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Transcription factors regulating terpene synthases in tomato trichomes
Spyropoulou, E.
Publication date 2012 Document Version Final published version
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Download date:03 Oct 2021 Transcription factors regulating terpene synthases in tomato trichomes
Transcription factors regulating terpene synthases in tomato trichomes Eleni Spyropoulou
Eleni Spyropoulou
Transcription factors regulating terpene synthases in tomato trichomes
ACADEMISCH PROEFSCHRIFT
ter verkrijging van de graad van doctor aan de Universiteit van Amsterdam op gezag van de Rector Magnificus prof. dr. D.C. van den Boom ten overstaan van een door het college voor promoties ingestelde commissie, in het openbaar te verdedigen in de Agnietenkapel op dinsdag 03 juli 2012, te 16:00 uur
door
Eleni Spyropoulou
geboren te Marousi, Griekenland
PROMOTIECOMISSIE
Promotor Prof. Dr. M.A. Haring Co-promotor Dr. R.C. Schuurink
Overige leden Prof. Dr. H.J. Bouwmeester Dr. M. Kant Dr. M. Prins Prof. Dr. A.K. Smilde Prof. Dr. A. Tissier
Faculteit der Natuurwetenschappen, Wiskunde en Informatica
Table of Contents
Page Chapter 1 5 General introduction
Chapter 2 37 Discovery and characterization of mono- and sesquiterpene synthases from Solanum lycopersicum
Chapter 3 69 A yeast-one-hybrid screen identifies a putative transcription factor that interacts with a tomato terpene synthase promoter
Chapter 4 96 Characterization of EMISSION OF TERPENES, a trichome-specific transcription factor from Solanum lycopersicum involved in terpene biosynthesis
Chapter 5 126 RNA sequencing of Solanum lycopersicum trichomes identifies transcription factors that interact with terpene synthase promoters
Chapter 6 166 General discussion
Summary 180 Samenvatting 181
ἓν οἶδα ὅτι οὐδὲν οἶδα Σωκράτης
Chapter 1
General Introduction
1. Plant secondary metabolites
2. Isoprenoid biosynthesis
3. Roles of isoprenoids
4. Specialized metabolite production and storage sites
5. Examples of specialized metabolites involved in the plant defense system
6. Regulation of isoprenoid biosynthesis
6.1 Regulation of plant defenses at the gene expression level 6.1.1 Trans-acting factors and cis-elements 6.1.2 Transcription factor families 6.2 Role of hormones in plant defenses 6.2.1 Jasmonic acid biosynthesis and regulation of downstream genes 6.3 Additional modes of regulation 6.4 Examples of transcription factors regulating terpenoid biosynthesis
7. Thesis outline
8. Account
1. Plant secondary metabolites
Plants produce a plethora of secondary metabolites- originally defined as such because their function was unclear, but they were known to be derived from the building blocks of primary metabolism like amino acids, lipids and sugars (Pichersky and Lewinsohn, 2011). However, given the accumulating evidence that secondary metabolites play important roles for example in the environmental adaptation of plants to both biotic and abiotic stress or in pollination (see sections 3 and 5), they have recently been termed “specialized” metabolites (Pichersky et al., 2006). Specialized metabolites can be divided into three main groups: phenolic compounds (phenylpropanoids, flavonoids), terpenoids/ isoprenoids and nitrogen- containing compounds (glucosinolates, alkaloids, cyanogenic glycosides) (Aharoni and Galili, 2011). A simplified scheme of the production pathways for some of these specialized metabolites is shown in Figure 1. Although different plant families can produce to some extend the same specialized metabolites, particular chemical classes are typically associated with a specific plant family- for example plants of the Solanaceae family are known to produce terpenoids, those of the Brassicaceae family accumulate glucosinolates, whereas Leguminous species produce typically flavonoids (Wu and Chappell, 2008).
Acetyl-CoA Pyr + GA-3P Aliphatic Aromatic PEP + E4P Amino Acids Amino Acids
MVA pathway MEP pathway Shikimate Pathway Alkaloids Glucosinolates Isoprenoids Complex Phenylpropanoids Alkaloids Complex Isoprenoids Flavonoids/ Complex Poliketids Flavonoids Figure 1. Simplified scheme of specialized metabolite production pathways. Precursor compounds are in italics, major classes of specialized metabolites are in bold. Pyr; pyruvate, GA-3P; glyceraldehyde 3-phosphate, MVA; mevalonate, MEP; methylerythritol, PEP; phosphoenolpyruvate, E4P; erythrose 4-phosphate. Modified from Aharoni and Galili, 2011.
Chapter 1 6 2. Isoprenoid biosynthesis
Plant isoprenoids (or terpenoids) constitute a large, structurally diverse class of chemical compounds, but the immediate precursors for all isoprenoid compounds are the simple C5-units isopentenyl diphosphate (IPP) and dimethallyl diphosphate (DMAPP; Fig.2). IPP and DMAPP can be synthesized through the methylerythritol (MEP) pathway in the plastids. Alternatively IPP can be synthesized and then converted to DMAPP through the mevalonate (MVA) pathway in the cytosol (Bouvier et al., 2005). However, exchange of isoprenoid precursors between plastids and the cytosol has been reported (e.g. Dudareva et al., 2005). For a detailed description of the enzymatic steps involved in the MEP and MVA pathways, see Figure 2 in Chapter 5. Condensation of these C5-units by prenyl diphosphate synthases leads to the formation of geranyl diphosphate (GPP) (or neryl diphosphate (NPP), the cis-isomer of GPP), farnesyl diphosphate (FPP) (or its cis-isomere (Z,Z)-FPP) and geranylgeranyl diphosphate (GGPP). Prenyl diphosphates are in turn converted to a variety of C10 monoterpenes, C15 sesquiterpenes and C20 diterpenes by enzymes of the terpene synthase (TPS) family (Wu et al., 2006). Terpenes may be modified by hydroxylation, dehydration, decarboxylation or ring formation. Such modifications may involve the activity of cytochrome P450s that catalyze a wide variety of chemical reaction steps in plant secondary metabolism, ranging from hydroxylation to ring formation and carbon- carbon bond cleavage (Morant et al., 2003). In addition, terpenes can also be decorated with other molecules thus forming the more complex isoprenoids. Modifications and decorations of the parental mono-, sesqui- and di-terpenes ultimately result in the thousands of different terpenoid structures found in nature (Aharoni et al., 2006). Finally, it is worth mentioning that isoprenoid biosynthesis also takes place in the mitochondria where for example the ubiquinones are produced (Aharoni et al., 2005).
Chapter 1 7 Acetyl-CoA Pyr + GA-3P
MVA MEP
IPPDMAPP IPP DMAPP
Sesquiterpenes FPP/ (Z,Z)FPPNPP/ GPP Monoterpenes
GGPP Diterpenes Brassinosteroids Dolichols Triterpenes Sterols Carotenoids Chlorophylls Gibberillins Abscisic Acid Figure 2. Simplified scheme of isoprenoid production pathways. Dashed lines indicate more than one enzymatic step. IPP; isopentenyl diphosphate, DMAPP; dimethallyl diphosphate, GPP; geranyl diphosphate, NPP; neryl diphosphate, FPP; farnesyl diphosphate, GGPP; geranylgeranyl diphosphate.
3. Roles of isoprenoids
Isoprenoids function in primary and specialized metabolism. In primary metabolism they serve various roles such as components of membranes (phytosterol), photosynthetic pigments (carotenoids and phytol side chain of chlorophyll) or hormones (gibberellins, brassinosteroids, abscisic acid), but the majority of plant isoprenoids function as specialized metabolites (Lange et al., 2000; Flores-Perez et al., 2010). These specialized metabolites are non-volatile or volatile chemicals that can be sequestered in the plant or emitted in the environment serving various roles (Langenheim, 1994). Non-volatile compounds can function as part of the plant’s direct defenses either by inhibiting or suppressing growth of pathogens- for example phytoalexins are well known for their antimicrobial activity (Ahuja et al., 2012), or by exhibiting toxic or deterrent effects against herbivores- for instance hydroxygeranyllinalool diterpene glycosides that are known to mediate resistance of Nicotiana attenuata plants
Chapter 1 8 against Manduca sexta (Heiling et al., 2010) or cucurbitacin C that is involved in spider mite resistance of cucumber (Balkema-Boomstra et al., 2003). Some volatile isoprenoids function as attractants of pollinators, seed dispersers and other beneficial animals, like the monoterpene linalool that is emitted from Clarkia breweri flowers (Pichersky et al., 1995). Many of the volatile isoprenoids however are emitted by herbivore-infested plants and serve as attractants of natural enemies (predators and parasitoids), thus providing indirect defense against herbivores. In this case these compounds can be released when feeding ruptures the structures in which they are stored (see section 4) or can be synthesized de novo at different time points after the onset of herbivore feeding (Pichersky and Gershenzon, 2002). Most of the volatile isoprenoids belong to the mono- and sesqui-terpene classes but also the C16-norterpene (E,E)-4,8,12-trimethyltrideca-1,3,7,11-tetraene (TMTT) and its C11-analog (E)-4,8-dimethyl-1,3,7-nonatriene (DMNT) are known to play a role in the plant’s indirect defenses (Ament et al., 2004; Lee et al., 2010). Induced terpene synthesis after wounding or herbivore attack has been reported from numerous plant species including tomato (van Schie et al., 2007), maize (Schnee et al., 2002; Schnee et al., 2006), poplar (Arimura et al., 2004a), lotus (Arimura et al., 2004b), cucumber (Mercke et al., 2004) and medicago (Gomez et al., 2005; Navia- Gine et al., 2009) and usually transcriptional activation of these terpene synthase genes correlates well with the emission of the terpene product (see section 5). Finally, volatile terpenes can play a role in plant-plant communication: when released from damaged plants they can induce defense responses in neighboring plants (Arimura et al., 2000).
