| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
|
First published online May 11, 2007; 10.1104/pp.107.096115 Plant Physiology 144:1680-1689 (2007) © 2007 American Society of Plant Biologists
Identification of a Bipartite Jasmonate-Responsive Promoter Element in the Catharanthus roseus ORCA3 Transcription Factor Gene That Interacts Specifically with AT-Hook DNA-Binding Proteins1,[W]Institute of Biology, Clusius Laboratory, Leiden University, 2333 AL Leiden, The Netherlands (D.V.E., M.S.S., J.W.K., J.M.); and Centro de Biotecnologia/Universidade Federal do Rio Grande do Sul, CEP 91501–970 Porto Alegre, Brazil (D.V.E., G.P.)
Jasmonates are plant signaling molecules that play key roles in defense against certain pathogens and insects, among others, by controlling the biosynthesis of protective secondary metabolites. In Catharanthus roseus, the APETALA2-domain transcription factor ORCA3 is involved in the jasmonate-responsive activation of terpenoid indole alkaloid biosynthetic genes. ORCA3 gene expression is itself induced by jasmonate. By loss- and gain-of-function experiments, we located a 74-bp region within the ORCA3 promoter, which contains an autonomous jasmonate-responsive element (JRE). The ORCA3 JRE is composed of two important sequences: a quantitative sequence responsible for a high level of expression and a qualitative sequence that appears to act as an on/off switch in response to methyl jasmonate. We isolated 12 different DNA-binding proteins having one of four different types of DNA-binding domains, using the ORCA3 JRE as bait in a yeast (Saccharomyces cerevisiae) one-hybrid transcription factor screening. The binding of one class of proteins bearing a single AT-hook DNA-binding motif was affected by mutations in the quantitative sequence within the JRE. Two of the AT-hook proteins tested had a weak activating effect on JRE-mediated reporter gene expression, suggesting that AT-hook family members may be involved in determining the level of expression of ORCA3 in response to jasmonate.
Jasmonic acid (JA) and its volatile methyl ester (MeJA), collectively called jasmonates, are fatty acid derivatives that are synthesized via the octadecanoid pathway (Turner et al., 2002  ; Wasternack et al., 2006). Jasmonates are involved in the regulation of a number of processes in plants, including certain developmental processes, senescence, and responses to wounding and pathogen attack (Turner et al., 2002; Wasternack et al., 2006). An important defense response that depends on jasmonates as regulatory signals is the induction of secondary metabolite accumulation (Gundlach et al., 1992; Memelink et al., 2001). JA induces secondary metabolism at the transcriptional level by switching on the coordinate expression of a set of biosynthetic genes (Schenk et al., 2000; Memelink et al., 2001).
The APETALA2 (AP2)-domain transcription factor ORCA3 (octadecanoid-derivative responsive Catharanthus AP2-domain) is a regulator of several genes involved in primary and secondary metabolism, including the strictosidine synthase (STR) gene, leading to terpenoid indole alkaloid biosynthesis in the plant species Catharanthus roseus (van der Fits and Memelink, 2000
The expression of the ORCA3 gene itself is rapidly induced by MeJA (van der Fits and Memelink, 2001
Different (Me)JA-responsive elements (JREs) have been identified in several plant promoters. The promoters of the PI-II (proteinase inhibitor) gene of potato (Solanum tuberosum; Kim et al., 1992 Here, we describe the identification of a bipartite (Me)JRE in the ORCA3 promoter, which is composed of an A/T-rich quantitative sequence determining the expression level and a G-box-like qualitative sequence switching on the (Me)JA response. Using yeast (Saccharomyces cerevisiae) one-hybrid screening, we have isolated four classes of DNA-binding proteins. One class characterized by a single AT-hook DNA-binding motif binds specifically to the quantitative (Me)JA-responsive sequence.
