APETALA2ETHYLENE RESPONSE FACTOR ... - Wiley Online Library

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Apr 5, 2011 - TOR OF JAZ (NINJA) and TOPLESS proteins, that blocks. MYC2 activity (Chini et al., 2007; Thines et al., 2007; Pauwels et al., 2010).
The Plant Journal (2011) 66, 1053–1065

doi: 10.1111/j.1365-313X.2011.04566.x

APETALA2/ETHYLENE RESPONSE FACTOR and basic helix–loop–helix tobacco transcription factors cooperatively mediate jasmonate-elicited nicotine biosynthesis Kathleen De Boer1,†,‡, Sofie Tilleman2,3,‡, Laurens Pauwels2,3,‡, Robin Vanden Bossche2,3, Valerie De Sutter2,3, Rudy Vanderhaeghen2,3, Pierre Hilson2,3, John D. Hamill1 and Alain Goossens2,3,* 1 School of Biological Sciences, Monash University, Melbourne, Vic. 3800, Australia, 2 Department of Plant Systems Biology, VIB, 9052 Gent, Belgium, and 3 Department of Plant Biotechnology and Genetics, Ghent University, 9052 Gent, Belgium Received 29 November 2010; revised 2 March 2011; accepted 4 March 2011; published online 5 April 2011. * For correspondence (fax +32 9 3313809; e-mail [email protected]). † Present address: Department of Anatomy and Developmental Biology, Monash University, Melbourne, Vic. 3800, Australia. ‡ These authors contributed equally to this work.

SUMMARY Transcription factors of the plant-specific apetala2/ethylene response factor (AP2/ERF) family control plant secondary metabolism, often as part of signalling cascades induced by jasmonate (JA) or other elicitors. Here, we functionally characterized the JA-inducible tobacco (Nicotiana tabacum) AP2/ERF factor ORC1, one of the members of the NIC2-locus ERFs that control nicotine biosynthesis and a close homologue of ORCA3, a transcriptional activator of alkaloid biosynthesis in Catharanthus roseus. ORC1 positively regulated the transcription of several structural genes coding for the enzymes involved in nicotine biosynthesis. Accordingly, overexpression of ORC1 was sufficient to stimulate alkaloid biosynthesis in tobacco plants and tree tobacco (Nicotiana glauca) root cultures. In contrast to ORCA3 in C. roseus, which needs only the GCC motif in the promoters of the alkaloid synthesis genes to induce their expression, ORC1 required the presence of both GCC-motif and G-box elements in the promoters of the tobacco nicotine biosynthesis genes for maximum transactivation. Correspondingly, combined application with the JA-inducible Nicotiana basic helix–loop–helix (bHLH) factors that bind the G-box element in these promoters enhanced ORC1 action. Conversely, overaccumulation of JAZ repressor proteins that block bHLH activity reduced ORC1 functionality. Finally, the activity of both ORC1 and bHLH proteins was post-translationally upregulated by a JA-modulated phosphorylation cascade, in which a specific mitogen-activated protein kinase kinase, JA-factor stimulating MAPKK1 (JAM1), was identified. This study highlights the complexity of the molecular machinery involved in the regulation of tobacco alkaloid biosynthesis and provides mechanistic insights about its transcriptional regulators. Keywords: Nicotiana, basic helix–loop–helix, ethylene response factor, nicotine, jasmonate, mitogenactivated protein kinase.

INTRODUCTION The plant kingdom produces hundreds of thousands of different small compounds that are often genus or family specific. These molecules, usually called secondary metabolites, display an immense variety of structures and biological activities that plants have tapped into over the course of evolution, and that is now harnessed by humans for industrial and medical applications. The cellular and genetic programs that steer the production of secondary metabolites can be launched rapidly when plants perceive particular ª 2011 The Authors The Plant Journal ª 2011 Blackwell Publishing Ltd

environmental stimuli. The jasmonate phytohormones (JAs) play a prominent and universal role in mediating these responses as they can induce synthetic pathways of molecules of a wide structural variety, encompassing all major secondary metabolite classes (Zhao et al., 2005; Pauwels et al., 2009). Changes in JA concentrations control the biosynthesis of nicotine and other pyridine alkaloids in tobacco (Nicotiana tabacum) together with other development- and 1053

1054 Kathleen De Boer et al. stress-related signals (Baldwin et al., 1996; Ohnmeiss et al., 1997). The expression of structural genes involved in nicotine biosynthesis is stimulated by methyl jasmonate (MeJA) in cultured cells or hairy roots of Nicotiana species (Imanishi et al., 1998; Goossens et al., 2003; Cane et al., 2005). These genes include those coding for ornithine decarboxylase (ODC), arginine decarboxylase (ADC) and putrescine N-methyltransferase (PMT), all three essential for the synthesis of the pyrroline ring, as well as aspartate oxidase (AO) and quinolinate phosphoribosyltransferase (QPRT), essential for the synthesis of the pyridine ring. Recently, a functional genomics screen identified two basic helix–loop–helix (bHLH) transcription factors (TFs) that function as positive regulators in the JA activation of nicotine biosynthesis in Nicotiana benthamiana (Todd et al., 2010). The NbbHLHs are related to the Arabidopsis (Arabidopsis thaliana) bHLH transcriptional activators MYC2, MYC3 and MYC4 that are essential elements of the conserved plant JA signalling module (Lorenzo et al., 2004; Fonseca et al., 2009; Ferna´ndez-Calvo et al., 2011). In the absence of JA hormone, the Arabidopsis JASMONATE ZIM-DOMAIN JAZ transcriptional repressor proteins bind MYC2 and recruit a co-repressor complex, composed of the NOVEL INTERACTOR OF JAZ (NINJA) and TOPLESS proteins, that blocks MYC2 activity (Chini et al., 2007; Thines et al., 2007; Pauwels et al., 2010). In the presence of bioactive JAs, CORONATINE INSENSITIVE1 (COI1) targets the JAZ proteins for degradation, which in turn releases MYC2. In tobacco roots, JA also triggers the COI1-mediated degradation of the JAZ repressors, resulting in the transcriptional activation of nicotine biosynthesis genes (Shoji et al., 2008). MYC2 primarily controls the first wave of JA-induced gene expression that reprograms cellular growth, development and metabolism in a species-specific manner (Lorenzo et al., 2004; Pauwels et al., 2009). This genetic switch involves multiple other TFs from diverse families such as the plantspecific APETALA2/ETHYLENE RESPONSE FACTOR (AP2/ ERF) TFs that include major regulators of specific metabolic pathways. WIN1 and members of the SHINE-clade control biosynthesis of cuticular wax in Arabidopsis (Aharoni et al., 2004; Broun et al., 2004; Kannangara et al., 2007). ORA47 activates the expression of the jasmonate (JA) biosynthesis gene LOX3 (Pauwels et al., 2008). LeERF2 controls ethylene production in tomato (Solanum lycopersicum) (Zhang et al., 2009). The octadecanoid-derivative-responsive Catharanthus AP2 domain (ORCA) TFs mediate JA-induced terpenoid indole alkaloid (TIA) biosynthesis in the Madagascar periwinkle (Catharanthus roseus) (Memelink et al., 2001). Overexpression of an ORCA member, ORCA3, in periwinkle cells is sufficient to induce several TIA biosynthetic genes (van der Fits and Memelink, 2000). In tobacco, the ERF family of AP2 domain TFs consists of at least 239 members (Rushton et al., 2008), and very recently it has been established that AP2/ERF factors also

