© 2007 The Author(s).
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RESEARCH PAPER |
Functional analysis of a RING domain ankyrin repeat protein that is highly expressed during flower senescence
1Department of Plant Sciences, University of California Davis, One Shields Avenue, Davis, CA 95616, USA
2Crops Pathology and Genetics Research Unit, USDA-ARS, One Shields Avenue, Davis, CA 95616, USA
* To whom correspondence should be addressed. E-mail: msreid{at}ucdavis.edu
Received 16 May 2007; Revised 27 July 2007 Accepted 30 July 2007
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Abstract |
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A gene encoding a RING zinc finger ankyrin repeat protein (MjXB3), a putative E3 ubiquitin ligase, is highly expressed in petals of senescing four o'clock (Mirabilis jalapa) flowers, increasing >40 000-fold during the onset of visible senescence. The gene has homologues in many other species, and the Petunia homologue is strongly up-regulated in senescing Petunia corollas. Silencing the expression of this gene in Petunia, using virus-induced gene silencing, resulted in a 2 d extension in flower life. In Mirabilis, a 2 kb promoter region, 5’ upstream of the MjXB3 gene, was isolated. The promoter sequence included putative binding sites for many DNA-binding proteins, including the bZIP, Myb, homeodomain-leucine zipper (HD-Zip), MADS-box, and WRKY transcription factors. The construct containing a 1 kb promoter region immediately upstream of the MjXB3 gene drove the strongest expression of the β-glucuronidase (GUS) reporter gene in a transient expression assay. In Petunia, GUS expression under the control of this heterologous promoter fragment was specific to senescing flowers. The Mirabilis promoter GUS construct was tested in other flower species; while GUS activity in carnation petals was high during senescence, no expression was detected in three monocotyledonous flowers—daylily (Hemerocallis ‘Stella d'Oro’), daffodil (Narcissus pseudonarcissus ‘King Alfred’), and orchid (Dendrobium ‘Emma White’).
Key words: Floral senescence, Mirabilis jalapa, senescence-associated genes, transcription factor, ubiquitin ligase
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Introduction |
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Flower senescence is a carefully co-ordinated process, clearly under genetic control, and considered to be an example of programmed cell death (for a review, see Rubinstein, 2000). As a model system, we have used the flowers of the four o'clock (Mirabilis jalapa). These ephemeral flowers show an intermediate pattern of senescence where exogenous ethylene accelerates floral senescence, but treatment with inhibitors of ethylene biosynthesis or action has little effect on flower longevity (Xu et al., 2007). In these flowers, it was demonstrated that early application of
-amanitin substantially delayed floral senescence, indicating the need for gene transcription in the process (Xu et al., 2007). Researchers have isolated many genes whose abundance changes during the onset of floral senescence, from systems as diverse as carnation, daylily, iris, daffodil, and Alstroemeria (Lawton et al., 1990; Valpuesta et al., 1995; Hunter et al., 2002; van Doorn et al., 2003; Breeze et al., 2004). Changes in transcript abundance of a number of genes during senescence of Mirabilis flowers were recently reported (Xu et al., 2007). Among the up-regulated genes identified in a differential screen, it was interesting to note one encoding an ankyrin repeat RING zinc finger protein, which was named MjXB3. Freemont et al. (1991) identified a novel cysteine-rich amino acid sequence motif common to a number of diverse proteins thought to interact with DNA, and named a human gene with this motif ‘RING1’ (Really Interesting New Gene 1). Ankyrin repeat RING domain-containing proteins have ubiquitin ligase activity, for which the RING domains are essential (Lorick et al., 1999; Stone et al., 2005). The ubiquitin ligase plays a key role in the E3 complex that targets individual proteins for proteolysis in the 26S proteasome. It appears that the complex includes one or more adaptor proteins that bind the target, and a ubiquitin ligase, which catalyses the attachment of ubiquitin, conjugated by E2 to the E3 complex, to the target protein (Thomann et al., 2005).