The ORCA3 Promoter Is Responsive to MeJA
ORCA3 mRNA accumulation in suspension-cultured C. roseus cells is rapidly induced by MeJA (van der Fits and Memelink, 2001
Localizing the JRE by 5' Deletion Analysis
To locate the JA-responsive region within the
Localizing the JRE by Internal Deletion Analysis
To narrow down the JA-responsive region in the ORCA3 promoter, an internal deletion analysis was performed in the context of the
A Fragment Containing the JRE Autonomously Confers MeJA-Responsive Transcriptional Activation
To determine whether regions in the The C fragment, which is a subfragment of D, confers a very low level of MeJA responsiveness, indicating that it contains part, but not all, of a functional JRE. The B fragment, which overlaps for a large part with the D fragment, does not carry any complete JRE. Finally, fragment A appears to carry a weak JRE distinct from the one in the D fragment.
Block scanning mutagenesis of the D region of the ORCA3 promoter (Fig. 4A ) was performed to pinpoint the exact location of the JRE. The activities of seven mutated versions of the tetramerized D fragment were analyzed in two independently transformed C. roseus cell lines for each construct. By comparing basal and MeJA-induced GUS gene expression for each mutant line to the expression conferred by the wild-type D tetramer, mutants M1, M4, and M5 were found to confer wild-type MeJA responsiveness (Fig. 4B). With mutation M2 and to some degree M3, the activation of GUS expression by MeJA treatment was significantly reduced, but expression was still induced. Mutant M6 and especially M7 conferred a basal expression level, which is actually reduced in response to MeJA. In each cell line, MeJA induced the expression of the endogenous ORCA3 gene to similar levels, and all cell lines expressed the CAT transgene at similar levels. These results revealed the presence of two different sequence elements in the D region. The first sequence is located in the region covered by mutations M2 and M3 around position –121 and seems to be necessary for a high level of expression, which we therefore call a quantitative sequence. The second sequence, situated in the region covered by mutations M6/M7, seems to be necessary to silence the basal expression level, and it activates transcription in response to MeJA. Therefore, this is a qualitative sequence, which acts as an on/off switch in response to MeJA.
Isolation of cDNAs Encoding Proteins Binding to Fragment D
In a first approach, the D region of the ORCA3 promoter was used as bait in a yeast one-hybrid transcription factor screening. Derivatives of yeast strain Y187, containing one or two copies of the D fragment fused to the HIS3 selection marker, were used in a one-hybrid screen for DNA-binding proteins with an expression library of C. roseus cells treated with MeJA, where cDNAs were cloned in a fusion with the GAL4 activation domain in the yeast expression vector pAD-GAL4-2.1. A total of 2.4 x 106 Y187-1D and 4.1 x 106 Y187-2D transformants were screened. Eighteen plasmids were isolated that conferred increased growth in Y187-1D and Y187-2D compared with the Y187 control, which corresponded to eight different mRNA species (Table I
). These eight species can be divided in three classes. The first class is represented by the single clone 2D81 encoding a protein with homology to a poorly characterized family of putative zinc-finger transcription factors (Kuusk et al., 2006
In another approach, a Y187-derived yeast strain containing four copies of the C element fused to the HIS3 selection gene was screened with an expression library of C. roseus cells treated with yeast extract, where cDNAs were cloned into the vector pACTII. Screening of 2.5 x 106 transformants yielded 87 colonies, of which 82 contained plasmids encoding single-repeat MYB transcription factors falling in four different related families (Table I).
To test whether mutation of the two distinct sequences within the D region of the ORCA3 promoter impaired binding of the DNA-binding proteins encoded by the one-hybrid clones, glutathione S-transferase (GST)-tagged proteins produced in Escherichia coli and D fragment variants were used in electrophoretic mobility shift assays. The 2D81 protein bound wild-type D fragment as well as all its mutated versions (Fig. 5 ), indicating that the encoded protein is not specific for the D element or that its binding site is located outside the mutated region. Both HD-Zip proteins 2D206 and 2D21 seemed to bind better to mutated D fragments M3 and M4 and showed reduced binding to mutated D fragment M1 compared to binding to the wild-type D fragment (Fig. 5; results not shown). The M1 mutation has no effect on MeJA responsiveness of the D region in Catharanthus cells. Binding of the four classes of single-repeat MYB proteins was impaired by mutation M4 (Fig. 5; results not shown), which has no effect on MeJA responsiveness of the D region in vivo. Binding of the zinc-finger, HD-Zip, and 1R-MYB classes of proteins was not affected by mutations in the two jasmonate-responsive sequences, suggesting that these proteins are not involved in the MeJA responsiveness or the level of expression conferred by the D fragment. All five AT-hook proteins did not bind to mutated D fragments M2 and M3 but did bind to the M1, M4, M5, M6, and M7 fragments (Fig. 5; results not shown). The importance of the M2/M3 region for binding of AT-hook proteins indicates that these transcription factors may be responsible for activation of gene expression mediated by the quantitative sequence.