play a prominent regulatory role in nicotine biosynthesis. In fact, it turned out that the NIC2 locus, one of the long-sought regulatory NIC loci that positively regulate nicotine biosynthesis in tobacco, comprised at least seven clustered AP2/ ERF encoding genes (Shoji et al., 2010). These NIC2-locus ERF TFs are close homologues of the C. roseus ORCA3 and showed both functional redundancy and divergence in their capacity to regulate nicotine biosynthesis. Similar to the involvement of ORCA3 in TIA biosynthesis, NIC2-locus ERFs activate most of the known structural genes in the tobacco nicotine biosynthesis pathway and bind a GCC motif in the PMT promoter (Shoji et al., 2010). In agreement with these findings, we previously showed that two JA-inducible AP2/ERF TFs from tobacco, ORC1 and JAP1, positively regulate the PMT promoter in a protoplast-based transient expression assay (Goossens et al., 2003; De Sutter et al., 2005). ORC1 and JAP1 both belong, similar to all the NIC2 AP2/ERFs, to group IX of ERF genes, and are also known as ERF221 (one of the NIC2 AP2/ERFs in clade 2-1) and ERF10 (clade 1), respectively (Rushton et al., 2008; Shoji et al., 2010). With this work, we demonstrate that the tobacco AP2/ERF factor ORC1/ERF221 can stimulate pyridine alkaloid biosynthesis in different Nicotiana species by inducing the expression of genes acting in all nicotine pathway branches. Our data indicate that ORC1/ERF221 acts in the JA signalling cascade leading to increased nicotine biosynthesis in tobacco, and that JA-modulated post-translational modification(s) can enhance ORC1 activity. RESULTS ORC1 stimulates nicotine biosynthesis in Nicotiana species We have previously shown that ORC1, and to a lesser extent JAP1, can promote induction of PMT in transfected tobacco BY-2 protoplasts (De Sutter et al., 2005). Nevertheless, the constitutive overexpression of ORC1 and JAP1, separately or together, did not result in nicotine production in BY-2 cell cultures (De Sutter et al., 2005; S. Tilleman and A. Goossens, unpublished data), indicating that the mere expression of these two AP2/ERF factors is not sufficient to switch on nicotine synthesis in cells that do not normally produce alkaloids. Therefore, we further investigated the regulatory potential of ORC1 and JAP1 by assessing the effects of their overexpression in stably transformed tobacco plants, in which nicotine and other pyridine alkaloids are normally synthesized, even in the absence of MeJA elicitation. Overexpression of ORC1 correlated with an increase in nicotine concentration along with higher QPRT transcript levels (Figures 1a and S1a), supporting its involvement in the regulation of tobacco alkaloid biosynthesis. In contrast, overexpression of JAP1 did not noticeably increase pyridine alkaloid levels, nor did it lead to a detectable increase in QPRT transcript levels in tissues of transgenic seedlings (Figures 1a and S1b).

ª 2011 The Authors The Plant Journal ª 2011 Blackwell Publishing Ltd, The Plant Journal, (2011), 66, 1053–1065

ERFs and bHLHs control nicotine biosynthesis 1055 levels and nicotine and anabasine levels were clearly increased in ORC1 overexpression lines (Figures 1b and S1c). These findings suggest that overexpression of ORC1, but not of JAP1, is sufficient to stimulate pyridine alkaloid biosynthesis in transgenic Nicotiana species, which correlates with the observation that ORC1 is more potent in activating PMT transcription in transfected tobacco BY-2 protoplasts (De Sutter et al., 2005). These findings are also in agreement with those of Shoji et al. (2010), who showed that gain- and loss-of-function of another clade 2-1 NIC2-locus ERF, i.e. ERF189, increased and decreased significantly the accumulation of nicotine in transgenic tobacco hairy roots, respectively.

Figure 1. Stimulation of pyridine alkaloid biosynthesis by overexpression of ORC1 in Nicotiana species. (a) Accumulation of nicotine in transgenic Nicotiana tabacum plants. (b) Accumulation of nicotine and anabasine in transgenic Nicotiana glauca hairy roots. The Y-axis shows the average alkaloid levels in transgenic seedlings and roots overexpressing ORC1 or JAP1, or transformed with an empty vector control (C). Error bars indicate the standard error (n = 3 independent transgenic lines per construct except for N. tabacum ORC1 lines where n = 2, see Figure S1). Statistical significance was determined by Student’s t-test (**P < 0.01).

Elicitation of ORC1 expression by MeJA occurs primarily in the roots of tobacco plants The role and activity of ORC1 was further investigated given its positive regulatory function. First, we analysed ORC1 expression in whole organs of hydroponically grown plants. Consistent with nicotine biosynthesis taking place in the roots of tobacco plants, the key biosynthetic genes PMT and QPRT are preferentially transcribed in root tissues (Shoji et al., 2000; Sinclair et al., 2000; Cane et al., 2005). In mock conditions, ORC1 transcript levels were lower in the leaves than in the roots (Figure 2). Moreover, the transcriptional induction of ORC1 upon MeJA elicitation was mainly confined to the roots, analogous to the biosynthetic genes PMT and QPRT (Figure 2). The MeJA induction of ORC1 in root was transient and peaked at 30 min after elicitation, whereas transcript levels of PMT and QPRT increased regularly over a period of 24 h. Hence, ORC1 was preferentially expressed in the same organ as the biosynthetic genes PMT and QPRT in planta. Furthermore, upon MeJA elicitation, the increase in ORC1 expression preceded the induction of PMT and QPRT, as expected for a potential regulator, and as

Next, we examined whether ORC1 and JAP1 could redirect metabolic fluxes in a heterologous Nicotiana species, the tree tobacco (Nicotiana glauca). This species was selected because, in contrast to tobacco, transformed N. glauca hairy root cultures show the capacity to produce not only nicotine but also significant levels of the pyridine alkaloid anabasine, the synthesis of which only requires nicotinic acid and cadaverine as precursors, and hence does not involve PMT (Sinclair et al., 2004; Goossens and Rischer, 2007). As seen in transgenic tobacco plants, overexpression of JAP1 in N. glauca roots did not increase the alkaloid content nor QPRT transcript levels in any of the lines examined (Figures 1b and S1c). In contrast, QPRT transcript

Normalized gene expression

10

10

PMT

120

QPRT

ORC1 Root EtOH

8

8

6

6

100

Root MeJA Leaf EtOH

80

Leaf MeJA 60

4

4 40

2

0

2

0

0.5

1

2

6

24

0

20 0 0

0.5

1

2

6

24

0

0.5 1

2

6

24

Time (h) Figure 2. Organ-specific expression of biosynthetic and regulatory genes in tobacco plants. Normalized expression of PMT, QPRT and ORC1 in roots and leaves of mock-treated (ethanol, EtOH) or methyl jasmonate (MeJA)-elicited Nicotiana tabacum plants measured by quantitative RT-PCR. The Y-axis shows the expression ratio relative to the normalized transcript levels of mock-treated roots at 0 h. Time after elicitation is shown in h (X-axis). Error bars indicate the standard error (n = 12).

ª 2011 The Authors The Plant Journal ª 2011 Blackwell Publishing Ltd, The Plant Journal, (2011), 66, 1053–1065

1056 Kathleen De Boer et al. previously observed in BY-2 cells (Goossens et al., 2003; De Sutter et al., 2005) and hairy roots (Shoji et al., 2010). ORC1 activates the expression of multiple genes involved in nicotine biosynthesis We previously showed that ORC1 transactivates NsPMT2 promoter activity in transient protoplast assays (De Sutter et al., 2005). To test whether ORC1 controls the transcription of additional nicotine biosynthesis genes, we made new promoter–reporter constructs for the tobacco ADC, ODC and QPRT genes. Gene duplication was taken into consideration because differential MeJA induction has been reported within the ODC and QPRT families and promoter sequences of distinct relatives are now documented in public databases for some families (Imanishi et al., 1998; Goossens et al., 2003; Xu et al., 2004; J. D. Hamill, unpublished data). ORC1 induces multiple tobacco genes involved in nicotine biosynthesis. The strongest transactivation was observed with the ProQPRT2 construct (more than 30-fold), whereas only a very small increase in reporter gene activity was detected with the ProQPRT1 construct (2.2-fold; Figure 3a). This finding correlates with the observation that expression

Normalized LUC activity

(a) 45 *** 40 35 30 5

***

0

C

ORC1

ProQPRT1

C

ORC1

ProQPRT2

Normalized LUC activity

(b) 30 25

***

20 15 10

*

5 0

** C

ORC1

ProADC1

C

ORC1

ProADC2

C

ORC1

ProODC

Figure 3. ORC1activates expression of multiple nicotine biosynthesis genes in BY-2 protoplast transient expression assays. (a) Transactivation of ProQPRT1-fLUC and ProQPRT2-fLUC. (b) Transactivation of ProADC1-fLUC, ProADC2-fLUC and ProODC-fLUC. Transactivation is expressed relative to the normalized luciferase (LUC) activity of the mock-treated control (C). Error bars indicate the standard error (n = 8 separate transfection events and measurements). Statistical significance was determined by Student’s t-test (***P < 0.001; **P < 0.01; *P < 0.05).

