JXB Advance Access originally published online on December 6, 2006
Journal of Experimental Botany 2007 58(3):533-544; doi:10.1093/jxb/erl229
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RESEARCH PAPER |
Characterization of two ethylene receptors PhERS1 and PhETR2 from petunia: PhETR2 regulates timing of anther dehiscence
1Department of Biological Sciences, National University of Singapore, Science Drive 4, Singapore 117543
2Temasek Life Sciences Laboratory, 1 Research Link, National University of Singapore, Singapore 117604
* To whom correspondence should be addressed. E-mail: dbskumar{at}nus.edu.sg
Received 25 April 2006; Revised 9 October 2006 Accepted 10 October 2006
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Abstract |
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Two cDNAs (PhERS1 and PhETR2) encoding ethylene receptor homologues were cloned and characterized from petunia. Genomic Southern blot analysis revealed that small families of PhERS1-related and PhETR2-related genes exist in petunia. PhERS1 and PhETR2 are constitutively expressed in stem, flower bud, flower, and roots, with a very low level in leaves. High levels of both PhERS1 and PhETR2 transcripts were detectable in petunia leaves treated with exogenous ethylene, while only PhETR2 mRNA increased after wounding and salt treatment. Arabidopsis plants transformed with a site-mutated PhERS1 in the potential ethylene-binding domain exhibited partial ethylene insensitivity, suggesting that the function of this domain is conserved in the two species. Transgenic petunia plants with decreased PhERS1 expression caused by double-stranded RNA inhibition (dsRNAi) exhibited the wild-type phenotype, but showed increased mRNA levels of PhETR2. This suggests functional compensation between the two genes. Antisense suppression of PhETR2 in petunia led to stomium degeneration and anther dehiscence before anthesis, indicating that PhETR2 regulates synchronization of anther dehiscence with flower opening. Tandem affinity purification (TAP)-tagged PhERS1 was transiently expressed in Nicotiana benthamiana leaves. Gel filtration analysis showed that TAP–PhERS1 forms a
300 kDa protein complex in vivo. Key words: Anther dehiscence, petunia, site-mutated ethylene receptor, TAP tag
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Introduction |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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The gaseous phytohormone ethylene affects many aspects of plant development from seed germination to organ senescence and responses to environmental stress (Abeles et al., 1992; O'Donnell et al., 1996). Ethylene perception and signal transduction in plants is initiated by ethylene receptors. In Arabidopsis, the well-characterized ethylene receptor gene family includes ETR1, ETR2, EIN4, ERS1, and ERS2 (Hua et al., 1995; Hua and Meyerowitz, 1998). These ethylene receptors are homologous to prokaryotic two-component signal transducers (Chang et al., 1993). A typical two-component regulator consists of at least two types of signal transducers, a sensor kinase which monitors specific stimulus and a response regulator (Stock et al., 1990; Parkinson and Kofoid, 1992). While ETR1, ETR2, and EIN4 contain the response regulator domain, ERS1 and ERS2 lack it. Homodimerization of ERS1 has also been observed in Arabidopsis (Hall et al., 2000). However, the mode by which signal transfer occurs in the receptors that lack the response regulator domain is still unknown.
Ethylene binds to hydrophobic membrane-spanning domains of the receptors at the N-terminus through a copper cofactor and inactivates the receptors (Hua and Meyerowitz, 1998; Rodriguez et al., 1999). Besides the negative regulation model, functional redundancy also exists between members of the receptor family in Arabidopsis and tomato. Single and double loss-of-function mutants of four ethylene receptors from Arabidopsis do not display the ethylene-related phenotype (Hua and Meyerowitz, 1998). Studies with tomato LeETR3 and LeETR4 showed that ethylene sensitivity caused by reduction in the LeETR4 mRNA level could be eliminated by crossing these lines with those overexpressing LeETR3 (Tieman et al., 2000). These data suggest the occurrence of functional compensation among the multiple ethylene receptors in a given species.
Petunia is one of the species well documented for its flower senescence responses to ethylene (Gubrium et al., 2000). Its components in the ethylene synthesis pathway such as 1-aminocyclopropane-1-carboxylic acid (ACC) synthase and ACC oxidase have been extensively studied (Tang et al., 1994; Linstrom et al., 1999). Furthermore, petunia has been used as a heterologous species to study the role of ethylene receptors from other species such as Arabidopsis and Brassica (Wilkinson et al., 1997; Shaw et al., 2002). Transgenic petunia expressing Arabidopsis etr1-1 and boers (an ERS1 homologue from Brassica) showed significant delays in flower senescence and flower abscission. However, very few studies have been carried out to examine the components in the ethylene perception and signal transduction pathway in petunia except the recent study on PhEIN2 (Shibuya et al., 2004). To date, four cDNA clones for putative ethylene receptors from petunia, including three ETR1 homologues (accession nos AF145972, AF145973, and AF145974) and one ETR3 homologue (accession no. AF145975), have been deposited in the GenBank database. Although no details regarding their functions are available, the extremely high similarity among the three ETR1 homologues suggests the presence of a complicated ethylene signal perception mechanism in petunia.
