© 2008 The Author(s).
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RESEARCH PAPER |
Tomato ethylene receptor–CTR interactions: visualization of NEVER-RIPE interactions with multiple CTRs at the endoplasmic reticulum
Plant Sciences Division, School of Biosciences, University of Nottingham, Sutton Bonington Campus, Loughborough LE12 5RD, UK
To whom correspondence should be addressed. E-mail: Donald.Grierson{at}Nottingham.ac.uk
Received 5 November 2007; Revised 16 January 2008 Accepted 16 January 2008
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Abstract |
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In the model plant Arabidopsis, members of a family of two-component system His kinase-like ethylene receptors have direct protein–protein interactions with a single downstream Ser/Thr kinase CTR1. These components of the ethylene signalling network found in Arabidopsis are conserved in the climacteric fruit tomato, but both the ethylene receptors and CTR1-like proteins (LeCTRs) in tomato are encoded by multigene families. Here, using a yeast two-hybrid interaction assay, it is shown that the tomato receptors LeETR1, LeETR2, and NEVER-RIPE (NR) can interact with multiple LeCTRs. In vivo protein localization studies with fluorescent tagged proteins revealed that the ethylene receptor NR was targeted to the endoplasmic reticulum (ER) when transiently expressed in onion epidermal cells, whereas the four LeCTR proteins were found in the cytoplasm and nucleus. When co-expressed with NR, three LeCTRs (1, 3, and 4), but not LeCTR2, also adopted the same ER localization pattern in an NR receptor-dependent manner but not in the absence of NR. The receptor–CTR interactions were confirmed by biomolecular fluorescence complementation (BiFC) showing that NR could form a protein complex with LeCTR1, 3, and 4. This suggested that ethylene receptors recruit these LeCTR proteins to the ER membrane through direct protein–protein interaction. The receptor–CTR interactions and localization observed in the study reinforce the idea that ethylene receptors transmit the signal to the downstream CTRs and show that a single receptor can interact with multiple CTR proteins. It remains unclear whether the different LeCTRs are functionally redundant or have unique roles in ethylene signalling.
Key words: BiFC, endoplasmic reticulum, Ser/Thr kinase, tomato ethylene signalling, two-component system His kinase
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Introduction |
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TopAbstractIntroductionMaterials and methodsImagingResultsDiscussionSupplementary dataReferences |
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Ethylene, a gaseous phytohormone, regulates many aspects of plant growth and development including seed germination, seedling growth, fruit ripening, organ senescence, and abscission, and is also involved in the reaction of plants to biotic and abiotic stresses such as wounding, pathogen attack, drought, and salinity (Abeles et al., 1992). The majority of our understanding regarding ethylene perception and signalling has been realized through the study of mutants in the model plant Arabidopsis thaliana, where ethylene is perceived by a family of membrane-bound receptors consisting of ETR1, ERS1, ETR2, ERS2, and EIN4 (Chang et al., 1993; Hua et al., 1995, 1998; Sakai et al., 1998). ETR1 is the founding member of the receptor family and has been localized to the endoplasmic reticulum (ER) membrane (Chen et al., 2002). It has been suggested that the ethylene-binding site is located in the first two transmembrane domains of the receptor and that the binding is mediated by a copper co-factor (Schaller and Bleecker, 1995; Rodriguez et al., 1999; Wang et al., 2006). The C-terminal half of the receptor, which shows sequence similarity to the bacterial two-component system His kinases, contains a His kinase domain and, in some cases, a receiver domain (in ETR1, ETR2, and EIN4). The Arabidopsis ethylene receptors are divided into two subfamilies. The subfamily I ethylene receptors (ETR1 and ERS1) contain all the conserved residues essential for His kinase activity, whereas the subfamily II receptors (ETR2, ERS2, and EIN4) lack some residues believed to be important for His kinase activity. Additionally, subfamily II receptors have an extra hydrophobic region in their N-termini, which could be a membrane-spanning domain or a signal sequence targeting the protein to the secretory pathway. Single loss-of-function (LOF) mutants of the ethylene receptors have little or no effect on ethylene signalling, whilst combinations of multiple receptor LOF mutants showed constitutive ethylene responses (Hua and Meyerowitz, 1998; Wang et al., 2003; Xie et al., 2006). This indicates that there is some functional redundancy among ethylene receptors. It also suggests that each receptor serves as a negative regulator of the ethylene signalling pathway because elimination of receptors activates the downstream ethylene responses.