of QPRT2, but not QPRT1, is stimulated by wounding or MeJA treatments in tobacco tissues (John D. Hamill, unpublished data). Furthermore, the transactivation of QPRT in tobacco protoplasts also matches the observations in stable transformed Nicotiana plants, in which overexpression of ORC1 triggered higher QPRT transcript levels (Figure S1). ORC1 also induced the promoters of the genes acting upstream of PMT in the nicotine biosynthetic pathway. ProADC2 was induced almost 20-fold and ProADC1 over fivefold. Only a minor increase (2.7-fold) was detected for ProODC (Figure 3b). In another report, the tobacco ERF189 was shown to regulate the expression of at least six genes involved in nicotine biosynthesis, including PMT, QPRT, ODC, N-methylputrescine oxidase (MPO), AO and quinolate synthase (QS) (Shoji et al., 2010). However, in contrast to ORC1, ERF189 efficiently activated ODC expression but did not appear to regulate ADC (Shoji et al., 2010). These previous results and our data suggest that the different ERF proteins encoded in the NIC2 locus are functionally divergent. The conserved G-box and GCC motif are both necessary, but not sufficient on their own, to relay ORC1 transactivation All transient expression assay experiments described above and previously (De Sutter et al., 2005) were performed with tobacco promoters of 650 bp or more. Prominent features that have been described for the N. tabacum PMT proximal promoters are a G-box (GCACGTTG) and a GCC motif (TGCGCCC), both of which are known to be essential sequences for JA or other stress-related responses in various plant species (Xu and Timko, 2004). Both elements are also present at similar positions in the Nicotiana sylvestris PMT2 promoter region tested previously (De Sutter et al., 2005), as well as in the promoters of QPRT2, ADC1 and ADC2 that respond strongly to ORC1 activation, but conspicuously not in those of QPRT1 and ODC that are poorly induced by the TF (Figure 3, Table 1). Guided by this observation, we tested a panel of native and mutant promoter sequences for their ability to respond to ORC1 in tobacco protoplast transient transactivation assays. The transcriptional activity of a minimal N. tabacum PMT1 promoter fragment (ProPMT1min; from )196 to )1 with respect to the translation initiation site) that contains both the G-box and the GCC motif was induced by ORC1 (Figure 4a) with similar efficiencies as the ProNsPMT2 promoter (De Sutter et al., 2005). Hence, the minimal PMT1 promoter element harboured all necessary cis elements to relay ORC1 induction. As expected because it is a known target of ERF189 and ORCA3 (van der Fits and Memelink, 2001; Shoji et al., 2010), deletion of the GCC motif in the minimal PMT1 promoter fragment (ProPMT1min-DGCC) abolished ORC1 transactivation activity (Figure 4a). But surprisingly, the sole deletion of the G-box (ProPMT1minDG) also dramatically reduced ORC1 activity, both with

ª 2011 The Authors The Plant Journal ª 2011 Blackwell Publishing Ltd, The Plant Journal, (2011), 66, 1053–1065

ERFs and bHLHs control nicotine biosynthesis 1057 Table 1 Presence of G-box and GCC motif in the promoters of tobacco alkaloid biosynthetic genes Gene

GCC motif

NtPMT1 NsPMT2 NtQPRT1 NtQPRT2 NtADC1 NtADC2 NtODC1 NtODC2

TGCGCCC TCCGCCC Not detected CCAGCCA TCGGCCT TCGGCCT Not detected Not detected

Positiona

G-boxb

)132/)126 )134/)128

)172/)167 )171/)166

CACGTT CACGTT Not detected AACGTG CACGTT CACGTT Not detected Not detected

)153/)147 )134/)128 )135/)129

Transcriptc

Position

)70 ND ND )82 ND ND )115 )108

)205/)200 )429/)424 )430/)425

a

Position relative to the start codon. The conserved sequences were searched over a length of 600 nucleotides upstream of the start codon. G-box located at a proximal distance upstream of the GCC motif. c Transcription initiation site relative to the start codon. ND, not determined. b

(c)

20

*** 15

10

**

**

50 40 30 20 10

ORC1 bHLH1

ORC1

C

ORC1

C

C

ORC1

5

60

Normalized LUC activity

(a) Normalized LUC activity

– –

+ –

– +

+ +

ProPMT1 min

ProPMT1 min ProPMT1 min ProPMT1 min ΔGCC ΔG

(d) ***

20 15 10 5

Pro PMT1 min

regard to the absolute expression levels and the transactivation effect (from 14.0-fold to 3.6-fold; Figure 4a). These findings suggest that the G-box element may also be involved in ORC1 function, directly or indirectly. In transactivation experiments with synthetic promoters containing four repeats of either the G-box or the GCC motif upstream of the Cauliflower Mosaic Virus 35S (CaMV35S) minimal promoter, neither the GCC motif nor the G-box alone were sufficient for ORC1 transcriptional induction

Pro4xGCC

Pro4xG

ORC1

C

ORC1

C

ORC1

C

ORC1

***

C

Normalized LUC activity

(b)

Pro4x G4xGCC

Normalized LUC activity

Figure 4. Importance of the G-box and the GCC motif for ORC1 activity. (a) Transactivation of intact and mutated minimal (min) ProPMT1 fragments. Mutated promoter fragments harboured 6-bp deletions at the G-box (DG) or the GCC motif (DGCC). (b) Transactivation of synthetic G-box (Pro4xG), GCC-motif (Pro4xGCC) and combination (Pro4xG4xGCC) promoter constructs. (c) Transactivation of ProPMT1MIN-fLUC by ORC1 and NbbHLH1. (d) Transactivation of ProQPRT2-fLUC by ORC1 and NbbHLH1. Transactivation is expressed relative to the normalized luciferase (LUC) activity of the mock-treated control (C). Error bars indicate the standard error (n = 8). Statistical significance was determined by Student’s t-test (***P < 0.001; **P < 0.01).

***

350 300 250 200 150 100 50

ORC1 bHLH1

– –

+ –

– +

+ +

ProQPRT2

(Figure 4b). A synthetic construct containing both the G-box and GCC motif tetramers was significantly – but more modestly than the ProPMT1min – induced by ORC1 (by 2.5fold) (Figure 4b). Our data suggest that the combined presence of both elements is necessary for optimal ORC1 activation in the regulatory cascade leading to the onset of nicotine biosynthesis, in agreement with the need for both elements for MeJA induction of the minimal ProPMT1 promoter fragment (Xu and Timko, 2004).

ª 2011 The Authors The Plant Journal ª 2011 Blackwell Publishing Ltd, The Plant Journal, (2011), 66, 1053–1065

1058 Kathleen De Boer et al. Finally, we searched for TFs that may act in combination with ORC1. NbbHLH1 is an interesting candidate: it is homologous to the MYC2 bHLH TF, it is induced by MeJA and it binds the G-box element of the PMT promoter in N. benthamiana, thereby relaying JA activation of nicotine biosynthesis (Todd et al., 2010). The full-length open reading frame (ORF) of the NbbHLH1 factor was transferred into the appropriate overexpression vector and tested for transient transactivation of the minimal PMT1 and the QPRT2 promoters. NbbHLH1 alone did not affect the transcriptional activity of ProPMT1min and induced ProQPRT2 6.7-fold (Figure 4c, d). However, when co-transfected, the combined activity of ORC1 and NbbHLH1 induced both promoters, up to 40-fold for ProPMT1min (Figure 4c) and almost 300-fold for ProQPRT2 (Figure 4d). These results indicate that the concerted action of both TFs leads to the optimal activation of the nicotine biosynthesis pathway.

