR1 receiver domain has several
382
functions that are not entirely redundant in the control of these three traits.
383
In the current study, we found that there are regions in the receiver domain of ETR1 that are
384
important for the control of ethylene-stimulated nutations and germination under salt stress. Using site-
385
specific mutagenesis of amino acids in the receiver domain we found that Q684, E730, and L734 are
386
required for a functional ETR1 protein in the control of ethylene-stimulated nutations, but not for the
387
other traits studied. Q684 is in a loop region, whereas, E730 and L734 are in the C-terminal tail of the
388
protein. When placed in the crystal model, all three amino acids residues fall along a single face of the
389
receiver domain (Fig. 9B). This surface of the receiver domain is modeled to face the kinase domain
390
(Mayerhofer et al., 2015). It is also noteworthy that ethylene-stimulated nutations are reduced by most
391
point mutations studied suggesting that the entire domain may be involved in the regulation of this
392
phenotype. We also identified two regions at or near the γ loop of the receiver domain that are important
393
in the control of germination on salt. One region is defined by D659 and C661 which are located just
394
before the γ loop and are needed to inhibit seed germination on salt. The other region is in the γ loop and
395
is defined by V665 and E666, which are located at the other end of the γ loop from D659 and C661 (Fig.
396
9B). Mutations in V665 and E666 result in a receptor that inhibits germination more than the wild-type
397
receptor. These mutations do not result in a hyper-functional receptor for the other traits studied. It is
398
interesting to note that the D659A transgene affects growth recovery after ethylene removal (Binder et al.,
399
2004), but C661A, V665A, and E666A do not (this study).
400
The mechanism by which these regions of the receiver domain affect receptor output is not known.
401
One possibility is that these regions are important for receptor-protein interactions. Bacterial two-
402
component receptors are modeled to form higher order clusters through direct interactions with adjacent
403
receptors where the signaling state of one receptor affects the signaling state of surrounding receptors
404
(Bray et al., 1998; Duke and Bray, 1999; Shimizu et al., 2003). Similar models have been invoked for
405
ethylene receptors (Binder and Bleecker, 2003; Binder et al., 2004; Binder et al., 2004; Grefen et al.,
406
2007; Gao et al., 2008; Gao and Schaller, 2009; Liu and Wen, 2012). The crystal structure of the ETR1
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407
receiver domain has been determined revealing a homodimer that is stabilized by the C terminus which
408
forms an extended β sheet (Müller-Dieckmann et al., 1999). However, in solution dimerization may not
409
occur (Hung et al., 2014) and a more recent structural model of the cytosolic domains of ETR1 indicates
410
that the receiver domain of each monomer in a receptor may not come close enough to dimerize
411
(Mayerhofer et al., 2015). None-the-less, the modeling shows that the C-terminal tail could have a role in
412
interactions with other proteins, including the receiver domains of adjacent receptor dimers (Mayerhofer
413
et al., 2015). It is thus possible that the Q684A, E730A, and L734A mutations interfere with these
414
interactions. Interestingly, the γ loop of ETR1 is in a different orientation from other structurally
415
characterized γ loops and has also been proposed to be involved in receptor-protein interactions (Müller-
416
Dieckmann et al., 1999). Thus, amino acids in the γ loop or C-terminal tail or both may mediate specific
417
interactions leading to the regulation of specific traits. Since these regions are on opposite sides of the
418
receiver domain (Fig. 9B), it is likely each region modulates interactions with different proteins. It is
419
currently unclear what receptor-protein interactions are mediated by these two regions of the receiver
420
domain, but based on prior receptor-protein interaction studies, some possibilities include other ethylene
421
receptors, CTR1, EIN2, His-containing phosphotransfer proteins, and response regulator proteins (Clark
422
et al., 1998; Urao et al., 2000; Cancel and Larsen, 2002; Gao et al., 2003; Hass et al., 2004; Grefen et al.,
423
2007; Gao et al., 2008; Scharein et al., 2008; Zhong et al., 2008; Bisson et al., 2009; Bisson and Groth,
424
2010).