The transient expression system combined with the recently established tandem affinity purification (TAP) method has been applied to characterize plant protein complexes (Rigaut et al., 1999; Rivas et al., 2002; Rohila et al., 2004). The TAP tag used refers to a fusion cassette including calmodulin-binding peptide (CBP), a TEV cleavage site, and the IgG-binding domain from protein A of Staphylococcus aureus (ProtA). The two high-affinity tags in the cassette, CBP and ProtA, allow efficient recovery of a fusion protein expressed at a low level in a complex mixture.
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Plant materials and treatments
Petunia (Petunia hybrida cv. Mitchell) plants were grown in the plant growth room maintained at 22±2 °C. For RNA analysis in different organs, leaves, stem, and roots were collected from plants at the vegetative stage when the plants were
10 cm in height. For wounding treatment, the leaves collected as above were cut into pieces of 2–3 cm2 and incubated in a box with high humidity (by floating the box in a container of water). For ethylene and salt treatment, petunia seedlings at the six-leaf stage were used. The seedlings were incubated in an airtight glass jar injected with 15 µl l–1 ethylene. Salt treatment was performed as described by Zhang et al. (2001). The wild-type and treated materials were immediately frozen in liquid N2 after collection, and stored at –80 °C until isolation of DNA and RNA. For Arabidopsis seedling triple response analysis, seeds were sown on MS medium containing 40 µg ml–1 kanamycin in the presence and absence of ACC, cold-treated at 4 °C for 4 d, and allowed to germinate in the dark at 23 °C for 72 h. The phenotypes of the apical hook and roots of the seedlings were observed and their hypocotyl lengths were measured.
Isolation of cDNAs for putative ethylene receptor homologues
RNA was isolated using an RNeasy Kit (QIAGEN) from both flowers and ethylene-treated leaves, separately. ERS and ETR homologues were isolated by reverse transcription–polymerase chain reaction (RT–PCR) from these tissues. To isolate the ERS homologue, degenerate primers ERS-5, 5'-GARTGYGCNYTNTGGATGCC-3' and ERS-3, 5'-STYCKCATYTCRTGRTTCAT-3' were used. To isolate the ETR homologue, degenerate primers ETR-5, 5'-GAAGGACCCCAATGGAGGTYKTCTYAC-3' and ETR-3, 5'-CCRTCYAAATCAGCAGGTGGAATCA-3' were used. The resulting PCR products were fractionated on a 1.0% (w/v) agarose gel and visualized by staining with ethidium bromide. The bands with the expected size of 0.5 kb were recovered, cloned into pGEM-T Easy vector (Promega), and sequenced (Prakash and Kumar, 2002). Based on the sequences obtained, primers were designed to isolate the full-length cDNAs by RACE (rapid amplification of cDNA ends). The primers used were ERS1 race-5, 5'-CATGGGCTCGCATGGACTCCTCCAGAAT-3' and ERS1-race 3, 5'-TGGAGGAGTCCATGCGAGCCCATGATCA-3' for the ERS homologue; and ETR race5, 5'-TTCCTCCACCTCCCTACTGCAACGTCTTT-3' and ETR race3, 5'-GCAGTAGGGAGGTGGAGGAAAGCCTGAG-3' for the ETR homologue. After analysis of the overlapping parts of each sequence, full-length cDNA corresponding to these two genes was isolated using the following gene-specific primers: PERS1f, 5'-GGGTGCACACGTGTGAAGTG-3'; PERS1r, 5'-GCATTAAGGTCGCTGGATAATTCAATA-3'; ETR-f, 5'-TTGGATTTTGCTCCCAAGAAACTTGT-3'; and ETR-r, 5'-GCAGCCAAGTCATATTTAGCATAA-3'. The full-length cDNAs were cloned into pGEM-T vector and sequenced from both ends. They were named PhERS1 and PhETR2. Similarly, partial length cDNA of two ETR1 homologues of petunia was isolated by RT-PCR to be used for expression analysis in the transgenic plants. Primers used for this were: ETR1f, 5'-GCTGCYATHTTRGARGAATCAATGAGG-3' and ETR1r, 5'-TASAMCCCTTSCCRASACCWTCACT-3' to clone the partial length cDNA; and ETR1race-5, 5'-AACGACGGCTTTCCGCAGCAGAACTGGG-3' and ETR1race-3, 5'-CCCAGTTCTGCTGCGGAAAGCCGTCGTT-3' for RACE. The sequencing data obtained were used to identify the cDNAs using a BLAST nucleotide homology search at the National Centre for Biotechnology Information (NCBI) website (http://www.ncbi.nlm.nih.gov/BLAST/). The protein sequences were predicted using the translate tool from ExPASy Proteomics Server (http://ca.expasy.org/). The multiple alignment of the homologues was performed using the program BOXSHADE (http://www.ch.embnet.org/software/BOX_doc.html).
Southern, northern and RT-PCR analysis
Genomic DNA was extracted from petunia leaves by the urea lysis method (Cone, 1989) and subjected to overnight digestion with different restriction enzymes (15 µg per enzyme), fractionated on a 1.2% agarose gel, and blotted onto a nylon membrane (Pall Biodyne, USA) (Zhang et al., 2004). The probes were labelled using the digoxigenin (DIG) PCR-labelling kit (Roche, Germany). For PhERS1, the probe used was the coding region from 924 to 1458 bp of the cDNA. For PhETR2, the full-length cDNA was used. Hybridization in DIG Hyb buffer (Roche, Germany) and chemiluminescent detection with CDP-star (Boehringer Mannheim, Indianapolis, USA) were performed according to the manufacturer's instructions. A series of post-hybridization washes was carried out as follows: 2x SSC with 0.1% SDS, 10 min at room temperature; 1x SSC with 0.1% SDS, 10 min at room temperature; 0.5x SSC/0.1% SDS 15 min at 65 °C; and 0.1x SSC/0.1% SDS 15 min at 65 °C.