(c)

6 5 4 3

*

2 1

hpORC1 MeJA





+



+

+

– –

ProNsPMT2



+

+

+

70

25

60 20 50 15

40 30

10

***

5

ORC1 JAZ1

– –

ProQPRT2

+ –

*** – –

ProPMT1 min

+ –

+ +

ProQPRT2

–Y–L

–Y–L–H

400

AD-NbbHLH1 DB-NtJAZ1

30 300

25 20

200

15

5

– + –

+ – –

+ + –

ProNsPMT2

+ + +

AD-AtMYC2

AD-NbbHLH1

100

– – –

AD-ORC1

AD

***

10

ORC1 MeJA MG132

+ +

(d)

35

20 10

– – –

– + –

+ – –

+ + –

+ + +

DB-AtJAZ1

Normalized LUC activity

(b)

We noted that the transcriptional activity of the ProNsPMT2 and ProQPRT2 promoters could be induced with MeJA in our protoplast transfection assays (Figure 5a), as it is in planta, indicating that JA perception still occurs in tobacco protoplasts. To assess whether ORC1 participates in this MeJA elicitation, we adapted our automated protoplastbased assay to score gene silencing effects in combination with hormonal treatment. Twenty-four hours after transfection with hairpin (hp) gene silencing constructs, BY-2 protoplasts were elicited with MeJA and fLUC reporter activity was measured after incubation for another 24 h. The ProNsPMT2 and ProQPRT2 activities were slightly and strongly reduced, respectively, upon transfection with the ORC1 hp construct (Figure 5a), suggesting that NIC2 AP2/ERF factors including ORC1 are involved in the

Normalized LUC activity

Normalized LUC activity

(a)

Jasmonate signalling modulates ORC1 activity

AD-ORC1 AD-AtMYC2 AD

ProQPRT2

Figure 5. Involvement of jasmonate (JA) signalling modules in the combined effect of methyl JA (MeJA)/ORC1 on the expression of nicotine reporter genes. (a) Inhibition of the MeJA inducibility of nicotine reporter genes by silencing of ORC1 in BY-2 protoplasts. ProNsPMT2-fLUC and ProQPRT2-fLUC transactivation was measured 24 h after mock ()) or MeJA (+) treatment, added 24 h after protoplast transfection with the hpORC1 silencing construct. (b) Transactivation of ProNsPMT2-fLUC and ProQPRT2-fLUC by ORC1 in the presence of MeJA and/or MG132. (c) Transactivation of ProPMT1MIN-fLUC and ProQPRT2-fLUC by combined overexpression of ORC1 and JAZ1. Transactivation is shown relative to the normalized luciferase (LUC) activity of the mock-treated control. Error bars indicate the standard error (n = 8). Statistical significance was determined by Student’s t-test (***P < 0.001; *P < 0.05). (d) NtJAZ1 interacts with NbbHLH1 but not with ORC1. Yeast cells were transformed with NtJAZ1 or AtJAZ1 fused to GAL4DB and NbbHLH1, ORC1 or AtMYC2 fused to GAL4AD, spotted in 10- or 100-fold dilutions on synthetic defined (SD) medium lacking Trp and Leu (–Y–L) or Trp, Leu and His (–Y–L–H) and grown for 2 days at 30C. The destination vector pGADT7 was used as a control.

ª 2011 The Authors The Plant Journal ª 2011 Blackwell Publishing Ltd, The Plant Journal, (2011), 66, 1053–1065

ERFs and bHLHs control nicotine biosynthesis 1059 MeJA-mediated activation of nicotine biosynthesis in tobacco and consistent with the effects observed by RNA interference (RNAi)-mediated suppression of the NIC2 AP2/ ERF genes in tobacco plants (Shoji et al., 2010). The action of ORC1 may be invoked, at least in part, through MeJA-mediated transcriptional induction of the ORC1 gene (Goossens et al., 2003), together with that of the other NIC2 ERF and the MYC2-like bHLH factors (Rushton et al., 2008; Shoji et al., 2010; Todd et al., 2010). In addition, however, we noted that in transient expression assays, transactivation of ProNsPMT2 and ProQPRT2 by ORC1 was augmented with MeJA elicitation (Figure 5b), suggesting that the JA signal enhances the transactivation potential of the ORC1 protein. This other level of regulation may rely on the JA-induced protein degradation of the JAZ repressor proteins by the 26S proteasome that is a key event in JA signalling (Shoji et al., 2008; Fonseca et al., 2009). Hence, we adapted our protoplast-based assay to score gene activation in the presence of the proteasome inhibitor MG132 in combination with hormonal treatment. MG132 clearly reduced the united MeJA/ORC1 transactivation of ProNsPMT2 and ProQPRT2 (Figure 5b), indicating the involvement of MeJA-induced protein degradation in the regulation of ORC1 activity by MeJA. The inhibitory effect of MG132 and the united effect of ORC1 and bHLH1 (Figure 4) prompted us to assess the possible interaction between ORC1 and other proteins known to participate in the JA signalling module, such as the JAZ repressors. Homologues of the Arabidopsis JAZ proteins have also been identified in Nicotiana species (Shoji et al., 2008). The NtJAZ1 ORF was transferred to the appropriate expression vectors for co-transfection with ORC1 in transient expression assays and transactivation of the minimal PMT1 and the QPRT2 promoters. NtJAZ1 reduced the ORC1 transactivation of both promoters (Figure 5c). This inhibitory effect is probably mediated by repression of the bHLH TF activity, as MYC2 and related bHLH factors are the targets of JAZ repressors in Arabidopsis (Ferna´ndez-Calvo et al., 2011). This hypothesis was supported by yeast two-hybrid (Y2H) analysis that indicated that NtJAZ1 interacted with NbbHLH1 but not with ORC1 (Figure 5d). Similarly, Arabidopsis JAZ1 did not interact with ORC1 whereas it could interact with NbbHLH1. Conversely, NtJAZ1 interacted with Arabidopsis MYC2 (Figure 5d). These results further support the model in which NIC2 ERFs interact with the JA signalling module composed of bHLH activators and JAZ repressors. Phosphorylation and a specific mitogen-activated protein kinase kinase enhance ORC1 activity Besides degradation of repressor proteins from TF complexes, phosphorylation is another key post-translational regulatory mechanism at play in phytohormone signalling. To determine whether phosphorylation events modulate

ORC1-mediated transcriptional activation of nicotine biosynthetic genes, we combined MeJA elicitation with the application of protein kinase inhibitors in transient expression assays. Application of the MAP kinase (MAPK) inhibitor PD98059 diminished the united effect of MeJA/ORC1 on the ProQPRT2-fLUC and ProNsPMT2-fLUC reporter constructs (Figure 6a), thus suggesting the involvement of a MAPK cascade in the potentiation of ORC1 by MeJA. In the light of these results, we noticed that a gene coding for a putative MAPK kinase (MAPKK) was previously cloned in a screen for potential regulators of tobacco alkaloid biosynthesis (Ha¨kkinen et al., 2007). This MAPKK corresponded to the tag C476 that was identified by cDNAamplified fragment length polymorphism (AFLP) profiling as co-induced with the nicotine biosynthetic genes and ORC1 in MeJA-elicited tobacco BY-2 cells (Goossens et al., 2003; Ha¨kkinen et al., 2007). The full-length ORF of the C476 MAPKK (EMBL accession CQ808961), hereafter renamed JA-FACTOR STIMULATING MAPKK1 (JAM1) was transferred into the appropriate overexpression vector and tested in transient expression assays. The sole overexpression of JAM1 did not affect the transcriptional activity of ProQPRT2 (Figure 6b). When tested in the absence of MeJA, ORC1 and JAM1 combined had no additive effect on ProQPRT2. However, a spectacular effect of ORC1 and JAM1 together was detected in the presence of MeJA, leading to more than a 1000-fold induction of ProQPRT2 (Figure 6b). Such a stimulatory effect was not detected between JAP1 and JAM1, regardless of the presence of MeJA (Figure 6b), suggesting a specificity of JAM1 for ORC1 and perhaps other NIC2 ERFs. A similar combined effect between ORC1, JAM1 and MeJA was observed with the ProPMT1min reporter construct: transactivation of ProPMT1min-fLUC by ORC1 increased from about 17-fold in the absence of MeJA and JAM1 to more than 80-fold in the joint presence of these two additional effectors (Figure 6c). Since ORC1 activity on the ProPMT1min reporter construct is dependent on both the GCC-motif and the G-box (Figure 4a,b), this suggests that the united effect of JAM1 and MeJA elicitation on ORC1 transactivation was mediated through the GCC motif and/or the G-box elements, and thus may involve the NIC2 ERF as well as the bHLH TFs. To verify whether the stimulatory effect of JAM1 and MeJA was restricted to the NIC2 ERF factors, we also assessed the effect of combining NbbHLH1, JAM1 and MeJA on ProQPRT2-fLUC and ProPMT1min transactivation. Co-transfection with JAM1/MeJA did not increase the transactivation potential of NbbHLH1 for ProPMT1min (data not shown). In contrast, a clear positive effect between NbbHLH1, JAM1 and MeJA was observed in the transactivation of the ProQPRT2-fLUC construct: transactivation of ProQPRT2-fLUC by NbbHLH1 increased from about sevenfold in the absence of MeJA and JAM1 to more than 130-fold in the joint presence of these two additional

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1060 Kathleen De Boer et al.