4. Specialized metabolite production and storage sites
Plants have evolved dedicated structures for the production and storage of (volatile) specialized metabolites. Plant volatiles are usually lipophilic substances
Chapter 1 9 with high vapor pressures and can be released from flowers, fruits, and vegetative tissue into the atmosphere, but also from the roots into the rhizosphere. In the flower petals, biosynthesis of plant volatiles takes place in specialized or nonspecialized epidermal and mesophyll cells, and their emission is in the vast majority tightly correlated with attraction of pollinators (Pichersky et al., 2006; Van Moerkercke et al., 2012). Also, roots contain secretory cells that can release volatiles, which play a role in the direct defense against microbial pathogens as well as in indirect defense, e.g., via the attraction of entomopathogenic nematodes (Rasmann et al., 2005; Wenke et al., 2010). Other common anatomical structures where plant specialized metabolites are stored include secretory cavities present in the skin of many fruits and special ducts, such as those found on evergreens, in which resins are stored in a mixture with volatile chemicals to keep the resin fluid but which can evaporate during exposure to air upon mechanical damage such that the resin hardens and seals the wound (Maffei, 2010). However, especially well studied are the glandular trichomes, which can be found on vegetative tissues of many plant species and which are the source of various specialized metabolites (Tissier, 2012). Glandular trichomes are classified in different types according to their shape and structure. They can be divided into peltate and capitate trichomes like those found in the Lamiaceae and Asteraceae families: the peltate trichomes consist of one basal cell, one stalk cell, and many secretory cells (typically 4–18) while the capitate trichomes comprise a basal cell, a single or multicellular stalk, and a head consisting of one or two cells (Werker, 2000; Maffei, 2010). Tomato trichomes in particular can be categorized as one of seven types (Luckwill, 1943): for example the glandular trichomes (type VI) of the cultivated tomato (Solanum lycopersicum) consist of a stalk and a four-celled “head” (Fig.3). These four cells are small and have a large wall-less subcellular cavity on top in which specialized metabolites are stored (Simmons and Gurr, 2005). Plants can possess non-glandular trichomes as well (like the type III trichomes of cultivated tomato). These non-glandular trichomes in combination with the sticky exudates of the glandular trichomes constitute a mechanical barrier
Chapter 1 10 to the movement and feeding of herbivores and thus play a role in the direct defense of the plant (Channarayappa et al., 1992; Simmons and Gurr, 2005). Primary metabolism in glandular trichomes is highly active to generate precursors for the specialized metabolic pathways on-site. Trichome cells of various plants, like for example Solanum species, contain chloroplasts, as well as a nucleus (Peterson and Vermeer, 1984; Pyke and Howells, 2002). Trichomes operate as a closed system with minimal import from the rest of the plant, usually being sucrose (Schilmiller et al., 2008). Cutin is often deposited in the wall of the lowest stalk cell of glandular trichomes in order to prevent the synthesized products to flow back into the plant (Fahn, 1988). Hence, trichome constituents, which can be autotoxic, are stored safely away from the other plant tissues in the subcuticular space of the gland cells. Finally, volatiles can be released when the head is ruptured by herbivore movement or be transported, actively or passively, out of the trichome into the air upon upregulation of their biosynthesis during indirect defenses (Gershenzon et al., 1992; Pichersky et al., 2006).
Figure 3. Cultivated tomato trichomes. (A) Glandular (type VI) and non-glandular (type III) trichomes found on a tomato stem (photograph by Jan van Arkel; IBED, UvA), (B) close-up of glandular trichomes consisting of a stalk and a four-celled “head” (from van Houten et al., 2012) and (C) 3D-reconstruction of multiple confocal images of a type VI trichome “head” to visualize autofluorescence (merge of GFP (emission 505-545nm) and RFP (emission 600-700nm) channels; images were obtained with a Zeiss LSM 510 confocal laser scanning microscope. Photograph by Erik Manders; SILS, UvA).
Chapter 1 11 5. Examples of specialized metabolites involved in the plant defense system
Although the main focus of this thesis is volatile terpenes, comparisons will be made with other specialized metabolites and their biosynthetic pathways. Therefore examples are given here, not only for terpenes involved in plant defenses, but also for other classes of specialized metabolites.
The phenylpropanoid eugenol is synthesized from phenylalanine (derived from the shikimate pathway) that is converted in several enzymatic steps to coniferyl alcohol, an intermediate in the synthesis of lignins, and subsequently to coniferyl acetate that serves as substrate for the (iso-)eugenol synthase (I/EGS). IGS and EGS have been cloned from basil glands and petunia and Clarkia breweri flowers (Koeduka et al., 2006; Koeduka et al., 2008). Apart from the role of these compounds as floral attractants of pollinators, they also seem to serve in defense against herbivores as they have been shown to have larvicidal activity against Spodoptera litura (Bhardwaj et al., 2010) and insecticidal activity against Tribolium castaneum and Sitophilus zeamais (Huang et al., 2002).
The fatty acid derivatives C11, C13, C15 methylketones are synthesized in high abundance in glandular trichomes of the wild tomato specie Solanum habrochaites glabratum, consisting mostly of 2-tridecanone and 2-undecanone (Antonious, 2001). They are produced in the plastids through the action of methylketone synthase 2 (ShMKS2) that hydrolyzes intermediates of fatty acid biosynthesis 3- ketoacyl-acyl carrier proteins, thus releasing 3-ketoacids. Subsequent decarboxylation by ShMKS1 of those 3-ketoacids leads to the formation of the methylketone products (Fridman et al., 2005; Ben-Israel et al., 2009; Yu et al., 2010). Direct toxicity of methylketones has been observed against spider mites (Chatzivasileiadis and Sabelis, 1997). There are several terpene compounds from various plant species that have been shown to play a role in the defense system either individually or as part of a volatile blend released by the plants and a few examples are presented here.
Chapter 1 12 However it must be noted that not for all terpene products, the respective terpene synthase has been identified. Linalool, synthesized in tomato glandular trichomes through the activity of the monoterpene synthase 1 (SlMTS1), has been shown to be emitted after wounding, in correlation with the increased transcript abundance after spider mite feeding or artificial wounding (van Schie et al., 2007). Furthermore the tomato monoterpenes p-cymene, γ-terpinene, β-phellandrene and α-myrcene, as well as the sesquiterpene zingiberene and its conversion product curcumene were shown to exert a repellent effect on whiteflies (Bleeker et al., 2009). Except for p-cymene, terpene synthases that produce these monoterpenes either as a single compound or as a mixture with others have been identified (for overview: Falara et al., 2011; Chapter 5) and recently the tomato terpene synthase responsible for the synthesis of zingiberene has been identified (Bleeker et al., submitted). Maize plants upon herbivory by lepidopteran larvae emit a mixture of compounds that attracts females of the parasitic wasp Cotesia marginiventris. This volatile bouquet is produced by a single terpene synthase (TPS10), which is induced in herbivore-damaged leaves, and produces various sesquiterpenes, but mainly (E)-β-farnesene and (E)-α- bergamotene (Schnee et al., 2006). In medicago, (E)-β-ocimene is constitutively produced in leaves by MtEBOS at low levels, but Spodoptera exigua feeding up- regulates its production (Navia-Gine et al., 2009). Finally, metabolite analysis of cucumber leaves with and without spider mite infestation lead to the identification of DMNT (deriving from 3S-(E)-nerolidol), (E)-β-ocimene and (E,E)-α-farnesene as the three major terpenes emitted from infested cucumber plants. Furthermore, transcriptome analysis of the same samples lead to identification of CsαFS, the enzyme responsible for the formation of (E,E)-α-farnesene from FPP. A 3S-(E)- nerolidol and (E)-β-ocimene synthase were not identified, however CsαFS could produce (E)-β-ocimene from GPP suggesting a dual role for this enzyme (Mercke et al., 2004).
Chapter 1 13 6. Regulation of isoprenoid biosynthesis
Plant defenses are costly and require resources otherwise used for growth and reproduction (Walters and Heil, 2007). Therefore, plants have evolved a complex, largely hormonal, signaling network to arrange defense and resource allocation (Pieterse et al., 2009). As it has been indicated in section 5, regulation of induced terpene biosynthesis occurs mostly at the transcript level of the terpene synthases, as well as on transcriptional regulation of precursor genes (discussed in Chapter 5), but also of downstream modifying enzymes, like the cytochrome P450s (Son et al., 1998; Luo et al., 2001). Activation of the specialized metabolite pathways in response to a stress signal is often triggered by phytohormones like jasmonic acid, ethylene, salicylic acid or abscisic acid (Nascimento and Fett-Neto, 2010).
6.1 Regulation of plant defenses at the gene expression level
The general principle for the activation of plant specialized metabolite biosynthesis involves a signal that initiates a transduction cascade leading to de novo biosynthesis or activation of transcription factors that in turn regulate the expression of specialized metabolite biosynthetic genes (Zhao et al., 2005). Regulation of gene expression in all eukaryotes involves sequences that flank the gene in question containing cis-regulatory elements (promoters) and proteins that recognize and can interact with these elements (trans-acting factors; Wray et al., 2003).
6.1.1 Trans-acting factors and cis-elements
Trans-acting factors (transcription factors; TFs) are DNA-binding proteins that can recognize and bind specific regulatory sequences, the so-called cis-elements, in the
Chapter 1 14 promoter of target genes and thus affect the rate of transcription initiation (Wray et al., 2003). Cis-elements are typically located adjacent to the promoter, but can also be found far upstream of it, in introns or even downstream of a gene, but DNA looping allows the gene regulatory proteins bound at any of these positions to interact with the proteins that assemble at the core promoter (Wray et al., 2003). In general, activating transcription of a gene involves decondensation of chromatin around the core promoter and some TF binding site(s), followed by the binding of general TFs that recruit the RNA polymerase II complex onto the basal promoter (Lee and Young, 2000). Apart from a DNA-binding domain, TFs usually have a transcription regulation domain (activating or repressing; Liu et al., 1999). Repressors inhibit transcription through various mechanisms, including competing with an activator for the DNA binding site, modifying chromatin structure or preventing recruitment of the transcription initiation complex (Lee and Young, 2000). Control of a cellular process (like response to a stress stimulus) requires the coordinate activation or repression of genes in an exact spatial and temporal pattern and such regulation can be achieved by interaction of a TF with specific cis- elements of the target gene(s) and by temporal and spatial expression of transcription factors themselves. Therefore, even TFs that share DNA-binding properties can control distinct biological processes (de Folter and Angenent, 2006). Such transcriptional regulation of TFs by upstream genes is part of the cells’ regulatory network that will not be discussed here.