Ubiquitin ligases have been demonstrated to participate in regulation of many functions in plants. Examples include SINAT5, which appears to be involved in auxin signalling (Xie et al., 2002), ATL2, which plays a role in plant defence (Serrano and Guzman, 2004), BRH1, which is part of the brassinosteroid response/pathogen response (Molnar et al., 2002), TED3/AtPex2p, which is involved in light signalling (Hu et al., 2002), and, most recently, the Arabidopsis RING-H2 gene, Xerico, which confers drought resistance when it is up-regulated, and appears to be involved in homeostasis of various plant hormones (Ko et al., 2006). Wang et al. (2006) found that a pathogen-induced type of programmed cell death required a ubiquitin ligase (XB3). MjXB3 might play a role in the co-ordination of the senescence programme, since it is up-regulated precisely when Mirabilis flowers become irreversibly committed to senescence (-aminitin no longer delays senescence; Xu et al., 2007). Here a study of the tissue- and stage-specific expression of this gene is reported, together with the effect of silencing its expression, using virus-induced gene silencing (VIGS), on floral longevity. In addition, the gene's promoter region, and its stage and tissue specificity, as indicated by transient expression of β-glucuronidase (GUS) fused to a promoter fragment, are described.
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Materials and methods |
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Plant materials
Four o'clock (M. jalapa), Petunia hybrida (‘Mitchell Diploid’), carnation (Dianthus caryophyllus L. ‘Imperial White’), daylily (Hemerocallis ‘Stella d'Oro’), Dendrobium orchid (Dendrobium ‘Emma White’), and daffodil (Narcissus pseudonarcissus ‘King Alfred’) plants were grown in the greenhouse at the University of California, Davis using standard growth conditions and cultural practices. Flowers and other tissues were harvested as needed.
Isolation of full-length MjXB3
The full-length sequence of MjXB3 was isolated by standard techniques using 3' and 5' rapid amplification of cDNA ends (RACE) with the Clontech kit (Clontech, Mountain View, CA, USA), and following the manufacturer's instructions. The sequence was analysed by the sequencing service of the College of Biological Sciences at UC Davis, and is deposited in GenBank as accession number EF470290.
MjXB3 expression analysis
Total RNA was extracted from Mirabilis flowers harvested at different stages of opening and senescence, as defined by Gookin et al. (2003), and from Petunia corollas at appropriate stages, using TRIzol Reagent (Invitrogen, Carlsbad, CA, USA). The isolated RNA was treated with RNase-free DNase (Promega, Madison, WI, USA) to remove any contaminating genomic DNA. First-strand cDNA was synthesized using 2 µg of total RNA, oligo(dT) primer, random hexamers, and PowerScript reverse transcriptase (BD Biosciences, Mountain View, CA, USA). This cDNA was used as template for semi-quantitative PCR. The abundance of 18S rRNA was used as an internal control. The primers for Mirabilis 18S rRNA were 5'-GATTCTATGGGTGGTGGTGCAT-3' and 5'-CTCAAACTTCCGTGGCCTAGAA-3'. MjXB3 transcripts were amplified using the primers 5'-TTGGAATTGTCACGAGGGAAGT-3' and 5'-CAAATCACAACCCGCATCTACA-3'. The primers for amplification of the Petunia MjXB3 homologue were 5'-CGGGATCCCGCTTAATGGAAGGACATGC-3' and 5'-GCTCTAGATCTTGCAGCCAAATGAAGTG-3'. These primers were used for both semi-quantitative RT-PCR analysis and real-time quantitative PCR analysis using SYBR Green PCR reagents as described by Chen et al. (2004) and Xu et al. (2007).
Virus-induced gene silencing (VIGS) of PhXB3
The pTRV1 and pTRV2 vectors for VIGS have been described in detail (Liu et al., 2002; Chen et al., 2004). A 284 bp fragment of the PhXB3 gene (expressed sequence tag clone CV293065) was PCR-amplified from Petunia cDNA using primers 5'-GGCTGCGATTATTAGGGCTGAA-3' and 5'-AACGCGAGGCATTGAGTCC-3'. As described in detail by Chen et al. (2004), the products were cloned into pTRV2-CHS to generate pTRV PhXB3/CHS constructs. Young Petunia plants were infected with Agrobacterium transformed with each of these pTRV PhXB3/CHS constructs, or with pTRV2 CHS (control), and flower longevity was determined as days from anthesis to complete wilting of the corolla (Chen et al., 2004).