Total RNA was extracted following the small-scale RNA extraction method (Verwoerd et al., 1989). For northern analysis, 25 µg of total RNA was loaded in each lane. The analysis was performed following Sambrook et al. (1989). For northern analysis of PhERS1, the probe used was a fragment including the 389 bp 5'-untranslated region (UTR) region and the following 91 bp of the coding region. For northern analysis of PhETR2, the probe used was a 3' 514 bp fragment containing 310 bp coding for the response regulator domain and the 204 bp 3'-UTR sequence. Hybridization, post-hybridization washes, and detection were performed as for Southern blot analysis.
In RT-PCR analysis, 1 µg of total RNA was treated with RNase-free DNase (Promega, USA) to destroy potential DNA contamination before carrying out first-strand cDNA synthesis using oligo(dT) primers. The cDNAs were first subjected to different cycles of PCR amplification to obtain an optimal cycle number to ensure the PCR products are in the exponential-linear phase. After optimization of the cDNA template to yield a similar band intensity to that for ubiquitin (internal control), these cDNAs were used for gene expression study. PCR was performed as follows, an initial denaturing step at 94 °C for 3 min, different numbers of cycles of amplification (28–34 cycles) of 94 °C for 30 s, 57 °C for 40 s, 72 °C for 45 s, and a 7 min final extension at 72 °C. The results were verified in at least two independent experiments. DyNAzymeII DNA Polymerase (Finnzymes, Espoo, Finland) was used for all the RT-PCRs. All the PCRs were performed in an MJ Research PTC 100 Thermal Cycler.
Full-length cDNA isolation and construction of chimeric genes
After sequence comparison, primers were designed to amplify the full-length sequence of these genes. To isolate the full-length ERS homologue, the two primers used were PMD-f, 5'-GGGTGCACACGTGTGAAGTG-3' and PMD-r, 5'-GCATTAAGGTCGCTGATAATTCAATA-3'; and for the ETR2 homologue, HK-f, 5'-TTGGATTTTGCTCTCCAAGAAACTTGT-3' and HK-r 5'-GCAGCCAAGTCATATTTAGCATAA-3' were used.
For generation of the PhERS1 construct, full-length PhERS1 cDNA including the 388 bp 5'-UTR and 182 bp 3'-UTR was amplified by primers containing BamHI and SacI restriction sites and cloned into pGEM-T vector. For the site-mutated PhERS1 construct, two PCRs were carried out with two sets of primers and the full-length PhERS1 cDNA as the template. The first reaction was with primer PMD-f and mutagenesis primer m-PhERS1-r, 5'-CAAGCTCCAGCAGAATGGAAAAGTAG-3'. The second reaction was with m-PhERS1-f, 5'-CTACTTTTCCATTCTGCTGGAGCTTG-3' and PMD-r primers. In these two PCR products, the predicted 37th amino acid proline was changed to leucine by the mutagenesis primers. A similar mutation in the ethylene receptor Never ripe (Nr) protein in tomato plants caused significantly delayed fruit ripening (Wilkinson et al., 1995). The resulting two fragments were ligated by PCR to generate the full-length mutated PhERS1 using PMD-f and PMD-r primers. Subsequently, mPhERS1 was cloned into pGEM-T vector and its sequence was confirmed.
The wild-type and site-mutated PhERS1 were subcloned into binary vector pBI121 (Clontech), introduced into plants under the cauliflower mosaic virus (CaMV) 35S promoter by Agrobacterium-mediated transformation. The double-stranded RNAi construct of PhERS1 (ds-PhERS1) was generated as described by Chuang and Meyerowitz (2000). A ß-glucuronidase (GUS) gene segment (nucleotides 787–1809) was used as the loop between the two PhERS1 fragments of the same sequence (390 bp 3'-UTR and the following 90 bp coding region) but in reverse directions in pBI121 under the 35S promoter. The antisense PhETR2 (APhETR2) construct was prepared with 1.1 kb of the cDNA flanked by BamHI and SacI sites. pGreen 0229 vector (Yu et al., 2004) was used for transient expression of TAP-tagged PhERS1 in Nicotiana benthamiana. The full-length PhERS1 cDNA from the start codon and just before the stop codon was inserted under the control of the 35S promoter. Thus, PhERS1 was fused with Prot A and CBP tags in the pGreen-TAP vector and the construct was named TAP–PhERS1. The above constructs were transferred into Agrobacterium tumefaciens (GV3101) by electroporation.
Agrobacterium-mediated petunia transformation and selection of transformants
Agrobacterium tumefaciens strain GV3101 colonies containing ds-PhERS1, mPhERS1, SPhERS1, and APhETR2 constructs were inoculated into MG/L broth (Jorgensen et al., 1996) (5 g l–1 mannitol, 1 g l–1 L-glutamic acid, 0.25 g l–1 KH2PO4, 0.10 g l–1 NaCl, 0.10 g l–1 MgSO4 · 7 H2O, 5 g l–1 tryptone, 2.5 g l–1 yeast extract, 1 mg l–1 biotin, pH 7.0) containing 50 mg l–1 kanamycin and 25 mg l–1 gentamycin, and grown at 28 °C overnight with shaking at 200 rpm. The OD600 of the culture was adjusted to 0.1–0.3 with MG/L broth before using in the transformation.