6.1.2 Transcription factor families
Transcription factors can be classified into different families according to their DNA binding domain. There are at least 64 TF families found in vascular plant genomes (Rushton et al., 2008) and binding of these proteins to the DNA bases commonly takes place through the secondary structure elements of a β-sheet or an
Chapter 1 15 α-helix (Yamasaki et al., 2008). The cis-elements TFs recognize and bind to are usually short stretches of 4-8 base pairs and with a few variations from a consensus can be characteristic for each TF family. However, specificity for the target promoters can partly be determined by adjacent sequences (Rushton et al., 2010; Figueroa et al., 2010). The first plant TF identified was a MYB domain protein required for the synthesis of anthocyanins in maize kernels (Paz-Ares et al., 1987). MYB TFs are involved in the regulation of an array of processes including secondary metabolism (e.g. flavonoid biosynthesis), cell fate and identity (e.g. trichome formation), development (e.g. anther development) or abiotic and biotic stress responses (e.g. drought stress and disease resistance; Dubos et al., 2010). DNA-binding specificity varies among different MYB proteins within and between plant species and only few DNA binding sites have been characterized functionally (Martin and Paz-Ares, 1997; Dubos et al., 2010). For example R2R3-MYB family members of group A bind to a CNGTT(A/G) motif (Prouse and Campbell, 2012). Another big family of plant TFs took its name from its founding member Apetala 2 that controls Arabidopsis flower and seed development (Jofuku et al., 1994). AP2 domain proteins are involved in regulating developmental processes as well as in abiotic stress acclimation and hormone-dependent signaling in response to, for example pathogens (Dietz et al., 2010). Members of the subgroup AP2/ERF have been implicated in defense responses mediated by jasmonates (see section 6.2) in a number of plant species (Rushton et al., 2008) including Catharanthus roseus (discussed in section 6.4). There are four subgroups in the family, each recognizing different core elements (for example the dehydration-responsive element (DRE) by the DREB subfamily or the GCC box (GCCGCC) by the ERF subfamily; Dietz et al., 2010). Basic helix-loop-helix (bHLH) transcription factors bind DNA through their N- terminal basic region, whereas the C-terminal HLH region functions as a dimerization domain (Toledo-Ortiz et al., 2003). They form one of the largest families of TFs in plants, however only a relative small number of bHLH genes
Chapter 1 16 have been studied in detail, some of which are involved in processes like anthocyanin biosynthesis, trichome differentiation or light signaling (Heim et al., 2003). These proteins typically bind to an E box motif (CANNTG) with, for example, MYC bHLH proteins recognizing a variation of the E box, known as the G box motif (CACGTG; Heim et al., 2003). Roles of Arabidopsis, Nicotiana tabacum and C. roseus MYC transcription factors are presented in following sections. Zinc finger (ZnF) transcription factors comprise different classes of TFs that all involve a sequence motif in which a zinc atom is bound by cysteine (C) and histidine (H) residues stabilizing the secondary structure of the “finger” (Klug, 2010). The zinc finger domain however, enables different proteins to bind not only DNA, but also bind or interact with RNA or other proteins (Ciftci-Yilmaz and Mittler, 2008). There are different classes of ZnF TFs such as the C2H2-type, DNA binding with one finger (Dof), WRKY and RING-finger (discussed in Chapter 4) that play a role in regulating various processes (Takatsuji, 1998). Consensus binding site for example the WRKY TFs is the W box (TTGACC/T) for almost all proteins investigated so far (Rushton et al., 2010). An overview of all transcription factor families and their characterized members from various plant species can be found in publicly available TF databases. Such databases facilitate comparative studies of transcriptional regulation in model plants like Arabidopsis (Guo et al., 2005), tobacco (Rushton et al., 2008), rice (Gao et al., 2006), poplar (Zhu et al., 2007) or 46 other plant species (Zhang et al., 2011a).
6.2 Role of hormones in plant defenses
Whereas plant resistance against immobile pathogens often is characterized by a hypersensitive response (HR), defense against herbivores is associated more with a decrease in tissue palatability (Anten and Pierik, 2010). Central in the organization
Chapter 1 17 of anti-herbivore defenses is the plant hormone jasmonic acid (JA) and its active derivative JA-isoleucine (JA-Ile), which rapidly accumulates during herbivory. The mode of action of JA has been studied in detail using JA biosynthesis- or perception-impaired mutant plants, which are often preferred by herbivores in choice tests while allowing for higher herbivore fitness (Howe and Jander, 2008). Accumulation of JA-dependent defense proteins and metabolites is often co- regulated by ethylene in a synergistic manner. In contrast, salicylic acid (SA) antagonizes the action of JA (Pieterse et al., 2009). SA is well known for its signaling role in defenses induced by biotrophic pathogens, but many stylet- feeding herbivores, like mites, whiteflies, and aphids, induce a cocktail of JA- and SA-related responses (Kant et al., 2008). Although it is not clear to which extent this mixed response is required for the plant to establish the appropriate defenses, the “decoy hypothesis” suggests that in some cases, the herbivore could benefit from a SA-mediated suppression of the JA defenses (Zarate et al., 2007). Finally, also the hormones auxin and abscisic acid (ABA) influence the properties of the signaling network mostly via antagonizing the action of JA and SA (Pieterse et al., 2009). The dynamics of this complex regulatory network, in which hormonal synergisms and antagonisms determine the final output of the defense response, depend largely on the type of herbivore as well as on the physiological status of the plant.
Since herbivory and wounding mainly elicit responses mediated by jasmonic acid as part of the plants’ (in)direct defenses, the focus in the next section will be on this hormone.
6.2.1 Jasmonic acid biosynthesis and regulation of downstream genes
The precursor for the biosynthesis of JA is α-linolenic acid released by lipases from chloroplast galactolipids. Activity of 13-lipoxygenase (LOX), allene oxide
Chapter 1 18 synthase (AOS) and allene oxide cyclase (AOC) enzymes converts α-linolenic acid to cis-(+)-12-oxophytodienoic acid (OPDA). In a parallel pathway dinor-OPDA can be formed from hexadecatrienoic acid by the same set of enzymes. OPDA (and dinor-OPDA) is translocated into the peroxisomes where OPDA reductase 3 (OPR3) catalyzes its reduction to oxo-pentenyl-cycloheptan-octanoic acid (OPC-8) that in turn undergoes three rounds of β-oxidation leading to jasmonyl-CoA (JA- CoA) formation. JA-CoA is then cleaved by a putative thioesterace yielding (+)-7- iso-JA that equilibrates to the more stable (-)-JA (Wasternack and Kombrink, 2010). In an uninduced situation transcription factors (TFs) are bound to the target elements in the promoters of JA-responsive genes, but their transcriptional activity is repressed by the binding of a repressor complex that in Arabidopsis consists of Jasmonate ZIM domain (JAZ), Novel Interactor of JAZ (NINJA) and Topless (TPL; Pauwels et al., 2010). There are 12 members of the JAZ family in Arabidopsis with functional redundancy, but also diverse tissue- and stage-specific expression patterns (Chini et al., 2009) and homologues are found in many plant species (Chico et al., 2008). In general, upon perception of a stress stimulus (-)-JA is converted to its active form JA-isoleucine (JA-Ile) through the action of JA amino acid conjugate synthase 1 (JAR1). High levels of JA-Ile lead to binding by the jasmonate receptor Coronatine Insensitive 1 (COI1) that is part of the Skp1/Cullin/F-box (SCF) complex and acts as an E3 ubiquitin ligase. The SCFCOI1 complex recruits the JAZ proteins that get polyubiquitinated and are directed to the 26S proteasome for degradation, releasing the repression of the TFs they were bound to, and thus activating expression of the downstream JA-responsive genes. Among the early JA-responsive genes are those encoding JAZ proteins, creating a negative feedback loop (Wasternack and Kombrink, 2010). Similar roles for COI1 homologues have been reported in other plant species, including tomato (Li et al., 2004) and the image emerges that a JAZ-mediated signaling web regulates JA- dependent functions, like specialized metabolite production or responses to abiotc and in addition developmental cues. In tomato, jasmonic acid-insensitive 1 (jai1;
Chapter 1 19 the homolog of the Arabidopsis COI1 gene) plants exhibited abnormal development of glandular trichomes and defense-related phenotypes like severely compromised resistance to spider mites (Li et al., 2004). The wound-induced systemic defense in tomato is believed to start with the interaction of systemin, an 18 amino acid peptide cleaved from the C-terminal region of the 200 amino acid precursor prosystemin, with a receptor in the plasma membrane, activating JA biosynthesis (Sun et al., 2011) in a cascade of events like those described above for Arabidopsis. However, in tomato no targets of JAZ proteins have been identified. AtMYC2 was until recently the only transcription factor described as a direct JAZ target (Chini et al., 2007), but two closely related bHLH TFs, AtMYC3 and AtMYC4 were shown to act additively with MYC2 in the activation of JA responses in Arabidopsis- i.e. playing a role in root growth and regulation of herbivory and pathogen responses (Fernandez-Calvo et al., 2011). In other plant species similar roles for JAZ and MYC proteins have recently been shown. For example in Nicotiana tabacum the biosynthesis of the alkaloid nicotine involves the action of NtCOI1, NtJAZ and NtMYC2 (Shoji and Hashimoto, 2011; De Boer et al., 2011; Zhang et al., 2011b) and in rice drought tolerance is conferred through a OsCOI1, OsJAZ and OsbHLH148 mediated cascade (Seo et al., 2011).