425
Our data also support a model where phosphotransfer through the receiver domain is important in the
426
control of certain traits. Support for this is that the D659A mutant affects both growth recovery after
427
ethylene removal (Binder et al., 2004) and germination on salt (this study). The down-stream target for
428
phosphotransfer from the ethylene receptors is unknown. Prior research has documented interactions
429
between ETR1 and both His-containing phosphotransfer proteins and response regulator proteins (Urao et
430
al., 2000; Hass et al., 2004; Scharein et al., 2008; Scharein and Groth, 2011) leading to a model where
431
phosphotransfer occurs from ETR1 to these proteins (Reviewed by: Mason and Schaller, 2005; Bishopp
432
et al., 2006; Shakeel et al., 2013). A direct demonstration of these proteins being down-stream targets of
433
ETR1 is still needed. However, it is noteworthy that some response regulator proteins affect seed
434
germination on salt (Mason et al., 2010) which supports the possibility that these proteins are downstream
435
targets for phoshorelay from ETR1. Since phosphorylation can affect bacterial receiver domain structure
436
leading to changes in dimerization and interactions with other proteins (Reviewed by: Gao and Stock,
437
2009; Bourret, 2010; Galperin, 2010; Capra and Laub, 2012), it is possible that both receptor-protein
438
interactions and phosphotransfer-induced changes in some of these interactions are involved in ETR1
439
sub-functionalization. Based on our data, such a phosphorylation-induced conformational change affects
440
the face of the receiver domain containing the γ loop, but not the face that includes the C-terminal tail.
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441
The exact output of the ethylene receptors is not yet known. The results presented above show that
442
ETR1 has multiple outputs via the receiver domain that modulate various traits. Future work will better
443
delineate ETR1 functions in the control of plant growth and development and identify the underlying
444
mechanisms for these functions.
445
446
Materials and Methods
447
Plant Materials and Chemicals
448
All mutants are in the Columbia background of Arabidopsis thaliana which was used as a wild-type
449
control. The etr1-6;etr2-3;ein4-4 triple mutants and the triple mutants transformed with gETR1, cETR1,
450
D659A, cetr1-ΔR, and 1114 have previously been described (Hall and Bleecker, 2003; Wang et al., 2003;
451
Binder et al., 2004; Binder et al., 2006; Kim et al., 2011). The D659A mutant was previously referred to
452
as getr1-[D] (Binder et al., 2004; Kim et al., 2011). ABA was from ACROS Organics (Belgium) and
453
norflurazon was from Fluka (Switzerland).
454
455
456
Homology Modeling
Three dimensional structural models of the ETR2 and EIN4 receiver domains were generated with
457
Molecular Operating Environment, version 2012.10 (MOE) using the ETR1 receiver domain as a
458
template structure (PDB 1DCF). This template was used because, of the possible templates identified by
459
MOE, it had the highest Z value. The ETR1 receiver domain protein sequence and each of the other two
460
receiver domain sequences were aligned with structural alignment enabled. For the alignment between the
461
ETR1 and ETR2 receiver domains a blosum35 substitution matrix was used and for the alignment
462
between the ETR1 and EIN4 receiver domains a blosum45 substitution matrix was used. The structures
463
were prepared and errors fixed both automatically with MOE and manually. The Protonate 3D function of
464
MOE was used to assign protonation states and homology models were then obtained using the
465
CHARMM27 force field (Foloppe and MacKerell, 2000). An ensemble of 10 possible structures for each
466
receiver domain was generated and the models ranked using MOE to calculate the Root-Mean-Square
467
Deviation values of atomic positions. These values ranged from 0.31 Å to 0.68 Å for EIN4 models and
468
0.36 Å to 0.90 Å for ETR2 models. The model for each receiver domain with the lowest value was then
469
superposed on the crystal structure of the ETR1 receiver domain and alterations in the positions of back-
470
bone carbons evaluated.
471
472
Plasmid Construction, Generation of Transgenic Lines, and RT-PCR
473
The cloning of gETR1 into pBluescript and pPZP211 has been previously described (Wang et al.,
474
2003). Several silent mutations were incorporated into pBluescript II SK- gETR1 and subsequently cloned
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475
into pPZP211-gETR1 with MfeI-HF and AflII (now called pPZP211-gETR1 silent) leading to a novel
476
avrII restriction site in gETR1. These mutations, along with the following ones, were made using LaTaq
477
polymerase (TaKaRa Bio. Inc.) according to the manufacturer’s instructions. The following point
478
mutations were made in pBluescript II SK-gETR1 and then cloned into pPZP211-gETR1 silent using
479
AflII and KpnI: E617A, N618A, C661A, V665A, E666A, N667A, Q681A, R682A, Q684A, E730A, and
480
L734A. All plasmid constructs and point mutations were confirmed by sequencing. Primers for the
481
generation of these mutants are listed in Supplemental Table S1
482
All of the transgene constructs created above were transformed into Agrobacterium tumefaciens strain
483
GV3101 pMP90 and then transformed into etr1-6;etr2-3;ein4-4 Arabidopsis plants using the floral dip
484
method (Clough and Bent, 1998). Two to three homozygous lines were identified for each transgenic line.