Plant transformation was carried out following the leaf disc co-cultivation method with some modifications for petunia (Horsch et al., 1985; Jorgensen et al., 1996). Fully expanded leaves were surface-sterilized with 15% (v/v) Clorox for 15 min, and rinsed three times in sterile, distilled water. These leaves were trimmed into segments of 1 cm2 in the presence of the Agrobacterium culture. The leaf discs were blotted dry on sterile C-fold paper towels and placed on shoot induction medium for 48 h in the dark. The shoot induction medium used was MS agar (Murashige and Skoog, 1962) supplemented with 100 mg l–1 acetosyringone and 2.5 µM benzyladenine (BA) (Jorgensen et al., 1996; Prakash and Kumar, 1997). After 2 d of co-cultivation, the explants were transferred to shoot induction medium with kanamycin (75 mg l–1) and cefotaxime (100 mg l–1). The shoots emerging from the cut ends after 3–4 weeks were excised and transferred to phytohormone-free MS medium with kanamycin (75 mg l–1) in sterile GA-7 vessels for rooting. The rooted shoots that were reaching the lid of the GA-7 were transplanted to soil and kept in the greenhouse for further studies.
Characterization of transgenic plants
The putative transgenic petunia plants were first subjected to PCR screening. The primers for neomycin phosphotransferase II (nptII) that were used to amplify an 800 bp fragment are 5'-GAACAAGATGGATTGCACGCAGGTTC-3' and 5'-AACTCGTCAAGAAGGCGATAGAAGGC-3'. PCR amplification was performed with an initial denaturation step at 94 °C for 4 min, followed by 35 cycles at 94 °C for 40 s, 62 °C for 40 s, and 72 °C for 40 s, and a final extension step at 72 °C for 7 min. Genomic Southern blot analyses were then performed for confirmation of transgene integration for the plants that gave positive bands in the PCR screening using the nptII probe as described above.
For expression analysis of different receptor homologues, the primers used were as follows: ETR1-1f, 5'-CTTTCAAGTGCTGATGGTAGAGCT-3' and ETR1-1r, 5'-CCCATACCACTATCTTGACATACTC-3' for PhETR1-1; ETR1-2 f, 5'-CTCT GATGAACTTGACACACGGG-3' and ETR1-2r, 5'-CACGGCTCCAATTACCATTTGC-3' for PhETR1-2; ERS1f, 5'-GAATGTGCTTTGTGGATGCC-3' and ERS1r, 5'-TCATTTCATGGTTCATCACAG-3' for PhERS1; and ETR2f, 5'-TGCAGTAGGGAGGTGGAGGAA-3' and ETR2r, 5'-ATCACATGATTCTGGTTGCCTG-3' for PhETR2.
Arabidopsis transformation, selection, and triple response assay
Agrobacterium-mediated transformation of Arabidopsis was carried out by the flower dip method (Clough and Bent, 1998). The T1 transgenic seedlings were selected on MS medium containing 40 mg l–1 kanamycin and transferred to soil to set seeds (T2). For the seedling triple response analysis, surface-sterilized T2 seeds were sown on MS medium containing 40 µg ml–1 kanamycin in the presence or the absence of 10 µM ACC, cold-treated at 4 °C for 4 d and allowed to germinate in the dark at 23 °C for 72 h. The transgenic lines were confirmed by PCR for the integrated nptII gene. The phenotypes of the seedlings and hypocotyl lengths were recorded.
Light microscopy
Anther tissues from antisense PhETR2 transgenic plants for light microscopy were prepared as described by Wolters-Arts et al. (1996) with some modifications. After fixation in 2% glutaraldehyde (post-fixation in 1% osmium tetroxide), anthers were dehydrated through an ethanol series and embedded in Spurr Resin (Sigma). The embedded tissues were sectioned at 2 µm thickness, stained with toluidine blue O (0.1% in 1% borax), and examined under a bright-field microscope.
Transient expression of TAP–PhERS1 in Nicotiana benthamiana leaves
Wild-type N. benthamiana were grown in a plant growth room at 22±2 °C. Agrobacterium strain GV3101 harbouring the TAP–PhERS1 construct was grown overnight at 28 °C in LB broth supplemented with 25 mg l–1 gentamycin, 50 mg l–1 kanamycin, and 10 mg l–1 tetracycline. The bacteria were centrifuged at room temperature at 3000 rpm for 15 min and resuspended in 10 mM MgCl2, 10 mM MES buffer, pH 5.6 and 100 µM acetosyringone. After incubation in this medium for 2–3 h at room temperature, the bacteria were infiltrated into the abaxial air spaces of the leaves of 3- to 4-week-old N. benthamiana with a 1 ml syringe without a needle. The leaves were harvested 2 d after infiltration, frozen immediately in liquid nitrogen, and stored at –80 °C.