6.3 Additional modes of regulation
There are additional levels on which gene expression can be regulated, which will be briefly presented here. Posttranscriptional regulation is known to be an important mechanism defining final concentration of the active gene product and it includes control of RNA (alternative) splicing, mRNA stability, or miRNA- mediated degradation (Blencowe et al., 2009). Additionally, protein-protein interactions or posttranslational regulation can significantly impact the regulatory activity of transcription factors. Transcriptional activity of for example some MYB TFs depends in vivo on protein-protein interactions, with homo- or hetero-
Chapter 1 20 dimerizations enabling DNA recognition with high affinity and specificity (Dubos et al., 2010). Posttranslational modifications of proteins include acetylation, hydroxylation, ubiquitination and more commonly phosphorylation, and such modifications can influence among others DNA binding affinity or protein stability by altering for example protein conformation (Vom Endt et al., 2002). Finally, epigenetic regulation (histone modifications and DNA methylation) can play a role in chromatin structure and so determine the transcriptional state and expression level of genes (Chinnusamy and Zhu, 2009).
6.4 Examples of transcription factors regulating terpenoid biosynthesis
There are relatively few transcription factors that regulate terpenoid biosynthesis identified to date, which are presented here. For an overview of TFs regulating (JA-induced) secondary metabolite biosynthesis see De Geyter et al., 2012.
The sesquiterpene phytoalexin gossypol is produced in the glands of cotton (Gossypium arboreum) aerial tissues from the biosynthetic precursor (+)-δ-cadiene. CAD1 ((+)-δ-cadiene synthase) catalyzes the first committed step towards the formation of gossypol and analysis of its promoter sequence revealed the presence of two W box cis-acting elements forming a palindrome (Xu et al., 2004). Degenerate primers were used to isolate WRKY cDNAs from a cotton cDNA library, but only one of the 10 fragments isolated had higher transcript levels in a glanded cotton cultivar than in a glandless cultivar. This cDNA was cloned full- length and designated GaWRKY1. CAD1 and GaWRKY1 had a similar spatial and temporal expression pattern. Furthermore, elicitor and jasmonate treatments induced transcription of both genes. GaWRKY1 was shown to localize to the nucleus and could bind to three tandem repeats of the W box palindrome of CAD1 in yeast and in vitro. Finally, in transgenic Arabidopsis plants it was shown that
Chapter 1 21 overexpression of GaWRKY1 strongly activated the CAD1 promoter (Xu et al., 2004). The sesquiterpene lactone endoperoxide artemisinin is produced in glandular trichomes of Artemisia annua with the first committed step for its biosynthesis being the cyclization of FPP by the amorpha-4,11-diene synthase (ADS). Analysis of the ADS promoter sequence revealed the presence of two reversely oriented W box cis-acting elements (Ma et al., 2009). A WRKY EST was identified in a glandular trichome cDNA library and was designated AaWRKY1. ADS showed the same expression pattern as AaWRKY1 and both were induced by elicitor and jasmonate treatments. Furthermore, AaWRKY1 was shown to localize to the nucleus and could bind to three tandem repeats of the W box of ADS in yeast and in vitro. Co-expression of 35S:AaWRKY1 and ADSp:GUS in stably double- transformed tobacco plants strongly activated GUS expression and finally transient overexpression of AaWRKY1 in A. annua leaves was shown to clearly activate expression of ADS, but also of the MVA pathway precursor 3-hydroxy-3- methylglutaryl coenzyme A reductase (HMGR) as well as of the downstream genes cytochrome P450 reductase (CPR) and artemisinic aldehyde reductase (DBR2). Notably FPS expression was not upregulated suggesting that FPS is not a rate- limiting factor in artemisin biosynthesis (Ma et al., 2009). The Nicotiana attenuata WRKY3 and WRKY6 were strongly induced in response to wounding and oral secretions (OS) of Manduca sexta caterpillars, respectively (Skibbe et al., 2008). However, neither of these TFs was transcriptionally induced by JA. It was proposed that they function upstream of the JA signal, since silencing WRKY3 and/or WRKY6 lead to reduced expression of JA-biosynthetic genes, as well as reduced levels of JA. Silenced plants were more vulnerable to herbivore feeding, which was associated with reduction in direct defense-related Trypsin Proteinase Inhibitors (TPI) protein activity as well as reduced emission of the indirect defense sesquiterpene cis-α-bergamotene. Exogenous application of JA- supplemented OS to the WRKY-silenced plants restored TPI activity levels and the amount of cis-α-bergamotene released (Skibbe et al., 2008).
Chapter 1 22 The common precursor for the synthesis of a range of terpenoid indole alkaloids (TIA) in Catharanthus roseus is 3α-(S)-strictosidine. Strictosidine is formed by condensation of secologanin (its terpenoid part, produced via multiple enzymatic conversions of the MEP pathway-derived monoterpene geraniol) with tryptamine (the indole moiety, produced via the shikimate pathway), through the action of the enzyme strictocidine synthase (STR; Fig.4). Tryptamine is formed by decarboxylation of tryptophan by the enzyme tryptophan decarboxylase (TDC; Memelink et al., 2001). A yeast-one-hybrid (Y1H) screen with the JA- and elicitor- responsive element (JERE) of the STR promoter led to the identification of two cDNAs encoding AP2/ERF domain proteins that were designated octadecanoid- derivative responsive Catharanthus AP2-domain (ORCA) 1 and 2. However only ORCA2 mRNA levels were induced by jasmonate and elicitor treatments (Menke et al., 1999). A genetic (T-DNA activation tagging) approach identified another AP2/ERF domain-containing TF that was named ORCA3. Overexpression of ORCA3 in transgenic C. roseus cell cultures induced primary (like 1-deoxy-D- xylulose 5-phosphate synthase; DXS) and secondary (like STR and TDC) metabolic pathway genes that are involved in TIA biosynthesis, all of which were shown to be methyl-jasmonate (MeJA)-inducible. Furthermore, upon addition of excess terpenoid precursor (loganin), TIA production was increased in the ORCA3 overexpressing cell lines (van der Fits and Memelink, 2000). Like ORCA2, ORCA3 was shown to specifically bind to the JERE fragment of the STR promoter. Moreover, treatment of C. roseus cell cultures with MeJA and/or a protein synthesis inhibitor (cycloheximide; CHX) lead to the discovery that ORCA3 induction by MeJA does not depend on de novo protein synthesis, but also that MeJA-induced STR and TDC expression does not require de novo synthesis of ORCA3 (or other TFs), therefore jasmonate most likely induces STR and TDC expression via modification (possibly phosphorylation) of pre-existing ORCA3 protein (van der Fits and Memelink, 2001). Expression of ORCA3 is itself rapidly induced by MeJA, which implies either autoregulation or regulation by upstream TF(s). By analyzing the ORCA3 promoter, a jasmonate-responsive element (JRE)
Chapter 1 23 was identified that consists of an A/T-rich quantitative sequence determining expression level and a G box-like qualitative sequence, likely to bind a bHLH TF, responsive to MeJA. Y1H screens identified (non MeJA-inducible) clones containing an AT-hook motif that could bind to the quantitative sequence, but none that could bind the qualitative sequence (Vom Endt et al., 2007). C. roseus bHLH MYC cDNA clones had been previously identified in a Y1H screen with a tetramer of the STR promoter G box (located upstream of the JERE element; Pre et al., 2000), and the one with highest homology to AtMYC2 was designated CrMYC2 and shown to be strongly induced by MeJA and that it could bind to the G box-like qualitative sequence of the ORCA3 promoter in vitro and in vivo. Surprisingly, although CrMYC2 could bind to the G box from the STR promoter, it could not activate gene expression via native STR promoter derivatives containing this same G box. CrMYC2 knock down lines showed strong reduction in the levels of MeJA- responsive ORCA2 and ORCA3 mRNA as well as alkaloid accumulation, but no effect on STR and TDC expression (Zhang et al., 2011c). Since MeJA induction of ORCA3 and CrMYC2 was equally fast and ORCA3 induction is insensitive to CHX (as is the CrMYC2 induction), it is probably not dependent on de novo CrMYC2 protein synthesis, but caused by activation of pre-existing CrMYC2 protein. This activation could involve degradation of JAZ proteins that, in analogy with the Arabidopsis system, repress CrMYC2 activity in the absence of MeJA (Zhang et al., 2011c). Additional complexity to the regulation of jasmonate-induced TIA biosynthesis in C. roseus brings the identification of three more classes of TFs involved in the process. The Y1H screen with a tetramere of the STR promoter G box allowed the identification of CrMYC TFs, but also of two TFs of the basic leucine zipper (bZIP) class (Pre et al., 2000), designated as G box-binding factors (CrGBF) 1 and 2, which were shown to be repressors of the STR gene expression via direct interaction with the G box (Siberil et al., 2001). Furthermore, Y1H screens of the elicitor-responsive element of the TDC promoter led to the isolation of three C2H2- type ZnF TFs that were designated zinc finger Catharanthus transcription factors
Chapter 1 24 (ZCT) 1, 2 and 3 and were shown to act as transcriptional repressors of TDC and STR promoter activity. Furthermore it was shown that yeast elicitor and MeJA increased ZCT mRNA levels and, interestingly, that the ZCT proteins could repress the activating activity of ORCA 2 and 3 on the STR promoter without competing for the same binding sites (Pauw et al., 2004). Finally, analysis of available promoters from the TIA pathway genes identified W box(es) in almost all of them and therefore degenerate primers were designed on conserved sequences of WRKY domains in order to identify mRNAs in MeJA-treated C. roseus tissues. One of the open reading frames obtained, designated CrWRKY1, was shown to be induced by MeJA and could bind to the TDC promoter sequence containing the W box in vitro and in yeast. Overexpression of CrWRKY1 resulted in much higher TDC, but also moderately higher ZCT levels. However, this strongly reduced the expression of transcriptional activators ORCA2, ORCA3 and to a lesser extend CrMYC2 and had no effect on STR expression. Furthermore, these overexpressing transgenic lines showed accumulation of TIA serpentine in the roots, where CrWRKY1 is highest expressed (Suttipanta et al., 2011).