Normalized LUC activity

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Figure 6. Involvement of mitogen-activated protein kinase (MAPK) phosphorylation in the methyl jasmonate (MeJA)/ORC1 combined effect on the expression of nicotine reporter genes. (a) Transactivation of ProNsPMT2-fLUC and ProQPRT2-fLUC by ORC1 in the presence of MeJA and/or PD98059. (b) Transactivation of ProQPRT2-fLUC by combined overexpression of JAP1/ORC1 and JAM1 in the presence of MeJA. (c) Transactivation of ProPMT1MIN-fLUC by combined overexpression of ORC1 and JAM1 in the presence of MeJA. (d) Transactivation of ProQPRT2-fLUC by combined overexpression of NbbHLH1 and JAM1 in the presence of MeJA. Transactivation is shown relative to the normalized luciferase (LUC) activity of the mock-treated control. Error bars indicate the standard error (n = 8). Statistical significance was determined by Student’s t-test (***P < 0.001; **P < 0.01; *P < 0.05).

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effectors (Figure 6d). These findings are also in agreement with the observation that the sole overexpression of NbbHLH1 did not affect the transcriptional activity of

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ProPMT1min but did induce that of ProQPRT2 (Figure 4c,d). Taken together, this suggests that the MeJA/JAM1 phosphorylation cascade may also stimulate the activity of bHLH

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ERFs and bHLHs control nicotine biosynthesis 1061

The molecular mechanisms involved in the regulation of Nicotiana sp. alkaloid biosynthesis have been studied for years and several potential regulatory proteins have been identified recently (Halitschke and Baldwin, 2003; Lou and Baldwin, 2006; Paschold et al., 2007; Rayapuram and Baldwin, 2007; Pandey and Baldwin, 2007; Ha¨kkinen et al., 2007; Shoji et al., 2008, 2010). Through previous transcriptomics-based screens, we identified several TFs and other proteins that are potentially involved in the MeJA-modulated regulation of pyridine alkaloid biosynthesis in tobacco BY-2 cells (Goossens et al., 2003; Ha¨kkinen et al., 2007; Morita et al., 2009). One such regulator is the TF ORC1/ ERF221, which we showed to induce the expression of PMT in tobacco protoplasts (De Sutter et al., 2005). A recent report demonstrated that ORC1/ERF221 is one of the seven AP2/ERF genes clustered in the NIC2 locus. Together, these NIC2 ERF TFs positively regulate the expression of genes involved in nicotine synthesis and transport (Shoji et al., 2010). Furthermore, ORC1 and other NIC2 TFs might possibly function as general activators of JA responsive physiologies that include, but are not limited to, alkaloid biosynthesis, as suggested by transcriptome analysis of nic1nic2 roots that indicated that the NIC loci regulate a complex network of stress response genes (Kidd et al., 2006; Shoji et al., 2010). In this study, we have further dissected the signal transduction cascades that regulate ORC1/ERF221 function. Figure 7 summarizes our main findings. ORC1 positively regulates the expression of multiple structural genes in the tobacco alkaloid synthetic pathway, like ERF189 in the same species or ORCA3 in periwinkle. This observation was initially based on transient expression assays in tobacco protoplasts. Furthermore, the stable overexpression of ORC1 was sufficient to stimulate pyridine alkaloid biosynthesis in at least two Nicotiana species, N. tabacum and N. glauca. This last result illustrates that pyridine alkaloid biosynthesis and AP2/ERF activity are not saturated, even in whole plants, and can be engineered by overexpression of a single TF, such as ORC1. Despite this analogy between the regulation of alkaloid biosynthesis in periwinkle and tobacco, the evolutionary forces that shaped the respective molecular machineries involved have resulted in functional specificities for the

? ?

JAM1

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Transcriptomics-based functional screens in stable and transient expression systems dissect the MeJA signalling cascades that regulate pyridine alkaloid biosynthesis in tobacco

SCF COI1

bHLH

DISCUSSION

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JAZ

factors, directly or indirectly. These results imply that a MAPK phosphorylation cascade is involved in the activation of nicotine biosynthesis by MeJA and in the potentiation of the complex that is composed of ERF and bHLH factors and that steers the expression of nicotine biosynthesis genes.

PMT, QPRT G-BOX

GCC-BOX

Figure 7. The jasmonate (JA)-signalling cascade that activates nicotine biosynthesis in tobacco. The methyl jasmonate (MeJA)-signalling cascade downstream of the SCFCOI1-mediated degradation of the JAZ repressor proteins potentiates ORC1 activity, at least partially by releasing the basic helix–loop–helix (bHLH) factors from the JAZ repressors. JAM1 may act downstream of the protein degradation event or in parallel to promote activity of ORC1, other NIC2 ethylene response factors (ERFs), and bHLH factors.

related TFs. This is exemplified by the observation that a GCC-motif element in the promoter of the periwinkle STR gene is sufficient to confer responsiveness to JA by recruiting the ORCA proteins, whereas deletion of the G-box from the STR promoter did not strongly affect the JA response (Menke et al., 1999). Nonetheless, the G-box element is found in most JA-responsive plant promoters and is responsible, for example, for binding of MYC2 and other bHLH transcriptional activators in Arabidopsis (Ferna´ndezCalvo et al., 2011). Both GCC motifs and G-boxes are also found in the proximal promoter sequences of several of the tobacco nicotine biosynthetic genes (Table 1), and the presence of both is required for induction of PMT1 by MeJA (Xu and Timko, 2004). In agreement with the latter observations, we found that the activity of ORC1 depends on the presence of both the GCC motif and the G-box in the proximal region of the PMT1 promoter, and that the combined application of ORC1 and G-box binding bHLH factors positively affects the induction of nicotine synthesis genes. Post-translational protein modification events potentiate the activity of ORC1, in particular MeJA-induced protein degradation and protein phosphorylation. The contribution of protein degradation can probably be attributed to the action of COI1 since both chemical inhibition of the 26S proteasome and co-transfection with the JAZ-repressor proteins antagonized ORC1 activity. The JAZ-repressor proteins are the targets of COI1 and bind bHLH TFs (Chini et al., 2007; Thines et al., 2007; Shoji et al., 2008; Fonseca et al., 2009; Ferna´ndez-Calvo et al., 2011). Hence, the JA-mediated release of the bHLH factors from the JAZ proteins and their interacting co-repressors such as NINJA

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1062 Kathleen De Boer et al. (Pauwels et al., 2010) enables them to intersect with the ERF factors and to activate the nicotine synthesis promoters. This finding agrees with the observation that JA-triggered, COI1-mediated degradation of JAZ repressors activates nicotine biosynthesis in tobacco roots (Shoji et al., 2008). Finally, we identified a component in the putative phosphorylation cascade that stimulates expression of a nicotine synthesis gene, namely a MAPKK protein, encoded by JAM1. Also in Arabidopsis, MAPK cascades are key relays of the respective JA signal transduction pathways and are involved in the control of MYC2 expression amongst others (Brader et al., 2007; Takahashi et al., 2007; Qiu et al., 2008). The complexity of the TF machines that activate nicotine biosynthesis in tobacco The phylogeny of the JA-responsive AP2/ERF and bHLH factors (Rushton et al., 2008), and the outcome of various functional screens in which multiple isoforms of both TF families were shown to positively regulate nicotine biosynthesis (De Sutter et al., 2005; Todd et al., 2010; Shoji et al., 2010), suggests that tobacco plants have developed a daunting repertoire of transcriptional regulators to control their secondary metabolism. Yet it is striking that for virtually none of the bHLH nor the NIC2 ERF activators identified so far, expression is exclusively confined to the roots, and that for many the corresponding transcripts are even encountered in all organs tested (Todd et al., 2010; Shoji et al., 2010; this study). This observation stands in sharp contrast to the almost exclusive root-specific pattern detected for many of the structural genes involved in nicotine biosynthesis. Similarly, MeJA induction of the expression of the structural nicotine synthesis genes is confined to the roots, whereas MeJA-mediated JAZ degradation and subsequent induction of bHLH and JAZ expression again occurs ubiquitously in the plant. Thus, as yet unrevealed functional specialization of the transcription factor machineries could account for different transactivation patterns, and, ultimately, for the exclusive confinement of the activity of key nicotine biosynthetic enzymes - and hence nicotine production itself-in tobacco root tissues. Functional specialization may rely on the formation of discrete protein complexes, involving specific protein–protein interactions with different TFs with distinct expression profiles or distinct affinity for particular promoter elements. In this context, an as yet unknown root-specific TF might confer root specificity to NIC2 ERF activity or a leafspecific TF might inhibit the function of NIC2 ERFs in the leaf. Alternatively, specific protein–protein interactions may not only influence the composition of particular transcription regulatory complexes but also control post-translational protein modifications. For instance, a MeJA signal cascade might stimulate the transcriptional activity of the clade 2-1 ERF ORC1 but not that of the clade 1 ERF JAP1 through differential post-translational modifications, e.g. MAPK-