The above leaf samples were ground in liquid nitrogen, then thawed in 2 vols of extraction buffer [20 mM TRIS–HCl, pH 8.0, 150 mM NaCl, 0.1% Igepal, 2.5 mM EDTA, 10 mM ß-mercaptoethanol, 1 mM phenylmethylsulphonyl fluoride (PMSF), 10 µM leupeptin, 2 mg l–1 aprotinin, 2 mg l–1 anti-pain, 1 µM E-64], filtered through four layers of Miracloth, and centrifuged at 5000 rpm for 10 min at 4 °C. The supernatant containing the total protein was either used directly for analysis or stored at –80 °C in aliquots.
SDS–PAGE and immunoblot analysis
Proteins were fractionated on a 12% SDS–polyacrylamide gel (Laemmli, 1970) using a Bio-Rad Mini-Protean gel apparatus. After electrophoresis, proteins were transferred to a PVDF membrane by electroblotting in a Trans-Blot Cell (Mini-Protean II system, Bio-Rad), in transfer buffer (25 mM TRIS base, 192 mM glycine, 20% methanol) at 100 V for 1.5 h at 4 °C. The membrane was then blocked in blotto [1x phosphate-buffered saline (PBS), 0.1% Tween-20, 5% low-fat dried milk] for 1 h at room temperature with gentle shaking. The membrane was washed briefly with washing buffer (1x PBS; 0.1% Tween-20) before incubation with the primary antibody with peroxidase–anti-peroxidase (PAP, Sigma), which was used to detect the Prot A fusion proteins. The PAP was diluted 1:5000 in blotto and binding was carried out for 2 h at room temperature followed by 5–6 washes (5 min each) with washing buffer. The hybridized antibodies were detected with anti-rabbit-IgG (Amersham) from the enhanced chemiluminescent (ECL) plus western blotting reagent pack and SuperSignal® West Pico Chemiluminescent substrate (Pierce). Protein molecular weight prediction was performed according to the protein analysis website (http://tw.expasy.org/).
Gel filtration analysis and purification of the protein complex
Gel filtration was carried out using an AKTA purifier system (Amersham) with a HiPrep 26/60 Sephacryl S-200 high resolution column (Pharmacia). Column equilibration and chromatography were performed in the extraction buffer. Fractions were collected every 1 ml. To concentrate the proteins, the eluate fractions were mixed with 20% trichloroacetic acid (TCA). The samples were incubated on ice for at least 30 min with occasional vortexing. The mixture was then centrifuged for 15 min at 13 000 rpm at 4 °C. The pellet obtained was then washed twice with cold acetone and air-dried for 5–10 min at room temperature. These individual concentrated eluates were dissolved in 1x SDS loading buffer, fractionated on a 12% SDS–polyacrylamide gel, and subjected to western blot analysis using the PAP antibody as mentioned above.
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Cloning of PhERS1 and PhETR2 cDNAs
Two partial cDNA fragments, each
0.5 kb, were isolated by RT-PCR using degenerate primers. Nucleic acid sequence alignment of these two fragments showed high similarity to ethylene receptors reported from other plant species. Subsequently, 5'/3' RACE PCRs were performed to obtain the 5' and 3' fragments of the cDNAs. For PhERS1, a cDNA of 2481 bp, with an open reading frame encoding 633 amino acids, was isolated. Although there were 423 bp of 5'-UTR in the PhETR2 fragment obtained by 5'-RACE, the full-length cDNA could not be amplified using the primer just behind the universal primer sequence (the primer provided in the RACE kit for isolation of the 5'-UTR up to the coding region). However, by using a downstream forward primer, a 2633 bp cDNA was isolated with a truncated 5'-UTR of 149 bp. It codes for 761 amino acids in its open reading frame and has a 3'-UTR of 204 bp. The gene organization in petunia was examined by genomic Southern blot analysis. There were at least two bands in each of the three lanes of the blot when the probe used was a conserved region in the ERS1 receptors (Fig. 1A), suggesting that this may be due to the presence of two closely related genes. However, the possibility that restriction sites within the introns of the gene might have led to the observation cannot be ignored. Similarly, when the blot was probed with full-length PhETR2, it showed more than three bands in the lanes containing genomic DNA digested by EcoRI, EcoRV, and SacI (Fig. 1B). Considering there is one occurrence each of EcoRI and SacI sites in the cDNA sequence, there should be at least two PhETR2-related genes in the genome. When the information from the two blots is taken together, it is clear that besides PhERS1 and PhETR2, the petunia genome contains other members of ethylene receptors or a set of receptors that share high nucleotide sequence similarity.
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The protein sequences corresponding to the two genes were compared with the ethylene receptors from other plant species. As shown in Table 1, the receptors that have high similarity to these two clones are from tobacco and tomato. For example, PhERS1 has 95% similarity to NTERS1 from tobacco (Terajima et al., 2001). Since petunia, tomato, and tobacco belong to the Solanaceae, this may indicate that the ethylene receptor genes have not diverged much within this family. Multiple alignments of the amino acid sequences with other reported homologues and the structural similarities between the receptors are shown in Fig. 2. PhETR2 contains the predicted ethylene sensor domain, a histidine kinase domain, and a response regulator domain. In contrast, PhERS1 does not contain the response regulator domain. They both contain a region that shows homology to the GAF domain (domains found in cGMP-specific and -stimulated phosphodiesterases, Anabaena adenylate cyclases and Escherichia coli FhlA), which is associated with cGMP binding (Aravind and Ponting, 1997).