485
RNA was extracted from 10 or more seedlings using the RNA Plant Extraction Kit from Qiagen and
486
DNA was cleaned using the Turbo DNase Kit from Ambion and PCR amplification was carried out using
487
the One-Step RT-PCR Kit from Qiagen using primers described previously (Kim et al., 2011). The
488
resultant amplification products were run on an 1.5% (w/v) agarose gel and detected with UV
489
illumination. Transcript levels of β-tubulin were analyzed as a control using primers described previously
490
(Gao et al., 2008).
491
492
493
High-Resolution, Time-Lapse Imaging and Analysis of Growth Rate and Nutation Angles
Arabidopsis seeds were surface sterilized with 70% alcohol for 30 seconds, placed on sterile filter
494
paper to dry, and then placed on agar plates containing 0.8% (w/v) agar, half-strength Murashige and
495
Skoog basal salt mixture, (Murashige and Skoog, 1962), pH 5.7 fortified with vitamins and with no added
496
sugar. Seeds were treated for 2 to 8 days at 4o C, treated with light for 4 to 8 h under continuous
497
fluorescent lights, and then allowed to grow in the dark for 2d on vertically orientated plates for time-
498
lapse imaging experiments. Time-lapse imaging of dark-grown Arabidopsis hypocotyls was carried out
499
using methods previously described with Marlin CCD cameras (Allied Vision Technology) using infrared
500
lighting (Binder et al., 2004; Binder et al., 2004; Binder et al., 2006; Kim et al., 2011). For growth
501
kinetics measurements, images were taken every 5 min. To measure growth response kinetics, seedlings
502
were grown in air for 1h followed by treatment for 2h with 10 µL L-1 ethylene to examine growth
503
inhibition kinetics. This was followed by a 5h treatment with air to examine growth recovery kinetics. For
504
treatment with silver, 100 µM AgNO3 was included in the agar. In these experiments, seedlings were
505
grown for 1h in air followed by treatment with 1 µL L-1 ethylene to examine growth inhibition kinetics as
506
described by McDaniel and Binder (2012). The growth rate of each seedling was analyzed using custom
507
software (Parks and Spalding, 1999; Folta and Spalding, 2001) and normalized to the growth rate in air
508
prior to application of ethylene. Growth rate in air was quantified from the first hour of measurements
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509
before ethylene was added. To measure nutational bending, seedlings were treated with 10 µL L-1
510
ethylene for 24h and images acquired every 15 min. The angles of each hypocotyl was measured
511
manually and nutation amplitude determined as previously described (Binder et al., 2006). All
512
experiments under all conditions were repeated in at least three separate experiments.
513
514
515
Seed Germination Experiments
Seed germination experiments were carried out according to the methods of Wilson et al. (2014).
516
Briefly, to reduce biological variation, seeds were collected from plants grown together under uniform
517
conditions (Hensel et al., 1993). Seeds were collected on the same day, stored in a desiccator for at least 3
518
weeks, mechanically sorted using the methods of Ellwell et al. (2011), and seeds between 250 and 300
519
µm in size were used. Seeds were surface sterilized with 70% (v/v) ethanol for 30s, dried, then placed on
520
agar plates as described above that contained either no salt or 150 mM NaCl. In some experiments, ABA
521
or the ABA biosynthesis inhibitor, norflurazon, was included at the indicated concentration. These were
522
prepared as 1000x stocks in ethanol and added after autoclaving. Solvent control plates contained 0.1%
523
(v/v) ethanol. The plates were sealed with porous surgical tape (3M) to prevent accumulation of ethylene
524
(Buer et al., 2003). Twenty seeds of a genotype were placed on a plate and three plates were used per
525
condition. Plates were kept vertically under long-day photoperiod (16h light/8h dark) at 20°C to 21°C
526
under 12-13 µmol m-2 sec-1 white light. The number of seeds that germinated on each plate was
527
determined every 12h for 7d. Germination was scored as the rupture of the testa (seed coat).