AT-Hook Proteins Are Weak Activators and Are Not Transcriptionally Induced by MeJA To test the hypothesis that the AT-hook proteins are activators of transcription via the D element, transactivation assays were conducted. Clones 2D173, 2D38M, and 2D7 were selected for these experiments, because they possibly contain the full-length coding regions, whereas the other two clones are obviously incomplete. As shown in Figure 6 , 2D7 and 2D173 caused 2- and 1.5-fold increases, respectively, in GUS reporter gene expression, whereas 2D38M had little or no effect.
To study whether the AT-hook proteins could possibly contribute to the MeJA responsiveness of the ORCA3 promoter as a result of increased protein abundance due to stimulation of AT-hook gene expression by MeJA, the mRNA levels corresponding to the AT-hook clones were determined in a MeJA time course. Although we were unable to obtain detectable hybridization with clone 2D7, mRNA species hybridizing to clones 2D38M, 2D173, 2D328, and 2D449 showed little or no variation in abundance after MeJA treatment within a 24-h time course (Fig. 7 ).
Using transgenic C. roseus cell suspensions harboring ORCA3 promoter-GUS fusions, we have demonstrated that MeJA-induced accumulation of ORCA3 mRNA occurs at the transcriptional level. By loss- and gain-of-function experiments, we located a 74-bp region within the ORCA3 promoter containing an autonomous JRE. Block scanning mutagenesis revealed two important sequences within the JRE: a quantitative sequence responsible for a high level of expression and a qualitative sequence that acts as an on/off switch in response to MeJA. Moreover, we identified by one-hybrid screening five clones of related AT-hook transcription factors, which bind specifically to the quantitative sequence in the ORCA3 JRE. Two of them were found to have a weak activating effect on D-mediated reporter gene expression, indicating that AT-hook family members are possibly involved in determining the level of expression conferred by the JRE in response to jasmonate.
A range of motifs has been found to be responsible for JA-induced transcription of various genes. A region containing an ACCGCC sequence, which binds the ORCA3 transcription factor (van der Fits and Memelink, 2001
Using the D and C fragments in one-hybrid screening, we have isolated four classes of DNA-binding proteins with possible roles in regulation of gene expression. Analysis of binding in vitro revealed that only the binding of the AT-hook class of proteins was affected by mutations in a sequence important for the jasmonate-responsive activity of the D region. The AT-hook proteins have impaired binding to mutated D derivatives M2 and M3. This region of the D fragment defines the quantitative JA-responsive sequence (Fig. 4). Therefore, one or more of the AT-hook proteins is probably responsible for the activity of the quantitative sequence. In accordance with this notion, two of the AT-hook proteins had an activating effect on D element-mediated gene expression. The weak transactivation effect observed is in line with the relatively mild effect of the M2 and M3 mutations on the D-mediated gene expression level. The AT-hook motif was first identified in the high mobility group I(Y) [HMG-I(Y)] nonhistone chromosomal proteins, which contain three copies of this motif (Aravind and Landsman, 1998 ). The AT-hook motif is a nine-amino acid sequence with a central highly conserved Gly-Arg-Pro core, which binds to the minor groove of AT-rich DNA. The HMG-I(Y) proteins are thought to coregulate transcription by modifying the architecture of DNA, thereby enhancing the accessibility of promoters to transcription factors. Following its original identification, the AT-hook motif has been found in a large number of proteins as single or multiple copies, often associated with one or more other DNA-binding domains (Aravind and Landsman, 1998). Plants have a class of AT-hook proteins, which is characterized by an N-terminal histone H1 globular domain followed by multiple (4–15) AT-hook motifs (Meijer et al., 1996). The pathogenesis-related HD protein from parsley (Petroselinum crispum) contains four AT-hook motifs combined with a HD and a zinc-finger-like PHD finger (Korfhage et al., 1994). The Catharanthus AT-hook proteins described here contain only a single copy of the AT-hook motif, which is followed by a conserved DUF296 thought to be a DNA-binding domain based on its invariable association with the AT-hook motif. Currently, all classes of plant AT-hook proteins are poorly characterized at the functional level, with a total absence of functional information for the single AT-hook/DUF296 class.