CTR1, a Ser/Thr protein kinase, acts downstream of the Arabidopsis ethylene receptors (Kieber et al., 1993). The carboxylic half of CTR1 shows high sequence similarity to the Raf family of mitogen-activated protein (MAP) kinases, suggesting that a MAP kinase phosphorelay cascade might be employed in ethylene signal transduction. It has been demonstrated that the N-terminus of CTR1 can interact directly with the subfamily I ethylene receptors (ETR1 and ERS1) in the yeast two-hybrid assay (Clark et al., 1998), whereas the subfamily II receptor ETR2 might also associate weakly with CTR1 (Cancel and Larsen, 2002). LOF mutations in CTR1 render plants showing constitutive ethylene responses, and its recessive nature indicates that CTR1 negatively regulates the ethylene signalling pathway. Notably, a missense CTR1 mutant ctr1-8, resulting from a Gly354 to Glu change in its CTR N-terminal (CN) motif, also develops constitutive ethylene responses (Huang et al., 2003). The CN motif-mutated CTR1-8 lacks the ability to interact with the ethylene receptors, suggesting that the protein–protein association between ethylene receptors and CTR1 is crucial to suppress the downstream ethylene responses.
Knowledge of the detailed ethylene signalling pathway defined in Arabidopsis enables comparative analyses to be carried out in other important crop species such as tomato where ethylene is critically involved in the fruit ripening process. In tomato, a family of six ethylene receptors (LeETR1–LeETR6; LeETR3 is referred to as NR for historical reasons) has been identified (Wilkinson et al., 1995; Zhou et al., 1996; Lashbrook et al., 1998; Tieman and Klee, 1999). The structure of the tomato ethylene receptor family is similar to that in Arabidopsis. Subfamily I tomato ethylene receptors LeETR1, LeETR2, and NR contain all the essential residues for His kinase function, whereas the subfamily II ethylene receptors (LeETR4–LeETR6) lack some conserved kinase residues. The Arabidopsis model shows that single ethylene receptor LOF mutants do not have a major effect upon ethylene signalling, possibly due to functional redundancy. However, this paradigm does not hold in the case of tomato, where reduction in either the LeETR4 or LeETR6 mRNA level causes an ethylene hypersensitive phenotype (Tieman et al., 2000). In addition, it has been observed that in transgenic tomato plants where NR expression is reduced by antisense inhibition, expression of LeETR4 increases proportionally. It appears, therefore, that somehow the tomato plant compensates for the loss of NR by increasing the expression of LeETR4. This phenomenon, referred to as functional compensation, has not been observed in Arabidopsis (Tieman et al., 2000; Kevany et al., 2007).