CrWRKY1
Shikimate pathway MEP pathway CrMYC2 DXS
Tryptophan Geraniol ORCA3 ORCA2 TDC STR Tryptamine Secologanin
CrZCT CrGBF
Strictosidine
TIA Figure 4. Regulatory network of terpene indole alkaloid (TIA) biosynthesis in Catharanthus roseus. Only key players mentioned in the text are shown here. Pathway enzymes are in boxes, transcription factors are in ovals. Dashed lines indicate more than one enzymatic step. Arrows indicate positive interactions, T-bars indicate negative interactions. For abbreviations see text.
Chapter 1 25 It becomes clear from the well-studied case of terpenoid indole alkaloid biosynthesis in Catharanthus roseus and what has been discussed so far, that regulators can form a dynamic network that controls the temporal- and tissue- specific expression of genes, with cross-talk between transcription factors with overlapping functions and (concurrent) induction of activators and repressors by elicitors and stress or developmental signals.
7. Thesis Outline
Chapter 1 gave a general introduction to isoprenoids and terpenes, how and where they are synthesized, the roles terpenes play in plants and how terpene biosynthesis is regulated. In Chapter 2, an overview of the terpene synthase family in tomato is given and a subset of mono- and sesqui-terpenes are further characterized, with a focus on genes that are expressed in the trichomes. Chapter 3 describes the dissection of the promoter of tomato monoterpene synthase 1 (SlMTS1) and the identification of a putative transcription factor that binds to a 207bp fragment proximal to the coding region. This TF was designated Emission of Terpenes 1 (SlEOT1) and further characterized in Chapter 4. In Chapter 5 an analysis of transcriptomic data from tomato stem trichomes is presented. Two transcription factors from this EST dataset are shown to interact with terpene synthase (described in Chapter 2) promoters in a transient assay in planta. Finally, in Chapter 6 results presented in this thesis are collectively discussed.
Chapter 1 26 8. Account
Chapter 1; parts have been published in: Alba JM, Allmann S, Glas JJ, Schimmel BCJ, Spyropoulou EA, Stoops M, Villarroel C & Kant MR. (2012). Induction and Suppression of Herbivore-Induced Indirect Defenses. In: Biocommunication of Plants, Signaling and Communication in Plants Vol 14, Springer, Witzany, Günther; Baluška, František (Eds.), pp 197-213. Chapter 2; parts have been published in: Bleeker PM*, Spyropoulou EA*, Diergaarde PJ, Volpin H, De Both MT, Zerbe P, Bohlmann J, Falara V, Matsuba Y, Pichersky E, Haring MA, Schuurink RC (2011) RNA-seq discovery, functional characterization, and comparison of sesquiterpene synthases from Solanum lycopersicum and Solanum habrochaites trichomes. Plant Mol Biol 77 (4-5):323- 336 (* PMB and EAS contributed equally to this paper) and Falara V, Akhtar TA, Nguyen TT, Spyropoulou EA, Bleeker PM, Schauvinhold I, Matsuba Y, Bonini ME, Schilmiller AL, Last RL, Schuurink RC, Pichersky E (2011) The tomato terpene synthase gene family. Plant Physiol 157 (2):770-789. Chapters 3 and 4; Spyropoulou EA, Haring MA, Schuurink RC (2012) Characterization of Solanum lycopersicum Emission of Terpenes 1, a trichome- specific transcription factor that regulates the Monoterpene Synthase 1 gene; manuscript in preparation and Spyropoulou EA, Haring MA, Schuurink RC (2011) Transcription factor modulating terpene biosynthesis (patent application nr. N2006928). Chapter 5; Spyropoulou EA, Haring MA, Schuurink RC (2012) RNA sequencing of tomato trichomes identifies a transcription factor involved in terpene biosynthesis; manuscript in preparation.
This project was funded by Technological Top Institute Green Genetics (grant nr. 1C002RP), KeyGene N.V., Enza Zaden, VCo and Takii.
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Chapter 1 36 Chapter 2
Discovery and characterization of mono- and sesquiterpene synthases from Solanum lycopersicum
Eleni A.Spyropoulou, Petra M.Bleeker, Michel A.Haring and Robert C.Schuurink
Department of Plant Physiology, Swammerdam Institute for Life Sciences, University of Amsterdam, Science Park 904, 1098 XH Amsterdam, The Netherlands
ABSTRACT
Terpenes constitute a large class of chemical compounds produced by almost all living organisms. In plants they have various functions, ranging from attracting pollinators to acting as defense compounds against pests. The recent release of the genomic sequence of tomato (Solanum lycopersicum) enabled the identification of (all) terpene synthases (TPS) involved in the production of these volatile compounds. Here we present the characterization of three novel sesquiterpene synthases and three novel monoterpene synthases from S.lycopersicum cv Moneymaker that are expressed in various organs. Two TPS genes, expressed predominately in trichomes, were induced by treatment with jasmonic acid. The products of these six terpene synthases were also analyzed in vitro. Our data contribute to the deciphering of the tomato terpenome.
INTRODUCTION
Terpenoids constitute a large class of chemical compounds produced by most, if not all, living organisms. Over 23,000 different terpenoid compounds have been characterized (Sacchettini and Poulter, 1997). Plants produce terpenoids that function in primary metabolism such as phytohormones (abscisic acid, gibberellins, cytokinins and brassinosteroids), are part of photosynthetic pigments (phytol and carotenoids), electron carriers (ubiquinone) or constitute structural components of membranes (phytosterol). However, the majority of plant terpenoids are secondary, or specialized metabolites, present only in a subset of plant lineages. They can be active as direct defensive compounds, such as phytoalexins that accumulate upon
Chapter 2 38 pathogen infection (Akram et al., 2008). In addition, volatile terpenoids can function as indirect defensive compounds by attracting predators or parasitoids of the attacking insect (Walling, 2000). The emission of different terpenoids is induced by insect herbivory (Kant et al., 2004; Olson et al., 2008; Navia-Gine et al., 2009). Two distinct pathways leading to the universal terpene precursors isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP) operate in plants. The mevalonate (MVA) pathway produces IPP in the cytosol, which can be converted to DMAPP by IPP isomerase. The 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway produces plastidial IPP and DMAPP. Head-to-tail elongation of IPP with DMAPP catalyzed by geranyl diphosphate (GPP) synthase or by neryl diphosphate (NPP) synthase lead to the formation of GPP and NPP (the cis-isomer of GPP; Croteau and Karp, 1979), respectively, which are the precursors for monoterpenes. (E,E)-Farnesyl diphosphate (FPP), and (Z,Z)-Farnesyl diphosphate (Kellog and Poulter, 1997) serve as precursors for sesquiterpenes, and are synthesized by (Z,Z)- and (E,E)-FPP synthases, respectively. (E,E,E)-Geranylgeranyl diphosphate (GGPP) synthase catalyzes formation of GGPP, the diterpene precursor. The MEP pathway provides precursors for the synthesis of monoterpenes and diterpenes in plastids, whereas sesquiterpenes are derived from precursors of the MVA pathway in the cytosol. However, exchange of precursor between cytosol and plastids has been reported. Snapdragon flowers can synthesize sesquiterpenes from plastidial isoprenes, indicating transport of IPP from the plastids to the cytosol (Dudareva et al., 2005). More recently it was shown that several sesquiterpenes from wild tomato (Solanum habrochaites) are synthesized in the plastids from plastidial (Z,Z)-FPP (Sallaud et al., 2009). The prenyl diphosphates are converted to terpenes by the action of terpene synthases (TPSs), a group of structurally and evolutionarily related enzymes (Chen et al., 2011). Induced terpenoid synthesis is often correlated with induced expression of terpene synthases (Herde et al., 2008; Navia-Gine et al., 2009; Zulak et al., 2009). Besides regulation at the level of terpene synthases, induction of
Chapter 2 39 precursor biosynthetic genes has also been reported (Kant et al., 2004; Ament et al., 2004). In vitro, sesquiterpene synthases can often produce monoterpenes when provided with GPP as substrate, and monoterpene synthases can produce sesquiterpenes when provided with FPP. Therefore, subcellular targeting of terpene synthases determines which substrate the terpene synthase encounters and thus which product is made in vivo. For instance, two nearly identical terpene synthases from snapdragon both catalyze the conversion of GPP to linalool and of FPP to nerolidol in vitro. However, only one of these linalool/nerolidol synthases has a transit peptide, localizing the protein to the plastids (Nagegowda et al., 2008). To be effective against pests and diseases, defensive terpenoid compounds are often produced at the surface of the plant. Trichomes, which are specialized secretory structures on the surface of leaves and stems, contain high levels of terpenes in many species (Chatzivasileiadis et al., 1999; Besser et al., 2009), as well as other secondary metabolites (Fridman et al., 2005; Ben-Israel et al., 2009). Furthermore, many investigations have shown that these compounds, including terpenes, are usually synthesized de novo in the trichomes (Olsson et al., 2009; Maes et al., 2011). New terpene synthases have been so far discovered by homology-based cloning (van Der Hoeven et al., 2000; van Schie et al., 2007; Portnoy et al., 2008; Jones et al., 2008), by random sequencing of cDNAs (van Der Hoeven et al., 2000; Wang et al., 2008) followed by bioinformatic searches of the resulting EST databases (Keeling et al., 2011) or by genome mining (Aubourg et al., 2002; Martin et al., 2010). However, terpene synthases that are less abundant in a particular organ or structure, or have low sequence similarity to known terpene synthases might not be identified by these methods. Lately the use of massive parallel pyrosequencing of transcripts, termed “RNA-seq”, is becoming more widely used (Wilhelm and Landry, 2009; Schilmiller et al., 2010), since its major advantage above the construction of EST databases is that quantitative expression levels of transcripts are better estimated due to the larger set of data. High-throughput sequencing has also been used for sequencing whole genomes of organisms. The determination of
Chapter 2 40 the sequence of the cultivated tomato genome made it possible to characterize the terpene synthase family (Falara et al., 2011). In combination with RNA-seq approaches (Bleeker et al., 2011) and the available volume of information about TPSs obtained so far by various other techniques, allowed us to construct a more complete picture about the biosynthesis of this very interesting class of compounds. Here we describe the identification and characterization of three novel sesquiterpene synthases and three novel monoterpene synthases from Solanum lycopersicum. Functional expression in E.coli provided information on the proteins encoded by these cDNAs. Determination of tissue-specific expression of the cultivated tomato terpene genes showed that the expression of most of them was highest in tissue containing trichomes. Furthermore, one sesquiterpene and one monoterpene synthase were induced by jasmonic acid treatment, suggesting their involvement in herbivore-induced terpenoid emission.