Unfortunately, in this one-hybrid screening, DNA-binding proteins able to bind specifically to the qualitative sequence were not found. Based on its similarity to the LAP promoter element (Boter et al., 2004 Our current model is that the quantitative sequence in the JRE is bound by one or more transcriptional activators, which confer a high level of ORCA3 expression in response to (Me)JA. The AT-hook transcription factors isolated in the one-hybrid screening are likely to fulfill the role of these activators (Fig. 8). The qualitative sequence, on the other hand, seems to bind a repressor in the noninduced state (Fig. 8B). Activation could be conferred by binding of an AT-hook protein to the quantitative sequence upon release of the qualitative sequence from repression (Fig. 8C), in which case the qualitative sequence would only be an off switch. Alternatively, and more likely in our opinion, the qualitative sequence could also bind an (bHLH-type) activator in the induced state (Fig. 8D). In the later case, repressor and activator could be different proteins or could be the same (bHLH) protein switching between two activity states.
Construction of ORCA3 Promoter-GUS Fusions
ORCA3 promoter deletions were generated by PCR or using internal restriction sites and cloned into reporter plasmid GusXX. Six copies of the Tcyt element were cloned into
Catharanthus roseus cell line BIX was transformed using Agrobacterium tumefaciens. Each transgenic cell line was a mixed population estimated to consist of thousands of independent transformants. Transgene expression level therefore reflected an average largely independent of T-DNA chromosomal position or copy number. For each construct, two independent mixed transgenic cell populations were generated and analyzed. Cell suspension cultures were treated 4 d after transfer with 10 µM MeJA (Bedoukian Research) diluted in dimethylsulfoxide (DMSO) at a final concentration of 0.1% (v/v). Control cultures were treated with DMSO only. In the CHX experiments, cells were treated with 100 µM CHX (Sigma) and/or 10 µM MeJA at final concentrations of 0.2% (v/v) DMSO.
A monomer and a dimer of the D fragment and a tetramer of the C fragment were fused to a TATA box-HIS3 gene. HIS3 gene constructs were integrated in the genome of yeast (Saccharomyces cerevisiae) strain Y187 by homologous recombination. After transformation of the cDNA libraries in the 1D, 2D, and 4C yeast strains, cells were plated on synthetic dextrose minimal medium lacking Leu and His supplemented with 5, 15, and 5 mM 3-amino-1,2,4-triazole (Sigma), respectively. Colonies were patched on medium containing 20 µg/mL X-
The inserts from selected one-hybrid plasmids (Table I) were cloned into pGEX-KG or pGEX-4T1. GST fusion proteins were isolated using glutathione Sepharose 4B beads (GE Healthcare) according to the manufacturer's instructions.
C. roseus cells of cell line MP183L were transformed by particle bombardment as described (Menke et al., 1999 Sequence data from this article can be found in the GenBank/EMBL data libraries under accession numbers EF025303 to EF025314.
The authors thank Ward de Winter for assistance with tissue culturing. Received January 18, 2007; accepted May 3, 2007; published May 11, 2007.
1 This work was supported by a Socrates/Erasmus grant (to M.S.S.). 
2 These authors contributed equally to the article. The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Johan Memelink (j.memelink{at}biology.leidenuniv.nl).