dependent phosphorylation. Although effective phosphorylation changes in NIC2 ERF and MYC2-like bHLH factors still need to be corroborated, relationships between such TFs and kinases have already been reported in several other plant species. For example, the DNA-binding affinity of the tomato (S. lycopersicum) AP2/ERF protein Pti4 is increased after phosphorylation by the Ser/Thr receptor kinase Pto (Gu et al., 2000). A rice (Oryza sativa) MAPK phosphorylates the AP2/ERF factor OsEREBP1, and thereby increases its GCCmotif-binding activity (Cheong et al., 2003). ORCA proteins may also be activated via phosphorylation (van der Fits and Memelink, 2001). Similarly, the MeJA-modulated MAPK-dependent phosphorylation cascade identified here may directly impinge on the conformation and properties of ORC1, other NIC2 ERFs and/or bHLH factors as well. Identification of the MAPK, and possibly other kinases, that act downstream of JAM1 will help us to better understand the putative phosphorylation events implicated in ERF and/ or bHLH potentiation and the ensuing onset of nicotine biosynthesis. EXPERIMENTAL PROCEDURES Nicotiana tabacum plant growth and treatment Wild-type N. tabacum SR1 were germinated in soil. At day 27, seedlings were transferred to a 32-L hydroponic chamber, containing 32 g of 10-30-20 Blossom Booster fertilizer (Scotts, http:// www.scottsprofessional.com/). The pH was set at 6.5, controlled daily, and adjusted with 1 M KOH, if necessary. Plants were grown in a greenhouse under the following conditions: 25C/16 h light, 20C/ 8 h dark. For elicitation, 3.2 mg MeJA in 32 ml of 10% ethanol solution was added to the 32-L hydroponic medium of 46-day-old plants. Control plants were treated with a mock volume of 10% ethanol. Roots and leaves were harvested separately at time 0 and then at 0.5, 1, 2, 6 and 24 h after elicitation. Per sample, organs from four plants were pooled, snap-frozen in liquid nitrogen and stored at )70C.

Plant and hairy root transformation Nicotiana tabacum SC58 and N. glauca plants were grown in an insect-free greenhouse as described previously (Sinclair et al., 2004; Cane et al., 2005). For the creation of transgenic N. tabacum plants, leaf discs were infected with Agrobacterium tumefaciens LBA4404 containing relevant binary vectors and, after co-cultivation on Murashige and Skoog (MS) agar (PhytoTechnology, http://www. phytotechlab.com/) containing 30 g sucrose L)1, 1 mg L)1 indole-3acetic acid and 0.5 mg L)1 6-benzylaminopurine for 3 days at 25C, transferred to MS agar with the same hormonal composition supplemented with 500 mg L)1 cefotaxim and 75 mg L)1 kanamycin sulphate (PhytoTechnology) for transgene selection. Transgenic regenerants were rooted in MS medium containing 25 mg L)1 kanamycin sulphate before being transferred to an insect-free greenhouse and selfed to produce T1 seeds. Transformed N. glauca root cultures were created with the methodology outlined previously (Chintapakorn and Hamill, 2003; Cane et al., 2005). Clonal root cultures were maintained in the dark at 22C, shaking at 100 r.p.m. in vessels containing 25 ml Gamborg B5 medium (PhytoTechnology) supplemented with 3% sucrose (pH 6), 25 mg L)1 kanamycin and 500 mg L)1 ampicillin, and subcultured every 3 weeks.

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ERFs and bHLHs control nicotine biosynthesis 1063 Alkaloid measurements Transgenic N. tabacum seedlings were grown for 8 weeks in vitro at 21C/16 h light on MS medium supplemented with 3% sucrose and 75 mg L)1 kanamycin sulphate, and solidified with 8 g L)1 agar. For each line the leaf and root tissue of approximately 50 seedlings were combined for analysis. Transgenic N. glauca root cultures were harvested for analysis 12 days after subculturing. Alkaloid concentrations within transformed N. tabacum plants and N. glauca root cultures were determined with HPLC analysis based on the protocols outlined in Cane et al. (2005).

Expression analysis One hundred milligrams of plant material (tobacco SR1 leaves and roots) was used for RNA extraction according to the Concert Plant RNA Reagent protocol (Invitrogen, http://www.invitrogen.com/). Single-stranded cDNA was prepared from this total RNA with SuperscriptII RT-polymerase (Invitrogen). Expression analysis in tobacco SR1 was verified by q-RT-PCR, run on a LightCycler 480 instrument with the LightCycler 480 SYBR Green I Master kit (Roche Applied Science, http://www.roche.com/). DCT relative quantification with gene normalization was carried out with the qBASE program (Hellemans et al., 2007). Primers for N. tabacum (see Table S1) were constructed based on sequence data from JAP1 (CQ808845), ORC1 (CQ808982), PMT (AF126812) and QPRT (AB038494). b-ATPase (U96496) was used as the reference gene (Reed and Jelesko, 2004). Transgene overexpression in transformed tobacco plants and tree tobacco root lines was verified by northern blot analysis as described (Chintapakorn and Hamill, 2003). DNA fragments used for molecular probes for TFs were recovered by RT-PCR on tobacco root RNA (see Table S1 for the primers). Molecular probe generation for Nicotiana rustica QPRT (accession number AJ243436) and Antirrhinum majus ubiquitin is described in Chintapakorn and Hamill (2003).

Generation of promoter and ORF constructs All constructs used were generated with Gateway technology (Invitrogen). Full-length ORFs of ORC1 (EMBL accession CQ808982), JAP1 (EMBL accession CQ808845), JAZ1 (EMBL accession AB433896) and JAM1 (EMBL accession CQ808961) were amplified between Gateway attB1 and attB2 sequences with the Platinum Pfx DNA polymerase (Invitrogen), and subsequently inserted into the Gateway vectors p2WG7 or pK7WG2D for ProCaMV35S driven overexpression (Karimi et al., 2002). For construction of the reporter constructs, the upstream sequences of N. sylvestris PMT2 (ProNsPMT2, 1742 bp; EMBL accession AB004323), N. tabacum ADC1 (ProADC1, 646 bp; EMBL accession AF127240), N. tabacum ADC2 (ProADC2, 704 bp; EMBL accession AF127241), N. tabacum ODC1 (ProODC1, 728 bp; EMBL accession AB031066), N. tabacum QPRT1 (ProQPRT1, 2128 bp; EMBL accession AJ748262) and N. tabacum QPRT2 (ProQPRT2, 2313 bp; EMBL accession AJ748263) were PCR amplified between the Gateway attB4 and attB3 recombination sites. Following Gateway BP and LR recombinations (Invitrogen), all promoter sequences were inserted into the Gateway destination vector pm43GWfl7 (De Sutter et al., 2005). The minimal PMT1 upstream sequences (ProPMT1min, EMBL accession AF126810), from)196 to)1 (with respect to the translation initiation site), were synthesized and inserted into the Gateway vector pGWL7 in front of the luciferase gene. Mutagenized versions of ProPMT1min were synthesized, harboured 6-bp deletions at the G-box (DCACGTT) or the GCC motif (DTGCGCC), and were also inserted into the Gateway vector pGWL7. Minimal CaMV35S promoter sequences, from )46 to )1 (with regard to the translation

initiation site), followed by the X enhancer sequences of tobacco mosaic virus, were PCR amplified from the Gateway vector pK7WG2D (Karimi et al., 2002), and flanked by a 5¢ PstI restriction site and a 3¢ Gateway attB2 recombination sequence. The PCR product was inserted into the SmaI-site of pBluescript KS(+). Subsequently, single-stranded oligonucleotides, containing four tandem copies of the ProPMT1 G-box (TGCACGTTGT) or GCC motif (TTCCGCCCT) sequences, 5¢ and 3¢ end flanked with BamH1 and PstI restriction sites, respectively, were developed. Additionally, the Gateway attB1 recombination sequence was linked upstream of the BamH1 site to facilitate further cloning. The sequences of the primers designed were respectively 5¢-CGA AGG ATC CAA AAA GCA GGC T [TGC ACG TTG TAA TA]4 CTG CAG AAT C-3¢ (G-box sense) and 5¢-CGA AGG ATC CAA AAA GCA GGC T [TTC CGC CCT AAT A]4 CTG CAG AAT C-3¢ (GCC motif sense). Complementary 5¢ and 3¢ end 17-mer oligonucleotides were annealed to the above single-stranded nucleotides and the partial double-stranded sequence was inserted upstream of the minimal CaMV35S promoter into the BamH1 and PstI sites of the pBluescript KS(+) vector. The resulting plasmid was transformed into DH5a Escherichia coli cells, allowing the natural repair system to create a complete double-stranded sequence. G-box and GCC motif synthetic promoter sequences were PCR amplified with Gateway attB sequences, after which they were inserted in front of the luciferase gene into the Gateway destination vector pGWL7. The construct comprising both the G-box and GCC motif tetramers in front of the luciferase gene was generated with a PCR-fusion/Gateway cloning procedure (Atanassov et al., 2009) using the above plasmids as the starting template for PCR.