MATERIALS AND METHODS
Hormone Treatment, RNA Isolation and cDNA synthesis
Tomato plants (Solanum lycopersicum cv Moneymaker C32) were grown in soil in a greenhouse with day/night temperatures of 23oC/18oC and a 16/8 h light/dark regime for 4 weeks. Jasmonic acid (JA; Duchefa, NL) was applied to plants by spraying 1mM solution made with tap water containing 0.05% SilwetL-77 (GE Silicones, VA, USA). Control plants were sprayed with tap water containing 0.05% SilwetL-77. Trichomes were collected 24h later at the bottom of a 50ml tube by vortexing stem pieces frozen in liquid nitrogen and subsequent removal of the stem pieces. All other tissues and organs analyzed were also frozen in liquid N2 and then
Chapter 2 41 the material was ground and total RNA was isolated using Trizol (Invitrogen, Paisley, UK). DNA was removed with DNAse (Ambion, Huntingdon, UK) according to the manufacturer’s instructions and cDNA was synthesized from 1.5μg RNA using M-MuLV H- Reverse Transcriptase (Fermentas, St. Leon-Rot, Germany).
Quantitative Real Time PCR
For Q-RT-PCR cDNA equivalent to 100ng total RNA was used as template in 20μl volume and reactions were performed in an ABI 7500 Real-Time PCR System (Applied Biosystems) using an Platinum SYBR Green qPCR SuperMix-UDG kit (Invitrogen, Paisley, UK) with the following cycling program: 2min 50oC, 7min 95oC, 45 cycles of 15sec at 95oC and 1min at 60oC, followed by a melting curve analysis. Primer pairs were tested for amplification kinetics and linearity with a standard cDNA dilution curve and new primers were designed if necessary. Expression levels were normalized using ACTIN (SGN-U579547) mRNA levels. Effect of JA in gene expression was analyzed in three biological replicates by T- test using PASW Statistics 17.0 (http://www.spss.com). The homogeneity of variance was tested by Levene’s test and values were log transformed before the analysis if necessary.
Production of recombinant proteins
SlTPS3, SlTPS7 and SlTPS8 open reading frames (ORF) were cloned from a mix of trichome, leaf, stem and root cDNA in pGEM-T easy (Promega, Madison, USA) and verified by sequencing. They were subsequently digested and re-cloned in pET32a (Novagen) downstream of the histidine (His) tag between restriction sites EcoRV (at the ATG site) and SacI (after the stop codon) for SlTPS3 and in pGEX
Chapter 2 42 KG (Guan and Dixon, 1991) downstream of glutathione S-transferase (GST) between restriction sites NcoI (at the ATG site) and SacI (after the stop codon) for SlTPS7. SlTPS8 was directionally cloned in pET200-TOPO downstream of the histidine (His) tag using the pET TOPO expression kit from Invitrogen (Paisley, UK). All constructs were maintained in E. coli TOP10 cells. For the expression analysis the constructs were transformed freshly into E. coli C41 (D3) cells (Dumon-Seignovert et al., 2004). Cells were grown in Terrific Broth with appropriate antibiotics to OD600 of 0.6 from a pre-culture inoculated from a single colony, incubated at 4oC for 30min and then induced with 1mM isopropyl-β-D- thiogalactoside (IPTG) for 16h at 16oC, shaking at 200rpm. Cells were harvested by centrifugation at 4500rpm for 15min at 4oC. The supernatant was removed and the pellets resuspended in 2-4ml of STS buffer (25mM Hepes-HCl pH 7.2, 10mM
MgCl2, 10% (v/v) glycerol) with added lysozyme (1mg/ml) and protease inhibitors (Complete, Roche, Basel, Switzerland). Cells were incubated on ice for 30min and subsequently sonicated (45sec, output control 3, duty cycle 30). Lysates were centrifuged at 4oC for 25min at 12,000rpm. The supernatants containing the bacterial crude extracts with recombinant proteins were aliquoted, snap frozen in o liquid N2 and stored at -80 C. Cloning of the sesquiterpene synthases and recombinant protein production has been described in Bleeker et al. (2011).
Functional expression analysis
Activity assays were performed for the monoterpene synthases in 20ml glass vials in a total volume of 1.5ml containing STS buffer (25mM Hepes-HCl pH 7.2,
10mM MgCl2, 10% (v/v) glycerol) including 5mM DL-Dithiothreitol (DTT), 150μl of crude protein extract and 1mM of precursors as substrate (GPP; geranyl diphosphate, NPP; neryl diphosphate, (E,E)-FPP; (E,E)-farnesyl diphosphate, (Z,Z)-FPP; 2z-6z-farnesyl diphosphate or GGPP; geranylgeranyl diphosphate;
Chapter 2 43 Echelon Biosciences Incorporated, Salt Lake City, USA). As negative control no precursor was used. For the sesquiterpene synthases activity assays were performed in 20ml glass vials in a total volume of 500µl containing 50mM HEPES pH7.2,
100mM KCl, 7.5mM MgCl2, 20µM MgCl2, 5% (v/v) glycerol, 5mM DTT with 50µl protein extract and either 2mM (E,E)-FPP, (Z,Z)-FPP, GPP or GGPP as substrate. Vials were immediately closed with a Teflon lined crimp cap and incubated at 30oC for 1h shaking at 150rpm. Enzyme products were sampled with a Solid Phase Micro Extraction (SPME) fiber for 10min after the vial had been agitated and heated to 50oC. The fiber was desorbed for 1min in an Optic injector port (ATAS GL Int., Zoeterwoude, NL), which was kept at 250oC. Compounds were separated on a DB-5 column (10m x 180μm, 0.18μm film thickness; Hewlett Packard) in an 6890 N gas chromatograph (Agilent, Amstelveen, NL) with a program set to 40oC for 1.5min, ramp to 250oC at 30oC per minute and 250oC for an additional 2.5min. Helium was used as a carrier gas, the column flow was set to 3ml per minute for 2min and to 1.5ml per minute thereafter. Mass spectra were generated with the ion source set to -70V at 200oC and collected with a Time-of- Flight MS (Leco, Pegasus III, St. Joseph, MI, USA) at 1850V with an acquisition rate of 20 scans per second. Because some terpenes, such as germacrenes, are prone to thermal conversion (Colby et al., 1998; Faraldos et al., 2007) the enzyme assays were also extracted with pentane which was injected at 50°C in the injection port. For this, 500µl lysate was assayed in the presence of 5mM DTT and either (E,E)-FPP, (Z,Z)-FPP, GPP or GGPP as a substrate, overlaid with 2ml pentane (Sigma). After one hour of incubation at 30°C the pentane layer was transferred to a 2ml glass vial and concentrated on ice with nitrogen gas, to a final volume of 50µl. Terpenoids were analyzed by injection of 2µl into the Optic injection port (ATAS GL Int.) at 50°C, subsequently heated to 275°C at a rate of 4°Cs-1 followed by gas-chromatography and mass-spectrometry as described by Bleeker et al. (2009). Boiled protein extract could not convert any of the precursors and was taken along as a negative control. Terpene products were identified using standards
Chapter 2 44 when possible or by comparing mass spectra and Kovats Indices (Adams, 2002). The enzymatic assays for SlTPS31 from cultivar M82 and the analyses of their products were performed as described in Falara et al. (2011).
List of primers used
Primer name Sequence 5' -> 3' SlTPS3_QF ATGGAGACGGACACGGAATTC SlTPS3_QR CAAACTAATTTTGGCTCCTCCATAG SlTPS7_QF GATTCTATTTAAACGGCATGAACATC SlTPS7_QR AGCAGATATAGAGTAGTGATGGAG SlTPS8_QF CCTCTAGGACACAAACATTCTTC SlTPS8_QR ACTATGCATACTGCACCCACTG SlTPS9_QF TCTAAATTTGACACGTGTGGCTG SlTPS9_QR CATCCCAGGAATTAACAACTCATATG SlTPS12_QF CGTTACTGGTTGAGTCTGTCG SlTPS12_QR CTTGATTATTTGAAATATTCCGGAGG SlTPS14_QF1 GGATAATCGAACTATTCATCAAGTG SlTPS14_QR ATGAACCAGCTTCAATACAACTTC SlTPS17_QF GTCATCTCTGATATACTAGTTGATCC SlTPS17_QR GATCTTTGACACGCTCACTCAAC SlTPS31_QF TGCGTTGTTAGTGGACTCTATTG SlTPS31_QR CCTAGCTAGCTACAGATTA SlACT_QF TCAGCACATTCCAGCAGATGT SlACT_QR AACAGACAGGACACTCGCACT
RESULTS
Phylogenetic analysis of the tomato terpene synthase genes
The nearly completed genomic sequence of S.lycopersicum has been recently analyzed and a total of 44 terpene synthase (TPS) genes were identified, out of which 29 appear to have uncompromised open reading frames (Falara et al., 2011). A recent classification of TPS genes divides them into seven clades: TPS-a, TPS-b, TPS-c, TPS-d (gymnosperm-specific), TPS-e/f, TPS-g and TPS-h (Selaginella moellendorffii-specific; Chen et al., 2011). In Figure 1 the phylogenetic tree of
Chapter 2 45 these 29 TPS is presented, grouped in the different clades. Tomato clade TPS-a genes encode mostly sesquiterpene synthases, whereas TPS-b, TPS-e/f and TPS-g genes typically encode monoterpene synthases. Clade TPS-c contains a diterpene synthase (TPS40; CPS1) and a second gene (TPS41) whose function is still unknown (Falara et al., 2011).