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Expression analysis of PhERS1 and PhETR2
Northern blot analysis showed that these two genes are constitutively expressed in stem, root, flower buds, and flowers (Fig. 3A). Expression of PhERS1 is low in leaves compared with that in other tissues. The mRNA level in PhETR2 in leaves is either less than the level of detection by northern blot or there is no expression in leaves at all. RT-PCR was then carried out to check the expression pattern of these genes in these organs (Fig. 3B), which showed that both PhERS1 and PhETR2 are expressed at low levels in leaves compared with that in the other organs.
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In addition to functioning in plant development, ethylene also plays a role in stress responses. To investigate the external factors regulating these two receptor genes, petunia seedlings were subjected to ethylene, wounding, and salt (NaCl) treatments. Both PhERS1 and PhETR2 were inducible by ethylene in the leaves (Fig. 3C). PhETR2 transcripts increased 1 h after wounding stress and continued to accumulate even after 6 h (Fig. 3C), while PhERS1 transcripts were not significantly affected by wounding. Both PhERS1 and PhETR2 mRNA levels increased after NaCl treatment in the range of 0–0.1 M (Fig. 3C). Higher concentrations of NaCl were required for induction of the expression level of PhETR2 compared with PhERS1.
Transformation of Arabidopsis with site-mutated PhERS1
Arabidopsis seedlings treated with ethylene will display a phenotype generally referred to as ‘triple response’, including inhibition of root and hypocotyl elongation, swelling of the hypocotyl and root, and exaggeration in the curvature of the apical hook (Guzman and Ecker, 1990). To investigate PhERS1 functions in the ethylene signalling pathway, two constructs containing the wild-type and site-mutated PhERS1 cDNA were generated. The phenotypes of the T3 seedlings are shown in Fig. 4. In the presence of ACC, transgenic seedlings harbouring the site-mutated PhERS1 (mPhERS1) showed only a weak triple response, because they lack the exaggerated twisted apical hook. The hypocotyls of 16 out of 20 mPhERS1 transgenic lines were longer than those of wild-type Columbia grown in MS basal medium supplemented with ACC (Table 2; Fig. 4A, D, E). Also, the seedlings transformed with wild-type PhERS1 displayed the characteristic triple response (Fig. 4F). The hypocotyl lengths, however, were somewhat longer than that of wild-type Columbia seedlings when incubated without ACC.
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Functional analysis of PhERS1 and PhETR2 in petunia
To investigate the involvement of PhERS1 and PhETR2 in plant growth and development, expression of PhERS1 and PhETR2 in transgenic petunia was modulated by expressing ds-PhERS1 and antisense PhETR2 (APhETR2) cDNA under the control of CaMV 35S promoter. These transgenic petunia lines have multiple copies of transgenes (Fig. 5A). There were no phenotypic differences between ds-PhERS1 transgenic and wild-type petunia. However, in the APhETR2 transgenic petunia flowers, early anther dehiscence was observed compared with that in the wild-type flowers. For a clearer understanding of the timing of anther dehiscence and flower opening in wild-type and APhETR2 transgenic plants, various developmental stages of flowers were identified as follows: stage 1, tightly closed flower bud; stage 2, just opened flower bud; stage 3, half opened flower; stage 4, fully opened flower (Fig. 6A). A close up of anthers from wild-type and APhETR2 flowers in these four stages are shown in Fig. 6B–I. At stage 3, the anthers were dehisced in APhETR2, and fresh pollen release had occurred, whereas in the wild type, anther dehiscence and fresh pollen release occurred only at stage 4 (Fig. 6D, I). These results indicate that PhETR2 plays a role in regulation of anther dehiscence timing.
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To analyse further if the development of anthers was affected, a histological study was performed on anthers in these stages. Except for the early degeneration of the stomium, the other parts of the anther in APhETR2 transgenic petunia flowers were similar to that of the wild type (Fig. 6J, K). This indicated that the cellular differentiation of the anthers was not affected by the down-regulation of PhETR2, but only stomium degeneration and anther dehiscence occurred before anthesis.
mRNA levels of ethylene receptors in transgenic petunia lines
Transgenic petunia plants were generated in order to examine the effect of down-regulation of the two ethylene receptors cloned. The transcript levels of several ethylene receptors were examined in APhETR2 (antisense suppression) transgenic plants. PhETR2 transcripts decreased in leaves of three independent APhETR2 transgenic lines (Fig. 7A). However, mRNA levels of other receptors, including PhETR1-1, PhETR1-2, and PhERS1, remained unchanged (Fig. 7A).
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Since no altered phenotype was observed related to the ethylene response in the ds-PhERS1 transgenic plant and functional redundancy has been suggested for the Arabidopsis and tomato ethylene receptor family, transcript levels of other receptor homologues were examined in a ds-PhERS1 transgenic line. Because PhERS1 and PhETR2 are expressed at low levels in leaf tissues, the changes in mRNA level may be more sensitive and thus easy for comparison in these tissues. In ds-PhERS1 leaves, PhERS1 transcripts were down-regulated as expected. PhETR1-1 and PhETR1-2 mRNA levels were similar to those in the wild-type plants (Fig. 7A). However, the PhETR2 transcript level was significantly increased in the ds-PhERS1 transgenic line. Also, R1 generation seedlings of the ds-PhERS1 line were screened by PCR and the mRNA levels of ethylene receptor homologues were examined (Fig. 7B). In two of the R1 progeny, 1-6 and 1-7, in which the PhERS1 mRNA level was reduced, PhETR2 mRNA increased, but mRNA levels of two PhETR1 homologues were not altered when compared with the wild type.