Protoplast transfection Protoplast preparation and transient expression experiments were carried out as described previously (De Sutter et al., 2005). For each construct, 2 lg plasmid DNA was added per transfection. MeJA (50 lM) or an equal volume of dimethyl sulphoxide (DMSO), the solvent of MeJA, was added immediately after transfection. The final DMSO concentration was 0.05%. In the case of inhibitor studies, MG132 (Z-leu-leu-leu-H(aldehyde), 50 lM; Peptide Institute Inc., http://www.peptide.co.jp/en/) or PD98059 [2-(2¢-amino-3¢-methoxyphenyl)-oxanaphtalen-4-one, 20 lM; Calbiochem, http://www.merckchemicals.be/) were added 1 h before MeJA treatment. Reporter gene expression was assessed 24 h after transfection. In all cases, protoplasts transfected with a p2GW7-GUS construct (De Sutter et al., 2005) were included as the control sample. All transactivation assays were conducted in an automated experimental set-up that involved eight separate transfection experiments and were performed at least twice.

Yeast-two-hybrid analysis The Y2H analysis was performed as described (Pauwels et al., 2010) with modifications. Gateway-compatible versions of pGADT7 and pGBKT7 (Clontech, http://www.clontech.com/) were used as destination vectors, co-transformed in the yeast strain PJ69-4A and selected on synthetic defined (SD)–Leu–Trp agar medium. Three individual transformants were randomly selected, grown for 1 day in selective liquid medium, dropped in 10 · and 100 · dilutions on SD– Leu–Trp and SD–Leu–Trp–His and allowed to grow for 2 days at 30C.

ACKNOWLEDGMENTS The authors thank Freya Lammertyn and Amparo Cue´llar Pe´rez for excellent technical assistance, and Martine De Cock for help in preparing the manuscript. This work was supported by the European FP7 project SMARTCELL (no. 222716), the Australian

ª 2011 The Authors The Plant Journal ª 2011 Blackwell Publishing Ltd, The Plant Journal, (2011), 66, 1053–1065

1064 Kathleen De Boer et al. Postgraduate Award from the Australian Research Council and the ARC research grant A19701779 (to KDB) and the Institute for the Promotion of Innovation by Science and Technology in Flanders with pre-doctoral fellowships (to ST and VDS). LP is a post-doctoral fellow of the Research Foundation Flanders (FWO).

SUPPORTING INFORMATION Additional Supporting Information may be found in the online version of this article: Figure S1. Transgene expression in transformed Nicotiana lines. Table S1. Oligonucleotide primers used for PCR. Please note: As a service to our authors and readers, this journal provides supporting information supplied by the authors. Such materials are peer-reviewed and may be re-organized for online delivery, but are not copy-edited or typeset. Technical support issues arising from supporting information (other than missing files) should be addressed to the authors.

REFERENCES Aharoni, A., Dixit, S., Jetter, R., Thoenes, E., Van Arkel, G. and Pereira, A. (2004) The SHINE clade of AP2 domain transcription factors activates wax biosynthesis, alters cuticle properties, and confers drought tolerance when overexpressed in Arabidopsis. Plant Cell, 16, 2463–2480. Atanassov, I.I., Atanassov, I.I., Etchells, J.P. and Turner, S.R. (2009) A simple, flexible and efficient PCR-fusion/gateway cloning procedure for gene fusion, site-directed mutagenesis, short sequence insertion and domain deletions and swaps. Plant Methods, 5, 14. Baldwin, I.T., Schmelz, E.A. and Zhang, Z.-P. (1996) Effects of octadecanoid metabolites and inhibitors on induced nicotine accumulation in Nicotiana sylvestris. J. Chem. Ecol. 22, 61–74. Brader, G., Djamei, A., Teige, M., Palva, E.T. and Hirt, H. (2007) The MAP kinase kinase MKK2 affects disease resistance in Arabidopsis. Mol. Plant Microbe Interact. 20, 589–596. Broun, P., Poindexter, P., Osborne, E., Jiang, C.-Z. and Riechmann, J.L. (2004) WIN1, a transcriptional activator of epidermal wax accumulation in Arabidopsis. Proc. Natl Acad. Sci. USA, 101, 4706–4711. Cane, K.A., Mayer, M., Lidgett, A.J., Michael, A.J. and Hamill, J.D. (2005) Molecular analysis of alkaloid metabolism in AABB v. aabb genotype Nicotiana tabacum in response to wounding of aerial tissues and methyl jasmonate treatment of cultured roots. Funct. Plant Biol. 32, 305–320. Cheong, Y.H., Moon, B.C., Kim, J.K. et al. (2003) BWMK1, a rice mitogenactivated protein kinase, locates in the nucleus and mediates pathogenesisrelated gene expression by activation of a transcription factor. Plant Physiol. 132, 1961–1972. Chini, A., Fonseca, S., Ferna´ndez, G. et al. (2007) The JAZ family of repressors is the missing link in jasmonate signalling. Nature, 448, 666–671. Chintapakorn, Y. and Hamill, J.D. (2003) Antisense-mediated down-regulation of putrescine N-methyltransferase activity in transgenic Nicotiana tabacum L. can lead to elevated levels of anatabine at the expense of nicotine. Plant Mol. Biol. 53, 87–105. De Sutter, V., Vanderhaeghen, R., Tilleman, S., Lammertyn, F., Vanhoutte, I., Karimi, M., Inze´, D., Goossens, A. and Hilson, P. (2005) Exploration of jasmonate signalling via automated and standardized transient expression assays in tobacco cells. Plant J. 44, 1065–1076. Ferna´ndez-Calvo, P., Chini, A., Ferna´ndez-Barbero, G. et al. (2011) The bHLH transcription factors MYC3 and MYC4 are targets of JAZ repressors and act additively with MYC2 in the activation of JA responses. Plant Cell, 23, 701– 715. van der Fits, L. and Memelink, J. (2000) ORCA3, a jasmonate-responsive transcriptional regulator of plant primary and secondary metabolism. Science, 289, 295–297. van der Fits, L. and 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. Fonseca, S., Chico, J.M. and Solano, R. (2009) The jasmonate pathway: the ligand, the receptor and the core signalling module. Curr. Opin. Plant Biol. 12, 539–547.