Figure 1. Phylogenetic tree of the 29 functional or potentially functional TPS genes in the tomato genome. Alignment and tree was created using Muscle (www.phylogeny.fr). Bootstrap values (1000 replications) are indicated at tree nodes. TPSs described in this chapter are in bold. Numbers in parenthesis indicate chromosome number.
Characterization of TPS-a clade sesquiterpene synthases from S.lycopersicum cv. Moneymaker
Analysis of the genomic sequence of S.lycopersicum showed that the majority of TPS genes in the tomato genome encode sesquiterpene synthases and most of these
Chapter 2 46 putative sesquiterpene synthases belong to the TPS-a clade. In this clade there are seven functional genes: SlTPS9 (previously shown to encode germacrene C synthase (van Der Hoeven et al., 2000), SlTPS12 (previously shown to encode β- caryophyllene/α-humulene synthase; Schilmiller et al., 2010), SlTPS14, SlTPS17, SlTPS31 (TPS31 was formerly known as LeVS2 (GenBank: AAG09949.1), but its function was not established), SlTPS32 and SlTPS36. To investigate in which tissues the active S. lycopersicum sesquiterpene synthases SlTPS9, SlTPS12, SlTPS14, SlTPS17 and SlTPS31 were transcribed (SlTPS32 and SlTPS36 have been described in Falara et al., 2011), we dissected mature S.lycopersicum plants for RNA isolation and subsequent quantitative RT-PCR. Expression of all terpene synthases was lowest in fruits (Fig.2). SlTPS9 and SlTPS17 displayed comparable expression patterns, with highest expression in stem trichomes (Fig.2a and 2d). SlTPS12 expression was highest in leaves (Fig.2b), probably in the leaf trichomes as shown for SlTPS12 in the cultivar M82 (Schilmiller et al., 2010). SlTPS14 was expressed mostly in the roots (Fig.2c). Expression of SlTPS31 was very low overall, but highest in stem trichomes and leaves (Fig.2e), possibly in their trichomes. Since jasmonic acid (JA) treatment resulted in the induction of SlMTS1 (SlTPS5) in stem trichomes (van Schie et al., 2007), we tested whether expression of terpene synthases SlTPS9, SlTPS12, SlTPS17 or SlTPS31 that are expressed in trichomes, was induced by JA treatment. Moneymaker plants that were 4 weeks old were treated with 1mM JA or sprayed with control solution, RNA was isolated after 24 hours, and the levels of transcripts were measured by Q-RT-PCR (Fig.2). Only the expression of SlTPS31 was significantly induced by JA treatment approximately 3- fold, comparable to the positive control SlMTS1 (data not shown). Interestingly, expression of SlTPS17 appeared to be reduced roughly 2-fold in JA-treated plants (p = 0.059).
Chapter 2 47
Figure 2. Tissue specific expression and JA induction of SlTPSs. Relative transcript levels for (a) SlTPS9, (b) SlTPS12, (c) SlTPS14, (d) SlTPS17 and (e) SlTPS31 as determined by Q-RT-PCR. Mean values (+SE) of 3 biological replicas are shown, normalized for Actin expression. L; leaf, WS; whole stem, BS; bald stem, T; stem trichomes, R; root, Fr; fruit, Fl; flower, C; control and JA; jasmonic acid induced stem trichomes. Asterisks indicate significant difference (* p < 0.05) according to T-test. ns; not significant.
Chapter 2 48 Proteins isolated from E.coli cells expressing the putative sesquiterpene synthases were assayed for their ability to convert (E,E)-FPP into sesquiterpenes. The protein encoded by the Moneymaker allele of SlTPS9 produced germacrene C, and minor amounts of germacrene A, B and D (Fig.3a), similar to the protein encoded by this locus in cultivar VFNT Cherry tomato. Recombinant SlTPS12 protein had β- caryophyllene/α-humulene synthase activity (Fig.3b), similar to the protein encoded by CAHS in cultivar M82. SlTPS17 produced mostly valencene from (E,E)-FPP and also an unidentified sesquiterpene, besides azulene and α- and β- farnesenes (Fig.3c). SlTPS31 predominantly made viridiflorene from (E,E)-FPP (Fig.3d).
Figure 3. Enzymatic activity of recombinant SlTPSs assayed with (E,E)-FPP. GC-MS chromatograms of products produced by (a) SlTPS9, (b) SlTPS12, (c) SlTPS17 and (d) SlTPS31 ectopically expressed in E.coli, assayed with (E,E)-FPP. SlTPS9 products were extracted in pentane, SlTPS12, 17 and 31 products were measured by Solid Phase Microextraction (SPME) sampling. Sesquiterpene peaks: 1 germacrene D; 2 germacrene A; 3 germacrene C 4 germacrene B; 5 β-caryophyllene; 6 α-humulene; 7 (E)-β-farnesene; 8 γ- gurjunene; 9 valencene; 10 (E,E)-α-farnesene; 11 viridiflorene; asterisk unidentified. The chromatogram shows the detector response for the terpene-specific ion mass 93 for SlTPS9-12-17 and total ion current for SlTPS31.
Chapter 2 49 Remarkably, all recombinant proteins that could use (E,E)-FPP as substrate could also use (Z,Z)-FPP as substrate (Fig.4). SlTPS9 made mostly germacrene C from both (E,E)-FPP as well as (Z,Z)-FPP. SlTPS12 made curcumene and β-bisabolene from (Z,Z)-FPP and SlTPS17 made various bisabolenes. SlTPS14 could use either (E,E)-FPP or (Z,Z)-FPP equally well to make mostly (Z)-γ-bisabolene or α- bisabolene, respectively (Falara et al., 2011).
Figure 4. Enzymatic activity of recombinant SlTPS9, 12 and 17 assayed with (Z,Z)-FPP. GC-MS chromatograms of sesquiterpenes formed by S. lycopersicum (Moneymaker) terpene synthases ectopically expressed in E. coli, assayed with (Z,Z)-FPP. SlTPS9 products were extracted in pentane, SlTPS12 and SlTPS17 products were measured by Solid Phase Microextraction (SPME) sampling. (a) SlTPS9 (b) SlTPS12 and (c) SlTPS17. Sesquiterpene peaks: 1 α-humulene; 2 germacrene A; 3 β-bisabolene; 4 germacrene C; 5 germacrene B; 6 γ-curcumene; 7 (Z)-γ-bisabolene; 8 (Z)-α-bergamotene; 9 (E)-γ- bisabolene; 10 (E)-α-bergamotene; asterisk unidentified. The chromatogram shows the detector response for the terpene-specific ion mass 93.
The sesquiterpene synthases were assayed for monoterpene synthase activity with GPP as substrate and could all convert this precursor to a range of most simple monoterpenes (Fig.5). SlTPS9 and SlTPS12 produced mostly β-myrcene, limonene
Chapter 2 50 and low amounts of terpinolene. In the assays with SlTPS17 additionally the monoterpenes (Z)-β-ocimene, (E)-β-ocimene and linalool were produced (Fig.5). No products were formed with GGPP as substrate (data not shown).
Figure 5. Enzymatic activity of recombinant SlTPS9, 12 and 17 assayed with GPP. GC-MS chromatograms of monoterpenes formed by S. lycopersicum (Moneymaker) terpene synthases ectopically expressed in E. coli, assayed with GPP. Products were measured by Solid Phase Microextraction (SPME) sampling. (a) SlTPS9 (b) SlTPS12 and (c) SlTPS17. Monoterpene peaks: 1 α-pinene; 2 β-myrcene; 3 δ-2-carene; 4 limonene; 5 α-terpinene; 6 terpinolene; 7 (E)-β-ocimene; 8 (Z)-β-ocimene; 9 γ-terpinene; 10 linalool; asterisk unidentified. The chromatogram shows the detector response for the terpene-specific ion mass 93.
Characterization of TPS-b clade monoterpene synthases from S.lycopersicum cv. Moneymaker
Homology searches of SlMTS1 on chromosome 1 of Solanum lycopersicum (http://solgenomics.net/) led to the discovery of seven additional monoterpene synthases (SlTPS1, 2, 3, 6, 7, 8 and 22) that all belong to clade b of the terpene synthases (TPS). In total, there have been 13 monoterpene synthases identified in TPS-b (Falara et al., 2011). SlMTS1 and SlMTS2 (renamed SlTPS5 and SlTPS4)
Chapter 2 51 have been previously characterized (van Schie et al., 2007). Eight more were non- functional or mutated (Falara et al., 2011), leaving SlTPS3, SlTPS7 and SlTPS8 on chromosome 1 to be further characterized. Q-RT-PCR was performed on cDNA from different S. lycopersicum cv. Moneymaker tissues in order to establish the expression pattern of these monoterpene synthases. SlTPS3 was predominately expressed in isolated stem trichomes and was also significantly induced by JA (Fig.6a). SlTPS7 was present in all the tissues examined except fruits, with highest expression in flowers and leaves and was not significantly induced by JA (Fig.6b). Finally, SlTPS8 was predominately expressed in roots and was not induced by JA in the trichomes (Fig.6c).