Unlike Arabidopsis, which only contains one constitutively expressed CTR1 gene, a family of four CTR1-like genes (LeCTR1–LeCTR4) has been identified in tomato (Lin et al., 1998; Leclercq et al., 2002; Adams-Phillips et al., 2004). Three tomato CTRs (LeCTR1, LeCTR3, and LeCTR4) show similar degrees of sequence homology to the Arabidopsis CTR1, whereas LeCTR2 appears to be more similar to an Arabidopsis MAP kinase kinase kinase EDR1, which is a negative regulator of disease resistance and ethylene-induced senescence (Frye et al., 2001). All four LeCTRs show sequence conservation of the N-terminal CN motif (Huang et al., 2003), which is important for the ethylene receptor–CTR1 interaction in Arabidopsis, whilst their C-termini contain all the conserved subdomains common to protein kinases. It has been shown that three tomato CTRs (LeCTR1, LeCTR3, and LeCTR4) are able to participate in ethylene signal transduction in Arabidopsis, as demonstrated by their ability to complement the AtCTR1 LOF mutants (Adams-Phillips et al., 2004). The Arabidopsis model suggests that all receptors could activate CTR1 to suppress downstream ethylene responses in the absence of ethylene. On the other hand, the multiple tomato ethylene receptors and CTRs are differentially regulated during development and in response to stimuli. It is therefore possible that specific tomato CTRs could interact with specific receptors and regulate different ethylene responses. To gain more insight into the tomato ethylene signalling mechanism, the protein–protein interactions between tomato ethylene receptors and CTRs have been characterized by testing protein association in yeast and by in vivo subcellular localization of fluorescent tagged proteins in onion epidermal cells.
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Materials and methods |
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Yeast two-hybrid assay
The tomato ethylene receptors (LeETR1132–754, LeETR2115–732, NR117–635, LeETR4135–761, LeETR5135–767, and LeETR6133–354) and the putative downstream kinases (LeCTR11–380, LeCTR250–700, LeCTR31–408, and LeCTR41–793) were cloned into yeast two-hybrid bait plasmid pEG202 and prey plasmid pJG4-5, respectively. Combinations of these constructs were transformed to yeast strain EGY48 carrying the reporter plasmid pSH18-34, and the yeast two-hybrid assay was carried out as previously described (Bartel and Fields, 1997).
Biolistic transformation
Transient gene expression in onion epidermal cells was carried out using a Biolistic PDS-1000/He Particle Delivery System (Bio-Rad) following the manufacturer's protocol. Gold particles (sphere 0.8–1.5 µm, AlfaAesar) were coated with 3 µg of plasmid DNA and fired into onion epidermal peels placed on MS medium using 1100 psi rupture discs (Bio-Rad) under a vacuum of 26–28 in Hg. The Petri dishes containing the onion peels were then incubated in the dark at room temperature prior to imaging. An incubation time of 12–14 h was used for the protein subcellular localization studies, and a shorter incubation time (6–8 h) was used for the biomolecular fluorescence complementation (BiFC) experiment to avoid self-association resulting from overexpression. The cyan fluorescent protein (CFP) variant Cerulean (Rizzo et al., 2004), the green fluorescent protein (GFP) variant EGFP (BD Clonetech), the yellow fluorescent protein (YFP) variant Venus (Nagai et al., 2002), and the red fluorescent protein mRFP1 (Campbell et al., 2002) were used in these experiments. The full-length cDNA of receptor NR and the genomic DNA of EIN3 were cloned to a CFP expression vector pDH51-GW-CFP (AM773751
[GenBank]
; S Zhong, unpublished data). The cDNAs encoding each LeCTR N-terminus (LeCTR11–560, LeCTR21–537, LeCTR31–566, and LeCTR41–541) were cloned to pDH51-GW-YFP (AM773752
[GenBank]
). In the BiFC constructs, the full-length NR and N-terminus of LeCTR were cloned as C-terminal fusions to the fluorescent protein fragments in vector pDH51-GW-YFPn (AM779183
[GenBank]
) and pDH51-GW-YFPc (AM779184
[GenBank]
), respectively. The CN motif-mutated LeCTR3 N-terminus (LeCTR3m) was generated by overlapping PCR using mismatch oligos: 5-CTGTCTGTTGAGCTTTGCAGGCAT-3 and 5-CCTGCAAAGCTCAACAGACAGGCT-3.