Goossens, A. and Rischer, H. (2007) Implementation of functional genomics for gene discovery in alkaloid producing plants. Phytochem. Rev. 6, 35–49. Goossens, A., Ha¨kkinen, S.T., Laakso, I. et al. (2003) A functional genomics approach toward the understanding of secondary metabolism in plant cells. Proc. Natl Acad. Sci. USA, 100, 8595–8600. Gu, Y.-Q., Yang, C., Thara, V.K., Zhou, J. and Martin, G.B. (2000) Pti4 is induced by ethylene and salicylic acid, and its product is phosphorylated by the Pto kinase. Plant Cell, 12, 771–785. Ha¨kkinen, S.T., Tilleman, S., Sˇwia˛tek, A. et al. (2007) Functional characterisation of genes involved in pyridine alkaloid biosynthesis in tobacco. Phytochemistry, 68, 2773–2785. Halitschke, R. and Baldwin, I.T. (2003) Antisense LOX expression increases herbivore performance by decreasing defense responses and inhibiting growth-related transcriptional reorganization in Nicotiana attenuata. Plant J. 36, 794–807. Hellemans, J., Mortier, G., De Paepe, A., Speleman, F. and Vandesompele, J. (2007) qBase relative quantification framework and software for management and automated analysis of real-time quantitative PCR data. Genome Biol. 8, R19.1–R19.14. Imanishi, S., Hashizume, K., Nakakita, M., Kojima, H., Matsubayashi, Y., Hashimoto, T., Sakagami, Y., Yamada, Y. and Nakamura, K. (1998) Differential induction by methyl jasmonate of genes encoding ornithine decarboxylase and other enzymes involved in nicotine biosynthesis in tobacco cell cultures. Plant Mol. Biol. 38, 1101–1111. Kannangara, R., Branigan, C., Liu, Y., Penfield, T., Rao, V., Mouille, G., Ho¨fte, H., Pauly, M., Riechmann, J.L. and Broun, P. (2007) The transcription factor WIN1/SHN1 regulates cutin biosynthesis in Arabidopsis thaliana. Plant Cell, 19, 1278–1294. Karimi, M., Inze´, D. and Depicker, A. (2002) GATEWAY vectors for Agrobacterium-mediated plant transformation. Trends Plant Sci. 7, 193– 195. Kidd, S.K., Melillo, A.A., Lu, R.-H., Reed, D.G., Kuno, N., Uchida, K., Furuya, M. and Jelesko, J.G. (2006) The A and B loci in tobacco regulate a network of stress response genes, few of which are associated with nicotine biosynthesis. Plant Mol. Biol. 60, 699–716. Lorenzo, O., Chico, J.M., Sa´nchez-Serrano, J.J. and Solano, R. (2004) JASMONATE-INSENSITIVE1 encodes a MYC transcription factor essential to discriminate between different jasmonate-regulated defense responses in Arabidopsis. Plant Cell, 16, 1938–1950. Lou, Y. and Baldwin, I.T. (2006) Silencing of a germin-like gene in Nicotiana attenuata improves performance of native herbivores. Plant Physiol. 140, 1126–1136. Memelink, J., Verpoorte, R. and Kijne, J.W. (2001) ORCAnization of jasmonate-responsive gene expression in alkaloid metabolism. Trends Plant Sci. 6, 212–219. Menke, F.L., Champion, A., Kijne, J.W. and Memelink, J. (1999) A novel jasmonate- and elicitor-responsive element in the periwinkle secondary metabolite biosynthetic gene Str interacts with a jasmonate- and elicitorinducible AP2-domain transcription factor, ORCA2. EMBO J. 18, 4455– 4463. Morita, M., Shitan, N., Sawada, K., Van Montagu, M.C.E., Inze´, D., Rischer, H., Goossens, A., Oksman-Caldentey, K.-M., Moriyama, Y. and Yazaki, K. (2009) Vacuolar transport of nicotine is mediated by a multidrug and toxic compound extrusion (MATE) transporter in Nicotiana tabacum. Proc. Natl Acad. Sci. USA, 106, 2447–2452. Ohnmeiss, T.E., McCloud, E.S., Lynds, G.Y. and Baldwin, I.T. (1997) Withinplant relationships among wounding, jasmonic acid, and nicotine: implications for defence in Nicotiana sylvestris. New Phytol. 137, 441–452. Pandey, S.P. and Baldwin, I.T. (2007) RNA-directed RNA polymerase 1 (RdR1) mediates the resistance of Nicotiana attenuata to herbivore attack in nature. Plant J. 50, 40–53. Paschold, A., Halitschke, R. and Baldwin, I.T. (2007) Co(i)-ordinating defenses: NaCOI1 mediates herbivore-induced resistance in Nicotiana attenuata and reveals the role of herbivore movement in avoiding defenses. Plant J. 51, 79–91. Pauwels, L., Morreel, K., De Witte, E., Lammertyn, F., Van Montagu, M., Boerjan, W., Inze´, D. and Goossens, A. (2008) Mapping methyl jasmonatemediated transcriptional reprogramming of metabolism and cell cycle progression in cultured Arabidopsis cells. Proc. Natl Acad. Sci. USA, 105, 1380–1385.

ª 2011 The Authors The Plant Journal ª 2011 Blackwell Publishing Ltd, The Plant Journal, (2011), 66, 1053–1065

ERFs and bHLHs control nicotine biosynthesis 1065 Pauwels, L., Inze´, D. and Goossens, A. (2009) Jasmonate-inducible gene: what does it mean? Trends Plant Sci. 14, 87–91. Pauwels, L., Ferna´ndez Barbero, G., Geerinck, J. et al. (2010) NINJA connects the co-repressor TOPLESS to jasmonate signalling. Nature, 464, 788–791. Qiu, J.-L., Zhou, L., Yun, B.-W., Nielsen, H.B., Fiil, B.K., Petersen, K., MacKinlay, J., Loake, G.J., Mundy, J. and Morris, P.C. (2008) Arabidopsis mitogen-activated protein kinase kinases MKK1 and MKK2 have overlapping functions in defense signaling mediated by MEKK1, MPK4, and MKS1. Plant Physiol. 148, 212–222. Rayapuram, C. and Baldwin, I.T. (2007) Increased SA in NPR1-silenced plants antagonizes JA and JA-dependent direct and indirect defenses in herbivore-attacked Nicotiana attenuata in nature. Plant J. 52, 700–715. Reed, D.G. and Jelesko, J.G. (2004) The A and B loci of Nicotiana tabacum have non-equivalent effects on the mRNA levels of four alkaloid biosynthetic genes. Plant Sci. 167, 1123–1130. Rushton, P.J., Bokowiec, M.T., Han, S., Zhang, H., Brannock, J.F., Chen, X., Laudeman, T.W. and Timko, M.P. (2008) Tobacco transcription factors: novel insights into transcriptional regulation in the Solanaceae. Plant Physiol. 147, 280–295. Shoji, T., Yamada, Y. and Hashimoto, T. (2000) Jasmonate induction of putrescine N-methyltransferase genes in the root of Nicotiana sylvestris. Plant Cell Physiol. 41, 831–839. Shoji, T., Ogawa, T. and Hashimoto, T. (2008) Jasmonate-induced nicotine formation in tobacco is mediated by tobacco COI1 and JAZ genes. Plant Cell Physiol. 49, 1003–1012. Shoji, T., Kajikawa, M. and Hashimoto, T. (2010) Clustered transcription factor genes regulate nicotine biosynthesis in tobacco. Plant Cell, 22, 3390–3409. Sinclair, S.J., Murphy, K.J., Birch, C.D. and Hamill, J.D. (2000) Molecular characterization of quinolinate phosphoribosyltransferase (QPRTase) in Nicotiana. Plant Mol. Biol. 44, 603–617.

Sinclair, S.J., Johnson, R. and Hamill, J.D. (2004) Analysis of wound-induced gene expression in Nicotiana species with contrasting alkaloid profiles. Funct. Plant Biol. 31, 721–729. Takahashi, F., Yoshida, R., Ichimura, K., Mizoguchi, T., Seo, S., Yonezawa, M., Maruyama, K., Yamaguchi-Shinozaki, K. and Shinozaki, K. (2007) The mitogen-activated protein kinase cascade MKK3-MPK6 is an important part of the jasmonate signal transduction pathway in Arabidopsis. Plant Cell, 19, 805–818. Thines, B., Katsir, L., Melotto, M., Niu, Y., Mandaokar, A., Liu, G., Nomura, K., He, S.Y., Howe, G.A. and Browse, J. (2007) JAZ repressor proteins are targets of the SCFCOI1 complex during jasmonate signalling. Nature, 448, 661–665. Todd, A.T., Liu, E., Polvi, S.L., Pammett, R.T. and Page, J.E. (2010) A functional genomics screen identifies diverse transcription factors that regulate alkaloid biosynthesis in Nicotiana benthamiana. Plant J. 62, 589–600. Xu, B. and Timko, M.P. (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. Xu, B., Sheehan, M.J. and Timko, M.P. (2004) Differential induction of ornithine decarboxylase (ODC) gene family members in transgenic tobacco (Nicotiana tabacum L. cv. Bright yellow 2) cell suspensions by methyljasmonate treatment. Plant Growth Regul. 44, 101–116. Zhang, Z., Zhang, H., Quan, R., Wang, X.-C. and Huang, R. (2009) Transcriptional regulation of the ethylene response factor LeERF2 in the expression of ethylene biosynthesis genes controls ethylene production in tomato and tobacco. Plant Physiol. 150, 365–377. Zhao, J., Davis, L.C. and Verpoorte, R. (2005) Elicitor signal transduction leading to production of plant secondary metabolites. Biotechnol. Adv. 23, 283–333.

ª 2011 The Authors The Plant Journal ª 2011 Blackwell Publishing Ltd, The Plant Journal, (2011), 66, 1053–1065