Analysis of the TAP–PhERS1 protein complex
PhERS1 lacks the response regulator domain of the classical two-component signal transduction elements. Therefore, in the present attempt to examine the possibility of PhERS1 forming a complex with other proteins in vivo, the TAP–PhERS1 was transiently expressed in N. benthamiana leaves. A band of 100 kDa was detected when proteins were extracted from infiltrated leaves after 2 d and immunoblot analysis was performed using PAP antibody (Fig. 8A). The predicted size of PhERS1 and the TAP tag protein are 75 kDa and 15 kDa, respectively. Thus, the 100 kDa band is consistent with the predicted size of the expressed fusion protein. Moreover, this band was absent in the proteins from the wild-type and water-infiltrated N. benthamiana leaves. Use of Agrobacterium at different cell densities (OD600=0.1, 0.2, and 0.5) for infiltration of N. benthamiana leaves did not show significant differences in the protein expression levels (Fig. 8A, B).
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To determine if any PhERS1-associated protein complex exists in vivo, the native protein extract from N. benthamiana leaves transiently expressing TAP–PhERS1 was subjected to gel filtration chromatography. An immunoblot analysis of the eluted fractions indicated the presence of such a protein complex (Fig. 8C). The estimated molecular mass of the PhERS1-associated complex when compared with the calibration curve ranged between 250 kDa and 350 kDa, peaking at
300 kDa. Taken together, the TAP-tagged PhERS1 forms a 300 kDa complex in vivo and the possible members of the protein complex have not been identified at this stage. |
Discussion |
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In this study, the molecular cloning of two cDNAs encoding ethylene receptors and the regulation of their expression by some external factors is reported. The deduced polypeptides of these two genes shared homology with the Arabidopsis ethylene receptors. PhETR2 shows 57.5% similarity with ETR2, but PhERS1 shared 70.8% sequence similarity with both ERS1 and ETR1, perhaps due to the fact that ERS1 and ETR1 belong to the same subfamily in Arabidopsis. Based on its high similarity to the ERS1 gene from Arabidopsis and other species, it was named PhERS1. The high sequence similarity among PhERS1, PhETR2, and the receptors from tomato and tobacco (Table 1) suggested that they may function in a similar way. The structures of these two receptors are different in that PhERS1 lacks the response regulator domain. It was suggested that such receptors without the regulator domain may use the domains of other proteins (Hua et al., 1998) as heterodimers. The conserved region and residues in the N-terminal and histidine kinase domains are similar between the two petunia clones. They both contain three transmembrane domains important for ethylene binding and the motifs similar to the bacterial histidine kinase domain. Also, the site mutation introduced into PhERS1 caused partial ethylene insensitivity in Arabidopsis, indicating a similar functional role for this receptor in the ethylene signal transduction pathway in the two species.
Northern blot analysis showed that these two receptors have similar expression patterns in different organs, with high levels of transcripts detected in flower buds and flowers. The very low expression level in leaves suggested the need for further investigation of the regulatory factors of these two genes. Various ethylene receptors in plants are differentially regulated by ethylene. Some are ethylene inducible, while others are not affected by exogenous ethylene treatment (Wilkinson et al., 1995; Hua et al., 1998). The present observation that exogenous ethylene induces PhERS1 expression was in agreement with that of other ERS homologues. Wounding and salt are two of the environmental stresses that can alter gene expression (van der Krol et al., 1999; Wang et al., 2004). For example, the tobacco NTHK1 gene (ethylene receptor) was positively regulated by these factors (Zhang et al., 2001). The present results showed that PhETR2 expression was also increased when petunia seedlings were experiencing these stresses. In wounded plants, ethylene will be produced and emitted (O'Donnell et al., 1996). It was not determined if the up-regulation of the PhETR2 mRNA level observed in the present study is a direct response to wounding or an indirect response to wound-induced ethylene emission. PhERS1, however, was unaffected by wounding and salt stress, demonstrating that individual ethylene receptors of a given family respond differently to different environmental stimuli.
Partial functional analysis of the two ethylene receptors of petunia, PhERS1 and PhETR2, was attempted by generating transgenic plants with down-regulated PhERS1 and PhETR2 expression levels. Despite the decrease in the corresponding transcripts, the dsRNAi PhERS1 transgenic plants showed the wild-type phenotype. While PhETR1 homologues were unaffected, PhETR2 transcript levels increased, suggesting that maybe it is a compensation for the decreased PhERS1 expression in these plants. Such functional compensation within a gene family, namely increasing the expression of one member to compensate the reduced expression of another member, has been reported in the tomato ethylene receptor family (Tieman et al., 2000) and several mammalian multigene families (Mulligan et al., 1998; Minkoff et al., 1999). Transgene positional effects may also be a cause of the increased PhETR2 transcript levels in two out of the seven dsPhERS1 R1 progeny examined, and therefore more transgenic lines should be analysed to address this in future experiments.