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Imaging |
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All images were obtained using a Leica TCS SP2 AOBS laser confocal scanning microscope. CFP was excited using a 458 nm laser and its emission was measured from 465 to 505 nm. The excitation wavelength for YFP is 514 nm and its emission was measured from 525 to 600 nm (525–560 nm when in the presence of mRFP1). For the BiFC experiments, the mRFP1 was exciting with the 514 nm laser and its emission was measured from 600 nm to 725 nm. Simultaneous imaging of CFP-YFP was achieved by using the line-by-line sequential scan mode to avoid signal bleed through between the CFP and YFP channels.
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Results |
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LeCTRs interact with ethylene receptors in the yeast two-hybrid system
The six tomato ethylene receptors were fused to the LexA DNA-binding domain in the bait plasmid pEG202, whilst LeCTRs were cloned into the prey plasmid pJG4-5. Different combinations of these constructs were co-transformed into yeast strain EGY48 carrying a LacZ reporter plasmid pSH18-34 and a chromosomally located LEU reporter gene. Both the β-galactosidase assay and the LEU activity assay indicated that ethylene receptors LeETR1 and LeETR2 interacted with all four LeCTRs, whilst NR interacted only with LeCTR1, LeCTR3, and LeCTR4 (Fig. 1). The subfamly II ethylene receptors (LeETR4, LeETR5, and LeETR6), however, showed no interaction with any of the LeCTRs.
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ER localization of LeCTR1, LeCTR3, and LeCTR4 is dependent on the NR ethylene receptor
In order to gain more insight into the ethylene receptor–CTR interaction, fluorescent protein-assisted localization experiments were carried out to investigate both the subcellular localization and possible association between NR (LeETR3) and the LeCTRs. The N-terminal domains of LeCTRs were fused to YFP under the control of the cauliflower mosaic virus (CaMV) 35S promoter. When transiently expressed in onion epidermal cells, all LeCTR–YFP fusion proteins were found in the cytoplasm and nucleus, and co-localized with the CFP, which served as a control for localization in the cytosol and nucleus (Haseloff et al., 1997). Co-expression of a nuclear transcription factor ETHYLENE INSENSITIVE3 (Chao et al., 1997) fused to CFP and LeCTR1–YFP confirmed the nuclear identification and location of the LeCTR–YFP proteins (Fig. 2H). An ER-targeted CFP, which has an N-terminal ER targeting sequence of the pumpkin 2S albumin and C-terminal ER retention signal HDEL (Matsushima et al., 2002), was also introduced into onion cells and showed a different localization pattern from those of the cytosolic LeCTRs, notably exclusion from the nucleus (Fig. 2E). To show that LeCTRs themselves could not be targeted to the ER, the LeCTR–YFP fusion proteins were simultaneously expressed with the ER-targeted CFP. Confocal projection images of the membrane regions in the co-transformed onion cells showed that the ER-targeted CFP highlighted the ER network, whereas YFP produced a diffuse image from the cytoplasm, which is surrounding the ER (Fig. 3A). When expressed alone without ethylene receptor, the LeCTR–YFP fusion proteins also generated the same diffuse image as the YFP control, suggesting a cytoplasmic localization (Fig. 3B–E). The separate localization of the proteins was confirmed by superimposing the fluorescent signals from CFP (false colour green) and YFP (false colour red) as merged images (Fig. 3).
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The full-length ethylene receptor NR fused to CFP (NR–CFP) was localized predominantly at the ER (Fig. 3F–I), when transiently expressed in onion epidermal cells under the control of a CaMV 35S promoter. When LeCTR–YFP was co-expressed with the NR–CFP constructs, LeCTR1, LeCTR3, and LeCTR4 were re-directed to the ER and co-localized with NR–CFP, as indicated by the precise reticulate pattern observed in both CFP and YFP channels and when superimposed (Fig. 3F, H, I). However, the LeCTR2–YFP still generated a more diffuse pattern from the cytoplasm and was not concentrated in the ER when co-expressed with NR–CFP (Fig. 3G). These observations showed that the ER localization of LeCTR1, LeCTR3, and LeCTR4 occurs only in the presence of the ethylene receptor NR. These in vivo subcellular protein localization results are thus in line with the yeast two-hybrid assay and suggest that NR associates directly with these three LeCTRs. Confirmation of these findings was sought by BiFC.