[W] The online version of this article contains Web-only data. www.plantphysiol.org/cgi/doi/10.1104/pp.107.096115 * Corresponding author; e-mail j.memelink{at}biology.leidenuniv.nl; fax 31–71–5275088.
Aravind L, Landsman D (1998) AT-hook motifs identified in a wide variety of DNA-binding proteins. Nucleic Acids Res 26: 4413–4421 Boter M, Ruíz-Rivero O, Abdeen A, Prat S (2004) Conserved MYC transcription factors play a key role in jasmonate signaling both in tomato and Arabidopsis. Genes Dev 18: 1577–1591 Brown RL, Kazan K, McGrath KC, Maclean DJ, Manners JM (2003) A role for the GCC-box in jasmonate-mediated activation of the PDF1.2 gene of Arabidopsis. Plant Physiol 132: 1020–1032 Chatel G, Montiel G, Pré M, Memelink J, Thiersault M, Saint-Pierre B, Doireau P, Gantet P (2003) CrMYC, a Catharanthus roseus elicitor- and jasmonate-responsive bHLH transcription factor that binds the G-box element of the strictosidine synthase gene promoter. J Exp Bot 54: 2587–2588 Chinnusamy V, Ohta M, Kanrar S, Lee B-H, Hong X, Agarwal M, Zhu J-K (2003) ICE1: a regulator of cold-induced transcriptome and freezing tolerance in Arabidopsis. Genes Dev 17: 1043–1054 Edwards DR, Mahadevan LC (1992) Protein synthesis inhibitors differentially superinduce c-fos and c-jun by three distinct mechanisms: lack of evidence for labile repressors. EMBO J 11: 2415–2424[ISI][Medline] Foster R, Izawa T, Chua NH (1994) Plant bZIP proteins gather at ACGT elements. FASEB J 8: 192–200[Abstract] Guerineau F, Benjdia M, Zhou DX (2003) A jasmonate-responsive element within the A. thaliana vsp1 promoter. J Exp Bot 54: 1153–1162 Gundlach H, Müller MJ, Kutchan TM, Zenk MH (1992) Jasmonic acid is a signal transducer in elicitor-induced plant cell cultures. Proc Natl Acad Sci USA 89: 2389–2393 He Y, Gan S (2001) Identical promoter elements are involved in the regulation of the OPR1 gene by senescence and jasmonic acid in Arabidopsis. Plant Mol Biol 47: 595–605[CrossRef][ISI][Medline] Kim S-R, Choi J-L, Costa MA, An G (1992) Identification of G-box sequence as an essential element for methyl-jasmonate response of potato proteinase inhibitor II promoter. Plant Physiol 99: 627–631 Korfhage U, Trezzini GF, Meier I, Hahlbrock K, Somssich IE (1994) Plant homeodomain protein involved in transcriptional regulation of a pathogen defense-related gene. Plant Cell 6: 695–708 Kuusk S, Sohlberg JJ, Magnus Eklund D, Sundberg E (2006) Functionally redundant SHI family genes regulate Arabidopsis gynoecium development in a dose-dependent manner. Plant J 47: 99–111[CrossRef][ISI][Medline] Mason HS, DeWald DB, Mullet JE (1993) Identification of a methyl jasmonate-responsive domain in the soybean vspB promoter. Plant Cell 5: 241–251[Abstract] Meijer AH, van Dijk EL, Hoge JHC (1996) Novel members of a family of AT hook-containing DNA-binding proteins from rice are identified through their in vitro interaction with consensus target sites of plant and animal homeodomain proteins. Plant Mol Biol 31: 607–618[CrossRef][ISI][Medline] Memelink J, Verpoorte R, Kijne JW (2001) ORCAnisation of jasmonate-responsive gene expression in alkaloid metabolism. Trends Plant Sci 6: 212–219[CrossRef][ISI][Medline] Menke FLH, Champion A, Kijne JW, Memelink J (1999) A novel jasmonate- and elicitor-responsive element in the periwinkle secondary metabolite biosynthetic gene Str interacts with a jasmonate- and