When the site-mutated PhERS1 (mutation to mimic the ethylene-insensitive nature) was introduced into Arabidopsis, transgenic plants treated with ACC showed a weak triple response compared with the wild-type seedlings (Guzman and Ecker, 1990). The morphogenic changes in the plants expressing site-mutated PhERS1 in the presence of ACC indicated partial ethylene insensitivity, suggesting a similar role for this receptor in the ethylene signal transduction pathway in the two species. However, introduction of wild-type PhERS1 into Arabidopsis resulted in a somewhat longer hypocotyl phenotype compared with wild-type Arabidopsis seedlings grown with or without ACC. Similarly, Arabidopsis etr1 loss-of-function mutants are reported to have a shorter hypocotyl phenotype in the presence of ethylene because etr1 was suggested to function ethylene independently in regulating cell elongation (Hua and Meyerowitz, 1998). Therefore, the altered hypocotyl length phenotype in the present study may suggest a role for PhERS1 in seedling growth in addition to its activity in ethylene perception.
The other petunia ethylene receptor, PhETR2, showed high amino acid homology to the tomato ethylene receptor LeETR4 gene. Transgenic tomato with reduced LeETR4 gene expression showed symptoms of extreme ethylene sensitivity (Tieman et al., 2000). No such ethylene sensitivity phenotype was observed in transgenic tomato when the expression levels of LeETR1 and LeETR3 were down-regulated, suggesting that LeETR4 is a unique member in the tomato ethylene receptor family. Similarly, because PhETR2 increased its expression level when PhERS1 was down-regulated, PhETR2 may be an important member in the petunia receptor family. Three independent antisense transgenic PhETR2 petunia lines that showed a decrease in the PhETR2 transcripts also exhibited anther dehiscence before flowers fully opened. Also, transcripts of the two PhETR1 homologues were not significantly affected in them.
Regulation of the timing of anther dehiscence by ethylene has been demonstrated in tobacco (Rieu et al., 2003). In ethylene-insensitive tobacco transformed by the Arabidopsis etr1-1 allele, a delay in anther dehiscence occurred. Tobacco flowers treated with the ethylene perception inhibitor 1-methylcyclopropene (MCP) also displayed delayed anther dehiscence, and ethylene treatment accelerated the dehiscence. Evidence was also provided that this effect of ethylene directly applies to the anther tissue (Rieu et al., 2003). However, the targets of ethylene are not known. Histological analysis of the anthers from antisense PhETR2 showed that cellular differentiation in the stomium was unaffected during development. The pollen grains were viable and seeds were set after self-pollination by hand. Only stomium degeneration and anther dehiscence occurred earlier compared with the wild type. It was shown that PhETR2 mRNA will increase upon ethylene treatment. In transgenic petunia with decreased PhETR2 mRNA, there may not be enough PhETR2 members to regulate the ethylene response in the anther and thus the early dehiscence occurred. It is possible that in petunia, PhETR2 is the key ethylene receptor to control the timing of anther dehiscence.
The presence of multiple ethylene receptors that have divergent structures and expression patterns in plants was suggested to be the mechanism through which plants co-ordinate different ethylene responses (Hua and Meyerowitz, 1998; Tieman and Klee, 1999). Of the two ethylene receptors isolated in the present study, PhERS1 lacks the receiver domain, which is supposed to accept the phosphate during signal transduction. In bacteria, the phosphate group can be transferred to a separate protein to transduce the signal (Parkinson and Kofoid, 1992). Also, the transgenic line with a decreased PhERS1 mRNA level showed the wild-type phenotype, without any alteration of floral senescence or anther dehiscence. To investigate the function of this kind of receptor without the response regulator domain, PhERS1 was fused with the TAP tag and transiently expressed in N. benthamiana leaves. Using gel filtration analysis, it was found that PhERS1 was part of a 300 kDa protein complex. As the predicted size of TAP–PhERS1 protein is 90 kDa, other as yet unidentified proteins in the 300 kDa complex may provide the response regulator domain and transduce the ethylene signal to downstream genes by interacting with PhERS1. In Arabidopsis, both ERS1 and ETR1 were shown to form membrane-associated, disulphide-linked dimers when transgenically expressed in yeast (Schaller et al., 1995; Hall et al., 2000). By yeast two-hybrid assay, it was also found that Arabidopsis ERS1 interacts with CTR1 physically in vitro (Clark et al., 1998). In petunia, it is also possible that a homodimer of PhERS1 or a heterodimer of PhERS1 with other receptors may form in this complex. Hence, isolation and characterization of the components in the protein complex will further our understanding of ethylene signal transduction through PhERS1.
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Acknowledgements |
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We thank Dr Harry J Klee, Department of Horticultural Sciences, University of Florida, Gainesville, USA, for the gift of wild-type petunia (diploid Petunia hybrida cv. Mitchell) seeds. This work was funded by a research grant (No. R-154-000-125-112) from the National University of Singapore.
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Abbreviations |
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ACC, 1-aminocyclopropane-1-carboxylic acid; CaMV, cauliflower mosaic virus; CBP, calmodulin-binding protein; DIG, digoxigenin; PAP, peroxidase–anti-peroxidase; PBS, phosphate-buffered saline; Prot A, protein A of Staphylococcus aureus; RACE, rapid amplification of cDNA ends; RT-PCR, reverse transcription-polymerase chain reaction; TAP, tandem affinity purification; UTR, untranslated region.
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