Biomolecular fluorescence complementation
Efforts to measure Forster resonance energy transfer between NR and multiple LeCTR proteins were not successful, possibly due to the weak fluorescence and the fast-moving nature of the NR protein (see Supplementary NR Movie S1 at JXB online). BiFC is an alternative experimental approach for in vivo visualization of protein–protein interaction, which is based on the formation of a functional fluorescent complex by the association of two non-fluorescent YFP fragments (Hu and Kerppola, 2003). The ethylene receptor NR was fused to the N-terminus of YFP (amino acids 1–154), whilst the N-termini of each LeCTR were linked to the C-terminal half of YFP (amino acids 155–238) and referred to as NR–YFPn and LeCTR–YFPc, respectively. If the YFPn and YFPc fragments were brought together by the NR–LeCTR interaction, a functional YFP fluorescent complex could be re-generated and therefore served as an indicator of the NR–LeCTR interaction. In onion epidermal cells co-transformed with combinations of these BiFC constructs, the NR–LeCTR1, NR–LeCTR3, and NR–LeCTR4 combinations generated strong BiFC signals, whereas the NR–LeCTR2 pair produced no fluorescence (Fig. 4). The specificity of the BiFC interaction was tested by introducing into LeCTR3 (referred to as LeCTR3m) an equivalent of the G to A mutation in the CN motif of CTR1-8, which is able to interfere with the interaction between the Arabidopsis ethylene receptor and the mutated CTR1-8 protein (Huang et al., 2003). The formation of the NR–LeCTR3m BiFC complex was severely weakened, as expected (Fig. 4E).
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Discussion |
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TopAbstractIntroductionMaterials and methodsImagingResultsDiscussionSupplementary dataReferences |
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Components of the Arabidopsis ethylene signalling network are conserved in tomato, but the complexity is greater. Tomato perceives ethylene with at least six putative receptors, and the ethylene signal is then transmitted to a family of downstream kinases (LeCTRs), whereas Arabidopsis has five receptors and only one downstream kinase (AtCTR1). This study focused on the protein–protein interactions between tomato ethylene receptors and the LeCTR proteins, and showed that a single ethylene receptor (NR) is capable of interacting with multiple LeCTRs at the ER.
The yeast two-hybrid assay showed positive interactions between LeCTRs (1, 3, and 4) and the subfamily I ethylene receptors (LeETR1, LeETR2, and NR) (Fig. 1). The NR–LeCTR interactions were further confirmed by co-localization and BiFC experiments (Figs 3, 4). The yeast interaction results also suggested that the tomato ethylene receptors LeETR1 and LeETR2 could associate with LeCTR2. LeCTR2 shares sequence similarity to Arabidopsis EDR1 (ENHANCED DISEASE RESISTANCE1), which regulates the Arabidopsis defence response, but is also involved in stress, cell death, and ethylene-induced leaf senescence (Tang et al., 2005). Further work will be required to establish whether or not LeCTR2 is involved in ethylene signalling. Interestingly, subfamily II ethylene receptors (LeETR4, 5, and 6) showed no detectable protein–protein interaction with LeCTR proteins in the yeast two-hybrid assay (Fig. 1). This is consistent with the findings that Arabidopsis CTR1 associated weakly with only one subfamily II receptor (ETR2) but interacted strongly with all subfamily I receptors (Clark et al., 1998; Cancel and Larsen, 2002). Although the Arabidopsis subfamily I receptors seem to play a more prominent role, tomato and Arabidopsis subfamily II receptors can bind ethylene and participate in ethylene signalling (Hua and Meyerowitz, 1998; Tieman et al., 2000; O'Malley et al., 2005; Kevany et al., 2007). It would be tempting to speculate that subfamily II receptors, which lack the conserved residues for His kinase activity and could not interact strongly with CTR (Cancel and Larsen, 2002), might require subfamily I receptors to pass the signal to the downstream CTR, for example by forming a heterodimer or multimer to interact with CTR.