elicitor-inducible AP2-domain transcription factor, ORCA2. EMBO J 18: 4455–4463[CrossRef][ISI][Medline] Neuteboom STC, Stoffels A, Hulleman E, Memelink J, Schilperoort RA, Hoge JHC (1993) Interaction between the tobacco DNA-binding activity CBF and the cyt-1 promoter element of the Agrobacterium tumefaciens T-DNA gene T-CYT correlates with cyt-1 directed gene expression in multiple tobacco tissue types. Plant J 4: 525–534[CrossRef][ISI][Medline] Pré M, Sibéril Y, Memelink J, Champion A, Doireau P, Gantet P (2000) Isolation by the yeast one-hybrid system of cDNAs encoding transcription factors that bind to the G-box element of the strictosidine synthase gene promoter from Catharanthus roseus. Int J Bio-Chromatogr 5: 229–244 Rouster J, Leah R, Mundy J, Cameron-Mills V (1997) Identification of a methyl jasmonate-responsive region in the promoter of a lipoxygenase 1 gene expressed in barley grains. Plant J 11: 513–523[CrossRef][ISI][Medline] Schenk PM, Kazan K, Wilson I, Anderson JP, Richmond T, Somerville SC, Manners JM (2000) Coordinated plant defense responses in Arabidopsis revealed by microarray analysis. Proc Natl Acad Sci USA 97: 11655–11660 Sibéril Y, Benhamron S, Memelink J, Giglioli-Guivarc'h N, Thiersault M, Boisson B, Doireau P, Gantet P (2001) Catharanthus roseus G-box binding factors 1 and 2 act as repressors of strictosidine synthase gene expression in cell cultures. Plant Mol Biol 45: 477–488[CrossRef][ISI][Medline] Solano R, Stepanova A, Chao Q, Ecker JR (1998) Nuclear events in ethylene signaling: a transcriptional cascade mediated by ETHYLENE-INSENSITIVE3 and ETHYLENE RESPONSE-FACTOR1. Genes Dev 12: 3703–3714 Turner JG, Ellis C, Devoto A (2002) The jasmonate signal pathway. Plant Cell (Suppl) 14: S153–S164 van der Fits L, Memelink J (2000) ORCA3, a jasmonate-responsive transcriptional regulator of plant primary and secondary metabolism. Science 289: 295–297 van der Fits L, Memelink J (2001) The jasmonate-inducible AP2/ERF-domain transcription factor ORCA3 activates gene expression via interaction with a jasmonate-responsive promoter element. Plant J 25: 43–53[CrossRef][ISI][Medline] Wasternack C, Stenzel I, Hause B, Hause G, Kutter C, Maucher H, Neumerkel J, Feussner I, Miersch O (2006) The wound response in tomato: role of jasmonic acid. J Plant Physiol 163: 297–306[CrossRef][ISI][Medline] Xu B, Timko M (2004) Methyl jasmonate induced expression of the tobacco putrescine N-methyltransferase genes requires both G-box and GCC-motif elements. Plant Mol Biol 55: 743–761[CrossRef][ISI][Medline]
|
|||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| ASPB Publications | PLANT PHYSIOLOGY | THE PLANT CELL | |
|---|---|---|---|
TOP
ABSTRACT
RESULTS
826 ORCA3 promoter extending from positions –826 to –53 relative to the translational start codon. In transgenic cells incubated with MeJA, expression of GUS was induced within 0.5 h and continued to rise up to 6 h. Accumulation of the endogenous ORCA3 transcript was also induced within 0.5 h of treatment with MeJA but thereafter followed different kinetics. To better understand the mechanisms behind these different kinetics, we compared GUS and ORCA3 mRNA accumulation in the presence of the protein synthesis inhibitor cycloheximide (CHX). ORCA3 mRNA accumulation is superinduced by a combination of MeJA and CHX (
CAT (Menke et al., 1999
-Gal (5-bromo-4-chloro-3-indolyl-