528
529
530
RNA Isolation and qRT-PCR
The transcript abundance of several Arabidopsis genes were examined using qRT-PCR. This
531
included gene transcripts for CRA1 and RAB18 which are ABA responsive (Lång and Palva, 1992;
532
Gliwicka et al., 2012). For this, total RNA was isolated from 25 mg (dry weight) of seeds that were sown
533
in the presence or absence of 1 µM ABA or 150 mM NaCl. RNA was isolated using to the methods of
534
Meng and Feldman (2010) as modified by Wilson et al. (2014). Transcript data were normalized to
535
At3g12210 (Dekkers et al., 2011) using the method of Livak and Schmittgen (2001) for each seed line for
536
each condition to obtain the relative amounts of target gene transcripts between plant backgrounds for
537
each treatment. These levels were then normalized to the levels observed in untreated wild-type seeds.
538
The primers used for the analysis of these transcripts have been previously described (Fujii et al., 2007;
539
Gliwicka et al., 2012).
540
541
542
Statistics
Data were analyzed using Student’s t test and considered statistically different at P < 0.05.
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Supplemental Material
The supplement contains the following figures and table.
546
Supplemental Figure S1. Time for 50% of point mutant seeds to germinate in response to NaCl.
547
Supplemental Figure S2. Time for 50% of chimeric receptor seeds to germinate in response to NaCl.
548
Supplemental Figure S3. Time for 50% of select point mutant seeds to germinate in response to ABA.
549
Supplemental Figure S4. Change in transcript abundance of RAB18 and CRA1 in response to NaCl and
550
551
552
553
norfluorazon.
Supplemental Figure S5. Time for 50% of select point mutant seeds to germinate on NaCl in response to
norflurazon.
Supplemental Table S1. Primers used for site-directed mutagenesis
554
555
556
557
Accession Numbers
Arabidopsis Genome Initiative accession numbers for genes studied in this article are: ETR1,
At1g66340; EIN4, At3g04580; ETR2, At3g23150; CRA1, At5g44120; RAB18.
558
559
560
Acknowledgements
The authors thank Jerome Baudry and Karan Kapoor for useful discussions on homology modeling
561
and Rachel Barker, Gabriel Dagotto, Katrina Deponte, Rebecca Murdaugh, Katherine Odom, David
562
Pease, Amanda Wehner, and Zack Zenn for technical help. This work was supported by a NSF grant
563
(IOS-1254423) to BMB.
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Figure Legends
876
Figure 1. The ETR1 receiver domain is not required for response to silver ions. (A) Domain structure
877
of full-length and truncated ETR1 receptor lacking the receiver domain used in this study. (B) Growth
878
kinetic profiles in the presence of 100 µM AgNO3 are shown for etr1-6;etr2-3;ein4-4 triple mutants
879
transformed with cDNA encoding for either a full-length ETR1 (cETR1) or a truncated transgene lacking
880
the receiver domain (cetr1-ΔR). For comparison, responses of wild-type and triple mutant seedlings are
881
shown. Seedlings were allowed to grow for 1h in air, followed by the addition of 1 µL L-1 ethylene (down
882
arrow). Growth rate was normalized to the growth rate in the air pre-treatment and represents the average
883
± SEM. Lines are a moving average calculated with a period of 2 using Microsoft Excel.
884
885
Figure 2. Receiver domain amino acids targeted for mutagenesis. (A) An amino acid sequence
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alignment of the receiver domains from ETR1, ETR2 and EIN4 was performed using Clustal W.
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Conserved residues are shaded gray. Amino acids targeted for mutagenesis to alanines in this study are
888
marked with open circles. Amino acid residues for each protein are numbered at the right. The closed
889
circle marks the conserved Asp (D659 in ETR1) involved in phosphotransfer that has previously been
890
mutated (Binder et al., 2004). (B) Homology models of the ETR2 and EIN4 receiver domains (black)
891
were generated as described in the “materials and methods”, and compared to the crystal structure of
892
ETR1 (white). Regions where the backbone carbons of the models diverge from the ETR1 structure are
893
marked with asterisks. (C) Transcript levels of receptor transgenes. The location of each point mutation is
894
labeled. All constructs were genomic DNA constructs and were transformed into the etr1-6;etr2-3;ein4-4
895
triple mutant background as described in the "materials and methods". RNA expression level for each
896
transgene was analyzed using RT-PCR. The transgene transcripts (marked with arrow) ran as a smaller
897
product than the etr1-6 product as we have previously described (Kim et al., 2011). Transcript levels for
898
β-tubulin in each plant line are shown as a control.