Sequence analysis
MjXB3 homologues were revealed by searching the Gene Indice database at TIGR using the BLAST program (http://www.tigr.org/tdb/tgi/plant.shtml). Comparison of multiple putative protein sequences was carried out using the ClustalX program. The cis-elements in the MjXB3 promoter region were identified by searching the PLACE (Plant Cis-acting Regulatory DNA Elements) database (http://www.dna.affrc.go.jp/PLACE/index.html) and the TFSEARCH (DNA Transcription Factor Binding Site Prediction) database (http://mbs.cbrc.jp/research/db/TFSEARCH.html).
Isolation of the MjXB3 promoter region
Genomic DNA was extracted from 1 g of Mirabilis leaf tissues as described by Rajaseger et al. (1997). The promoter region was isolated using vectorette PCR as described by Ko et al. (2003). Briefly, 60 µg of genomic DNA was digested overnight using BamHI, SpeI, and EcoRI (20 µg of DNA per endonuclease). The digested DNA was then ligated to the appropriate vectorette: vectCTAG for the BamHI-digested DNA; vectGATC for the SpeI-digested DNA; and vectTTAA for the EcoRI-digested DNA. Promoter fragments were amplified using primary primers and nested primers designed for the vectorette sequences and for the upstream untranslated region of MjXB3. The isolated promoter sequence is deposited in GenBank as EF470291.
MjXB3 promoter fragments of different length were amplified using the specific primers based on the promoter sequences. For the 914 bp promoter fragment, the primers 5'-CCAAGCTTCTAGTGATTTATGAATCTCCAATAC-3' and 5'-GGATCCTTGATGATGTGTGTGACTCT-3' were used.
Plasmid promoter constructs
35S::GUS
The GUS gene was excised from the vector pDU96.3432 by BamHI and SacI digestion and inserted into the multiple cloning sites (MCS) of pGSA1403.
SAG12::GUS
The SAG12 promoter was amplified from Arabidopsis genomic DNA with the primers 5'-GTTTGTACTTGGTACCTTTGG-3' and 5'-TTGTTTTAGGAAAGTTAAATG-3'. The 35S promoter in the 35S::GUS construct was replaced by the SAG12 promoter.
MjXB3::GUS
The 35S promoter in the 35S::GUS construct was replaced with MjXB3 promoter fragments of different lengths (–1734 bp and –914 bp), by digesting the 35S::GUS construct with BglII and XhoI, and ligating the fragments to the digested vector.
Constructs were transformed into Agrobacterium strain LBA4404.
Transient expression assay
Agrobacterium strain LBA4404 transformed with different constructs was cultured for 24 h in 3 ml of YEP medium containing 10 mg l–1 chloramphenicol. The cultures were centrifuged, and the pelleted bacteria were resuspended in an inoculation buffer (10 mM MgCl2, 200 µM acetosyringone, 10 mM MES, pH 5.2). Petals and leaves were injected with the Agrobacterium suspension using needle-less syringes. Roots were submerged in the Agrobacterium suspension for 2 d.
GUS staining
GUS staining was carried out as described by Rodrigues-Pousada et al. (1993). Tissues were submerged in ice-cold 90% acetone and placed on ice for 15 min. After discarding the acetone, the tissues were washed three times with a rinse solution [50 mM NaPO4 pH 7.2, 0.5 mM K3Fe(CN)6, 0.5 mM K4Fe(CN)6]. The staining solution [the rinse solution plus 1 mM X-Gluc (Fisher Scientific, Hampton, NH, USA)] was then added, and infiltrated into the tissue under vacuum for 1 min. Tissues were kept in the staining solution at 37 °C overnight before recording the staining pattern. The remaining chlorophyll in the leaf tissues was further removed with 90% acetone at 20 °C overnight.
Heterologous assays
Twelve carnation petals were detached from fully open flowers and inoculated with Agrobacterium transformed with constructs containing GUS driven by the 35S, SAG12, or MjXB3-914 promoters. Half of the carnation petals were placed for 24 h in a sealed container ventilated (30 l h–1) with air containing 1 µl l–1 ethylene, then the GUS staining pattern was determined for all the petals. Three orchid flowers were harvested and inoculated with the same promoter–GUS constructs, then treated with ethylene as above until the flowers started to wilt. GUS activity in the wilting and control petals was then visualized as described above.