To test the significance of the NR–LeCTR interactions in vivo, ethylene receptor NR and LeCTRs tagged with fluorescent proteins were transiently expressed in onion epidermal cells. The protein localization results indicated that the tomato ethylene receptor NR was targeted to the ER (Fig. 3), which is in agreement with the reported Arabidopsis ETR1 localization (Chen et al., 2002). LeCTRs expressed on their own were localized to the cytoplasm and nucleoplasm (Fig. 2). This is expected since all LeCTRs lack ER targeting signals or retention sequences, and small soluble proteins (e.g. GFP) synthesized in the cytosol are able to diffuse into the nucleus (Haseloff et al., 1997). The dark spherical areas observed inside the nuclei are probably nucleoli (Fig. 2), since fluorescent proteins are normally excluded from the nucleoli. When LeCTR1, LeCTR3, and LeCTR4 were simultaneously expressed with the ethylene receptor NR, they were co-localized to the ER, suggesting that NR could interact with LeCTR1, 3, and 4, and this association is responsible for their ER localization (Fig. 3). A membrane topology model for CmERS1, which is a homologue of NR in melon, has recently been proposed (Ma et al., 2006). In this model, the N-terminal domain of CmERS1 is anchored to the ER membrane with the N-terminus facing the ER lumen, whereas the C-terminal His kinase domain, which is likely to be the binding site of CTR, is exposed to the cytosol. If NR has the same membrane topology, LeCTR proteins would interact with the NR C-terminus on the cytosolic side of the ER membrane. It should be noted that overexpression of the LeCTR proteins using a CaMV 35S promoter is likely to saturate all the CTR-binding sites of the endogenous onion ethylene receptors at the ER and result in failure of the abundant LeCTR proteins to find interacting receptor partners, thus releasing them into the cytosol and nucleus as overexpression artefacts. However, when a tomato ethylene receptor NR was co-expressed, those free LeCTR proteins would be retrieved and anchored back to the ER. This is in agreement with the observation that the CTR1 protein became soluble in multiple ethylene receptor LOF mutants (Gao et al., 2003). The fluorescence from LeCTR2–YFP (Fig. 3G) was more diffuse even in the presence of receptor NR, suggesting it could not interact with NR, as was confirmed by the yeast two-hybrid assay (Fig. 1).
The BiFC assay showed that onion cells co-expressing NR–YFPn with three LeCTR–YFPc constructs were able to generate YFP fluorescence (Fig. 4). This suggests that the NR and these LeCTR proteins (1, 3, and 4) were in sufficiently close association to enable the YFP fragments to reconstitute a functional fluorescent complex. However, BiFC could generate false-positive results due to the irreversible nature of the association between the two YFP fragments, especially when they were overexpressed. Even with the negative controls, it is difficult to rule out the possibility that some of the BiFC fluorescence might be generated by the self-association between the two YFP fragments. In this study, BiFC images were recorded 6–8 h after biolistic transformation to minimize overaccumulation of fluorescent protein. However, prolonged incubation for >24 h did lead to a false-positive BiFC signal in all negative controls tested (see Supplementary material S2 at JXB online). Therefore, the BiFC results alone are not sufficient to prove the interaction between NR and LeCTR proteins without confirmation from the yeast two-hybrid assay (Fig. 1) and the protein localization experiments (Fig. 3). In the BiFC experiments, mRFP1 was used as a transformation control and highlighted the cytoplasm and nucleus, whereas the BiFC fluorescence produced a nucleus exclusion image pattern (see, for example, Fig. 4A) similar to that of the ER-targeted CFP (Fig. 2E) and did not co-localize with the cytosolic mRFP1. This supports the suggestion that the NR–LeCTR interactions take place in a membrane system and possibly at the ER. However, the NR–LeCTR BiFC signal was not sufficient to be recorded at high magnification to produce the cortical ER images, perhaps because the fluorescence generated by the BiFC complex is weak, even though a bright YFP variant Venus, which was estimated to be 13-fold more efficient than the enhanced yellow fluorescent protein (EYFP) in BiFC (Shyu et al., 2006), was used in these experiments. Thus, during the BiFC assay, the pinhole of the laser scanning confocal microscope had to be enlarged to achieve maximal sensitivity at the expense of confocality.