899
900
Figure 3. Growth, nutations, and growth recovery are differentially rescued by etr1 point mutant
901
transgenes. The etr1-6;etr2-3;ein4-4 triple mutants were transformed with genomic DNA encoding a
902
mutant etr1 transgene containing the indicated point mutation. For comparison, data from wild-type, etr1-
903
6;etr2-3;ein4-4 triple mutants, and triple mutants transformed with a wild-type genomic ETR1 (gETR1)
904
transgene are included. (A) The growth rates in air of triple mutants transformed with the indicated
905
receptor are plotted. Growth rate in air was determined from the first hour of growth kinetic
906
measurements prior to the introduction of ethylene. Data represents the average growth rate ± SEM. (B)
907
Nutations in response to 10 µL L-1 ethylene were measured in etr1-6;etr2-3;ein4-4 triple mutants
908
transformed with the indicated receptor. The average peak nutation amplitude ± SEM is plotted. (C-E)
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909
Growth kinetic profiles for triple mutants transformed with receptors are shown. Seedlings were allowed
910
to grow for 1h in air, followed by the addition of 10 µL L-1 ethylene (down arrow). Ethylene was removed
911
2h later (up arrow) and seedlings grown in air for an additional 5h. Growth rate was normalized to growth
912
rate in the air pre-treatment and represents the average ± SEM. Lines are a moving average calculated
913
with a period of 2 using Microsoft Excel. In panels A and B, data were analyzed with t tests and increases
914
caused by the transgene over the etr1-6;etr2-3;ein4-4 triple mutants were considered statistically
915
significant for P < 0.05 (*).
916
917
Figure 4. The rapid germination on salt of etr1;etr2;ein4 triple mutants is differentially reversed by
918
etr1 point mutant transgenes. Germination time-courses of etr1-6;etr2-3;ein4-4 triple mutants
919
transformed with genomic DNA encoding a mutant etr1 transgene containing the indicated point mutation
920
were determined in the absence (A) or presence (B-D) of 150 mM NaCl. For comparison, data from wild-
921
type, etr1-6;etr2-3;ein4-4 triple mutants, and triple mutants transformed with a wild-type genomic ETR1
922
(gETR1) transgene are included in each panel. The percent of seeds that germinated was determined every
923
12h. All experiments were performed in triplicate. The average percent seed germination ± SD at each
924
time is plotted for each seed line.
925
926
Figure 5. The rapid germination on salt of etr1;etr2;ein4 triple mutants is not reversed by an ETR1-
927
EIN4 chimeric receptor transgene containing the EIN4 receiver domain. Germination time-courses
928
of etr1-6;etr2-3;ein4-4 triple mutants transformed with cDNA for a chimeric ETR1-EIN4 receptor
929
containing the receiver domain of EIN4 (1114) were determined in the presence and absence of 150 mM
930
NaCl. Germination time-courses of wild-type, etr1-6;etr2-3;ein4-4 triple mutants, and triple mutants
931
transformed with cDNA for full-length ETR1 (cETR1) or a truncated ETR1 lacking the receiver domain
932
(cetr1-ΔR) are included for comparison. All experiments were performed in triplicate. The average
933
percent seed germination ± SD at each time is plotted for each seed line.
934
935
Figure 6. ABA phenocopies the effects of NaCl. Germination time-courses of etr1-6;etr2-3;ein4-4 triple
936
mutants transformed with genomic DNA encoding a mutant etr1 transgene containing the indicated point
937
mutation were determined. For comparison, data from wild-type, etr1-6;etr2-3;ein4-4 triple mutants, and
938
triple mutants transformed with a wild-type genomic ETR1 (gETR1) transgene are included. Seeds were
939
germinated in the presence or absence of 1µM ABA. All experiments were performed in triplicate. The
940
average percentage of seed germination ± SD at each time is plotted for each seed line.
941
942
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Copyright © 2015 American Society of Plant Biologists. All rights reserved.