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Results |
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MjXB3 encodes a RING zinc finger ankyrin repeat protein homologous to ubiquitin ligases
The full-length sequence of MjXB3, with an open reading frame (ORF) of 1341 bp, was isolated. The deduced polypeptide encoded by this gene is highly homologous to proteins from diverse plant species. A search of the plant genome databases of the Institute for Genomic Research (TIGR) revealed that sequences homologous to MjXB3 have been isolated from Arabidopsis, rice, and other species (Fig. 1). The homologous sequence from rice (XB3) encodes the Xa21-binding protein 3 from rice (Nodzon et al., 2004; Wang et al., 2006), which is a ubiquitin ligase and required for a pathogen-induced programmed cell death. All the homologous sequences showed high conservation of amino acids in the RING zinc finger and ankyrin repeat domains. Beyond these domains, the sequences diverged.
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MjXB3 and its Petunia homologue were specifically expressed in senescing flowers
Semi-quantitative RT-PCR showed that MjXB3 was highly expressed in senescing Mirabilis flowers, but not in flower buds or open flowers (Fig. 2A). Transcripts of MjXB3 were detected at low levels in young and old leaves; expression was higher (comparable with that in senescing flowers) in stems and roots. RT-PCR of RNA extracted from Petunia flowers at different stages showed that the Petunia MjXB3 homologue was also highly expressed in senescing flowers, but not in flower buds or open flowers (Fig. 2B).
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Effect of silencing PhXB3 in Petunia
To date it has not been possible to develop an efficient transformation/regeneration protocol for Mirabilis, so studies of the function of the gene isolated from Mirabilis were conducted in Petunia, using the highly homologous Petunia sequence, and a VIGS protocol. A tobacco rattle virus (TRV) construct bearing a fragment of the Petunia chalcone synthase (CHS) gene was used as a vector for VIGS in a purple-flowered Petunia (Chen et al., 2004). White sectors or flowers result from silencing of CHS and serve as a reporter for silencing of target genes whose fragments are included in the same vector (Chen et al., 2004). The longevities of wild-type, of white flowers from Petunia plants infected with TRV bearing a CHS fragment, and of purple flowers from plants infected with TRV bearing PhXB3/CHS fragments were not significantly different, averaging 10.5 d. In contrast, the life of silenced (white) flowers from the phxb3/chs plants was significantly longer, averaging 12.4 d (Table 1). This effect on floral longevity was associated with a reduction in transcript abundance (to <5% of the control level) only in the silenced plants (Table 1).
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Isolation of the MjXB3 promoter
A 2 kb promoter region upstream of MjXB3 was isolated and sequenced. Analysis of the sequence using the PLACE database revealed putative binding sites for various transcription factors, including the bZIP, Myb, homeodomain-leucine zipper (HD-Zip), MADS-box, and WRKY transcription factors (data not shown). Several elements that are over-represented in light-responsive gene promoters were also found in the MjXB3 promoter.
Transient assay of the activity of the MjXB3 promoter
Two different length fragments of the MjXB3 promoter region (starting at –1734 bp and –914 bp) were cloned and linked to the GUS reporter gene, and used to infect different tissues at various physiological stages. Fusions of GUS with the 35S promoter and the SAG12 promoter were used as controls. Strong GUS expression was detected in both fresh and senescing Petunia flowers inoculated with the 35S::GUS fusion construct (Fig. 3). The SAG12::GUS construct drove expression strongly in the senescing flowers, but also drove some expression in the fresh flowers. In contrast, the MjXB3::GUS fusions, regardless of length, drove GUS expression only in senescing flowers. The strongest expression was seen when GUS was driven by the –914 bp fragment (Fig. 3). The MjXB3(–914)::GUS construct was therefore used in subsequent experiments.
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The 35S promoter drove GUS expression in young leaves, senescing leaves, and roots of Petunia plants (Fig. 3). SAG12 drove expression in senescing leaves and roots, and weak expression in young leaves, while the MjXB3 promoter (–914 fragment) did not drive obvious GUS expression in any of the tissues tested (Fig. 3) except the senescing petals.
As in Petunia, the MjXB3 promoter drove GUS expression in ethylene-treated carnation petals that had inrolled, but little, if at all, in the untreated ones (Fig. 4). GUS expression driven by the SAG12 promoter was very strong in the ethylene-treated carnation petals, but was also apparent in the untreated ones. The 35S promoter drove GUS expression in both ethylene-treated and untreated petals, but to a higher level in the treated ones.