In the current Arabidopsis ethylene signalling model, ethylene receptors activate CTR1 to suppress the downstream ethylene response in the absence of ethylene, whilst ethylene binding deactivates the receptors and switches on the downstream signalling events. However, little is known about the actual signalling events immediately after CTR. It has been reported that EIN2 and EIN2-like proteins could function downstream of CTR (Alonso et al., 1999; Zhu et al., 2006). Additionally, it has been shown that a novel class of membrane protein, GREEN-RIPE, could modulate ethylene sensitivity specifically in tomato fruit tissue (Barry and Giovannoni, 2006). Protein localization studies indicated that the tomato LeEIN2 was targeted to the ER, as was the receptor–CTR complex, whereas GREEN-RIPE was located at the Golgi apparatus (Resnick et al., 2006; Zhou et al., 2007). It has been recently reported that the ethylene receptor ETR1 could also be found at the Golgi in Arabidopsis root hairs and co-localized with RTE1, which is the Arabidopsis homologue of GREEN-RIPE (Dong et al., 2008). Therefore, it is possible that the ethylene receptor–CTR complex might not be exclusively anchored to the ER membrane but could also be targeted or translocated to other parts of the endomembrane system. It is therefore important to establish how and why ethylene signals are transmitted from the endomembrane system to the nucleus, where ethylene responses are eventually determined by EIN3 and ERF transcription factors (Chao et al., 1997; Solano et al., 1998).
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Supplementary data |
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Additional information can be found at JXB online.
Movies S1. Ethylene receptor NR–EGFP at the ER membrane. The full-length cDNA of NR was fused to EGFP in cloning vector pDH51-GW-EGFP (AM773753 [GenBank] ). The NR–EGFP construct was expressed in onion epidermal cells under the control of the CaMV 35S promoter. Time series images were recorded showing the dynamic movement of NR–EGFP at the ER network.
Figure S2. Self-assembly of the BiFC negative control pairs after prolonged expression. Confocal images of onion epidermal cells overexpressing fluorescent protein fragments 24 h after the biolistic transformation. The YFP N-terminus co-expressed with the YFP C-terminus fused to LeCTR1 (A), LeCTR2 (B), LeCTR3 (C), and LeCTR4 (D). NR–YFPn co-expressed with the CN motif-mutated LeCTR3 (E). The red fluorescent protein mRFP1 was included as transformation control.
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Acknowledgements |
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We thank Harry Klee and Jim Giovannoni for generously providing receptor and CTR clones, Roger Tsien for supplying mRFP1, Atsushi Miyawaki for providing the YFP Venus, and Rachel Hackett for helpful discussion. Part of this work was funded by BBSRC.
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Footnotes |
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* These authors contributed equally to this work.
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Abbreviations |
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CaMV, cauliflower mosaic virus; CFP, cyan fluorescent protein; BiFC, biomolecular fluorescence complementation; CTR1, constitutive triple response1 protein; ER, endoplasmic reticulum; GFP, green fluorescent protein; LOF, loss-of-function; MAP, mitogen-activated protein; mRFP1, monocistronic red fluorescent protein; NR, never-ripe ethylene receptor protein; YFP, yellow fluorescent protein.
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References |
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