943
Figure 7. ABA changes the transcript abundance of RAB18 and CRA1. The levels of transcript for
944
CRA1and RAB18 were measured using qRT-PCR. For this, seeds were germinated for 2d in the presence
945
or absence of 1µM ABA and mRNA extracted from wild-type and etr1-6;etr2-3;ein4-4 triple mutants as
946
well as triple mutants transformed with the indicated mutant transgene. Data were normalized to the
947
levels of At3g12210 in each seed line to determine the relative transcript level for each gene. These were
948
then normalized to levels of the transcript in untreated wild-type seeds. The average ± SEM for two
949
biological replicates with three technical replicates each is shown. aABA caused a statistically significant
950
increase in transcript levels (P < 0.05). bStatistically different from wild-type seeds in the same condition
951
(P < 0.05). All P values were calculated by t test.
952
953
Figure 8. Norflurazon reduces differences in germination between select point mutants. Germination
954
time-courses of etr1-6;etr2-3;ein4-4 triple mutants transformed with genomic DNA encoding a mutant
955
etr1 transgene containing the indicated point mutation were determined. For comparison, data from wild-
956
type, etr1-6;etr2-3;ein4-4 triple mutants, and triple mutants transformed with a wild-type genomic ETR1
957
(gETR1) transgene are included. Seeds were germinated on 150 mM NaCl in the absence or presence of
958
the indicated concentrations of the ABA biosynthesis inhibitor norflurazon. All experiments were
959
performed in triplicate. The average percentage of seed germination ± SD at each time is plotted for each
960
seed line.
961
962
Figure 9. Models for ETR1 receiver domain output. (A) The ETR1, ETR2, and EIN4 receptors form
963
homodimers with each monomer containing an ethylene-binding, GAF, kinase, and receiver domain as
964
labeled. The conserved His (H) and Asp (D) amino acid residues involved in phosphotransfer are shown.
965
The receiver domain is predicted to have several roles in the control of several phenotypes. In this model,
966
two outputs require ETR1 His-kinase activity where transphosphorylation from one monomer to a
967
conserved His in the other monomer occurs. This phosphate then transfers to conserved Asp residues in
968
the receiver domains of ETR1, ETR2, and EIN4. In this model, growth recovery after the removal of
969
ethylene is stimulated by phosphotransfer to all three receptors. By contrast, germination on salt is only
970
controlled by phosphotransfer to the receiver domain of ETR1 which acts to inhibit germination.
971
Ethylene-stimulated nutations require the ETR1 receiver domain, but are stimulated via a
972
phosphotransfer-independent mechanism. (B) Two views are shown of the tertiary structure of the
973
backbone carbons of the ETR1 receiver domain based on the published crystal structure (Müller-
974
Dieckmann et al., 1999). The positions of the amino acid residues determined to have the largest effects
975
on ethylene-stimulated nutations (Q684, E730, L734) and germination on salt (D659, C661, V665, E666)
976
are shown as space-filled representations.
977
-29-
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Copyright © 2015 American Society of Plant Biologists. All rights reserved.
978
Table 1: Summary of rescue of traits by ETR1 transgenes
Phenotype Rescueda
etr1-6;etr2-3;ein4-4
transformed with:
Growth in
air
Growth recovery
Ethylene-
after ethylene
stimulated
Germination rate
b
nutations
during salt stressd
b
removal
c
gETR1
+++
+++
+++
+++
E617A
+++
+++
+
+++
N618A
+++
++++
+
+++
D659A
+++e
+e
+++f
+
C661A
+++
++
+
+
V665A
++
++
+
+++++
E666A
++
+++
+
+++++
N667A
+++
+++
+
++
Q681A
++
++++
+
++++
R682A
++
++
+
+++
Q684A
++
++++
-
++
E730A
++
++++
-
++
L734A
++
+++
-
++
979
980
a
981
b
982
rescue, ++; 30-50% rescue, +; no rescue, -.
983
c
984
comparable, +++; recovery time slightly slower, ++; and recovery time very slow and only slightly faster
985
than the etr1-6;etr2-3;ein4-4 mutants,+.
986
d
987
65% slower, +++++; 20- 65% slower, ++++; comparable, +++; 15-35 % faster, ++; > 35% faster, +.
988
Based on data from eBinder et al. (2004) and fKim et al. (2011).
Rescue for each mutant transgene was scored relative to the rescue obtained with the gETR1 transgene.
Results of growth in air and nutations (Fig. 3) were scored as follows: >70% rescue, +++; 50-70%
Results for growth recovery (Fig. 3) were scored as follows: recovery time faster, ++++; recovery time
Results for the time for 50% germination on salt (Supplemental Figure S1) were scored as follows: >
989
990
991
-30-
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