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The 35S and SAG12 promoters drove GUS expression in senescing corollas of daffodil, daylily, and orchid, although the level of GUS expression was relatively low in orchid (Fig. 5). However, GUS expression could not be detected in any of the corollas that had been inoculated with the MjBX3(–914)::GUS construct (Fig. 5).
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Discussion |
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The deduced MjXB3 protein shows high homology to RING zinc finger domain-containing ankyrin repeat proteins, especially to ubiquitin ligases, suggesting a role in ubiquitination of specific proteins, thereby targeting them for degradation in the proteasome (Ciechanov and Schwartz, 1998; Lorick et al., 1999). This hypothesis is supported by the increase in flower longevity resulting from silencing expression of this gene (Table 1). The 20% increase in the life of Petunia corollas where this gene had been silenced suggests that its expression may be part of the senescence network, although senescence processes can continue in its absence.
RING zinc finger domain-containing ankyrin repeat proteins have been suggested to play a regulatory role in a number of different phases of plant growth and development (Huang et al., 2006; Ko et al., 2006; Wang et al., 2006). Among the RING zinc finger genes identified in the complete genome sequence of Arabidopsis thaliana, MjXB3 is most similar to XBAT31 (At2g28840). The function of XBAT31 remains unknown, but another XB3 homologue, XBAT32, is expressed in root cortical cells flanking lateral root primordia, as these cortical cells enter programmed cell death (Kosslak et al., 1997; Nodzon et al., 2004). Importantly, MjXB3 shows high similarity to the ubiquitin ligase (XB3) that is required for a pathogen-induced type of programmed cell death in rice (Wang et al., 2006).
When driven by MjXB3 promoter fragments, the GUS reporter gene showed a senescence-specific expression pattern. Although RT-PCR detected MjXB3 expression in stems and roots at a similar level to that in senescing flowers (Fig. 2A), transient GUS expression suggests that MjXB3(–914) is specific to senescing flowers (Fig. 3). It could be that RT-PCR experiments detected the expression of other MjXB3 homologues. There are, for example, five putative XB3 homologues in the Arabidopsis genome (Nodzon et al., 2004). The SAG12 promoter has been reported to drive GUS expression in senescing but not young leaves in Arabidopsis, and therefore was used as a control in this study (Noh and Amasino, 1999). Since SAG12 did drive some GUS expression in fresh Petunia and carnation corollas, it seems that the SAG12 promoter is not as senescence specific in flowers as the MjXB3(–914) promoter.
It was intriguing to note that the MjXB3 promoter was decorated with binding elements for a range of DNA-binding factors, including the WRKY, bZIP, Myb, HD-Zip, and MADS-box transcription factors. In other systems, transcription factors in each of these families have been shown to play a role in the regulation of senescence (Fernandez et al., 2000; Hinderhofer and Zentgraf, 2001; Miao et al., 2004; Xu et al., 2007). The number of DNA-binding elements on the promoter is consistent with the network model of senescence control suggested by He et al. (2001).
The fact that programmed cell death induced by a pathogen required a ubiquitin ligase (Wang et al., 2006), and the data presented here, suggests that ubiquitin ligases are an important common feature of programmed cell death in plants.
It was interesting to find that the MjXB3 promoter did not drive gene expression in the senescing corollas of the ethylene-independent daylily and daffodil, nor in the senescing petals of the ethylene-dependent Dendrobium ‘Emma White’ orchid, although 35S and SAG12 promoters did. It seems that the promoter, although effective in heterologous dicotyledons, is not active in monocotyledonous species. Many senescence-associated genes have been isolated from flowers of daylily and iris (Valpuesta et al., 1995; van Doorn et al., 2003). Searching for MjXB3 homologues in these species and isolation and analysis of their promoters may provide more insights into the regulation of senescence in ethylene-independent monocotyledonous flowers and useful tools to manipulate their longevity genetically.
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Acknowledgements |
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We appreciate the assistance of Dr Dinesh-Kumar Savithramma, Yale University, in providing the TRV constructs. Goldsmith Seeds generously donated seeds of blue-flowered hybrid Petunia cultivars. We gratefully acknowledge the generous financial support of the American Floral Endowment and the USDA Agricultural Research Service.
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