Figure 6. Tissue specific expression and JA induction of SlTPSs. Relative transcript levels for (a) SlTPS3, (b) SlTPS7 and (c) SlTPS8 as determined by Q-RT-PCR. Mean values (+SE) of 3 biological replicas are shown, normalized for Actin expression. L; leaf, WS; whole stem, BS; bald stem, T; stem trichomes, R; root, Fr; fruit, Fl; flower, C; control and JA; jasmonic acid induced stem trichomes. Asterisks indicate significant difference (* p < 0.05) according to T-test. ns; not significant.
Chapter 2 52 The enzymatic activity of the proteins was investigated in crude E.coli extracts. Recombinant SlTPS3 catalyzed the formation of β-myrcene, terpinolene and limonene as well as camphene, geranial and linalool when using GPP as substrate. The same products were produced when using NPP as substrate except geranial and linalool (Fig.7a). SlTPS7 recombinant protein produced β-myrcene and limonene as well as (dihydro) carveol and linalyl-acetate (the acetate ester conversion of linalool) from GPP. When provided NPP as substrate, SlTPS7 produced the same compounds as with GPP including two unidentified terpenes (Fig.7b). Finally, the full-length SlTPS8 cDNA was also expressed in E.coli and the resulting protein was found to produce 1,8-cineole, as well as β-myrcene and limonene from GPP and NPP (Fig.7c).
Figure 7. Enzymatic activity of recombinant SlTPSs. GC-MS chromatograms of products produced by (a) SlTPS3, (b) SlTPS7 and (c) SlTPS8 ectopically expressed in E.coli,
Chapter 2 53 assayed with GPP and NPP and measured by Solid Phase Microextraction (SPME) sampling. Monoterpene peaks: 1 camphene; 2 β-myrcene; 3 limonene; 4 terpinolene; 5 geranial; 6 linalool; 7 (dihydro) carveol; 8 linalyl-acetate; 9 1,8-cineole; asterisk unidentified. The chromatogram shows the detector response for the terpene-specific ion mass 93.
Both (E,E)-FPP and (Z,Z)-FPP could be used as substrate by SlTPS7, which produced (E)-α-bergamotene, (E)-β-farnesene and (E,E)-α-farnesene with the canonical FPP and (Z,Z)-α-farnesene with zFPP (Fig.8a). SlTPS8 could only use (Z,Z)-FPP as substrate, producing (Z)-α-bergamotene, γ-curcumene and α- bisabolene (Fig.8b). SlTPS3 did not form any products when assayed with (E,E)- and (Z,Z)-FPP and none of the monoterpene synthases tested could use GGPP as substrate (data not shown).
Figure 8. Enzymatic activity of recombinant SlTPSs. GC-MS chromatograms of products produced by (a) SlTPS7 and (b) SlTPS8 ectopically expressed in E.coli, assayed with zFPP or FPP and measured by Solid Phase Microextraction (SPME) sampling. Sesquiterpene peaks: 1 (Z,Z)-α-farnesene; 2 (E)-α -bergamotene; 3 (E)-β-farnesene; 4 (E,E)-α-farnesene; 5 (Z)-α-bergamotene; 6 γ-curcumene; 7 α-bisabolene. The chromatogram shows the detector response for the terpene-specific ion mass 93.
Chapter 2 54 An overview of all the products produced by the TPSs when assayed with the various precursors is given in Table 1.
Table 1. TPS products of in vitro enzymatic assays. Terpene products of the various TPSs assayed with GPP, NPP, FPP or zFPP. GGPP was assayed for all TPSs except SlTPS31 and could not be used as substrate by any of the enzymes. ND; not detected, NA; not assayed.
TPS nr GPP NPP FPP zFPP 3 camphene camphene ND ND
limonene limonene terpinolene terpinolene geranial linalool 7 (E) (Z,Z) limonene (dihydro)carveol (E) (dihydro)carveol linalyl-acetate (E,E)- linalyl-acetate 8 ND (Z) limonene limonene 1,8-cineol 1,8-cineol 9 NA germacrene D germacrene A germacrene C germacrene A limonene germacrene B germacrene C germacrene B terpinolene 12 NA limonene (E) (Z) terpinolene 17 NA (Z) limonene valencene (Z) (E) (E,E) (E) (Z)
linalool terpinolene 31 NA NA viridiflorene NA
DISCUSSION
The presence and biosynthesis of monoterpenes and sesquiterpenes in S.lycopersicum has been previously investigated (Colby et al., 1998; van Der Hoeven et al., 2000; van Schie et al., 2007; Bleeker et al., 2009; Schilmiller et al., 2010). Van Schie et al. (2007) used a degenerate primer approach to identify two
Chapter 2 55 monoterpene synthases (LeMTS1 and LeMTS2, renamed SlTPS5 and SlTPS4). SlTPS5 catalyzed the formation only of linalool from GPP and (E)-nerolidol from FPP, whereas SlTPS4 generated several monoterpene products from GPP and had no sesquiterpene synthase activity. Colby et al. (1998) isolated a cDNA from the cultivar VNFT Cherry and showed that it encodes germacrene C synthase and van der Hoeven et al. (2000) showed that synthesis of germacrene C, β-caryophyllene and α-humulene was controlled by a locus on chromosome 6. Using the VFNT cDNA of germacrene C as a probe to screen cDNA libraries, van der Hoeven et al. (2000) also isolated two cDNAs, designated SSTLE1 and SSTLE2, that were very similar to VNFT germacrene C. Based on the sequences of both cDNAs, we can conclude that they are different alleles (or one of them may be a cloning artifact) of SlTPS9, which we now know to be located on chromosome 6 (Falara et al., 2011). Subsequently, Schilmiller et al. (2010), using a proteomic approach, identified a protein sequence in S.lycopersicum cv. M82 trichomes that catalyzed the formation of β-caryophyllene and α-humulene, and was therefore designated as CAHS (β- caryophyllene/α-humulene synthase). Our data show that SlTPS12 encodes CASH. With the recent mining of the tomato genome more TPSs were identified, some of which have been characterized here. The primarily root-expressed SlTPS14 (Fig.2c) was shown to encode a bisabolene synthase (Falara et al., 2011). Bisabolene has been reported before to be the product of root (-specific) sesquiterpene synthases (Ro et al., 2006; Köllner et al., 2008; Valdez-Calderon et al., 2011). In Arabidopsis, AtTPS12 and AtTPS13 form mainly (Z)-γ-bisabolene and are shown to be wound-inducible, suggesting an involvement in the protection of the Arabidopsis roots from bacterial and/or fungal infections. However, (Z)-γ- bisabolene could not be detected in Arabidopsis tissues or headspace and it was suggested that it could be escaping detection due to minute amounts or further metabolized (Ro et al., 2006). In maize, the β-bisabolene synthases ZmTPS6 and ZmTPS11 are shown to be expressed both in roots and leaves and induced in the roots by herbivore damage in the leaves. Furthermore, β-bisabolene is only
Chapter 2 56 detected in the roots, suggesting a long-distance signaling cascade (Köllner et al., 2008). In S. lycopersicum bisabolene has not been detected, at least in the headspace or leaf extracts (Bleeker et al., 2009). Here, we have only checked induction of TPS expression by JA (a hormone that induces defenses similar to those of wounding) in stem trichomes, where the bisabolene synthase SlTPS14 is not expressed, but it could be conceived that in tomato it has a similar function as in Arabidopsis or maize. SlTPS17 gave multiple products, but mainly valencene (Fig.3c), which is typically a flavor and aroma compound of citrus plants (Sharon- Asa et al., 2003). SlTPS17 is almost exclusively expressed in the trichomes and was down-regulated by JA, although not at a statistically significant level (p=0.059; Fig.2d). Even though nothing has been reported about an effect of valencene in the tomato defense system, it has been shown that transgenic Citrus sinensis fruit peel with a reduced accumulation of limonene was less attractive to the citrus pest medfly compared to control fruit (Rodriguez et al., 2011). It is not improbable that terpene biosynthesis is down-regulated as part of the plant’s response to pests and pathogens. Finally, SlTPS31, which is a very lowly expressed gene in all the tissues (Fig.2e), makes viridiflorene as a single product (Fig.3d). Viridiflorene has been described before as part of the essential oil mixture from various plant species like Atriplex, exhibiting, as a mix, antioxidant and cytotoxic activity (Ho et al., 2010; Rodriguez et al., 2010; Beattie et al., 2011). SlTPS31 was up-regulated in stem trichomes by treatment with JA, indicating a potential role in the plant’s induced defenses.
Interestingly, the sesquiterpene synthases tested here (SlTPS9, 12 and 17) accepted both (E,E)-FPP (Fig.3) and (Z,Z)-FPP (Fig.4) as substrates. Our observations of sesquiterpene synthases reacting with substrates other than the canonical (E,E)-FPP adds additional weight to previous intimations of the flexibility of these enzymes. For example, Jones et al. (2011) reported a cytosolic sequiterpene synthase from sandalwood able to use both (E,E)- and (Z,Z)-isomers of FPP to produce similar compounds. Such observations from multiple species suggest that these properties
Chapter 2 57 of the enzyme are not in vitro artifacts, but might have in vivo relevance. However, whether (Z,Z)-FPP is available in the cytosol of the Solanum trichomes is not yet known, although evidence has been presented that S.habrochaites accession LA1777 produces (Z,Z)-FPP in the plastids (Sallaud et al., 2009). The sesquiterpene synthases described here were also able to convert GPP to (mono) terpene products (Fig.5) indicating a level of plasticity of the active pocket of the protein. While some sesquiterpene synthases are restricted to the use of a single substrate even with regard to precursors of the same size ((E,E)-FPP or (Z,Z)-FPP; Besser et al., 2009), there are examples of terpene synthases that can accommodate both GPP as well as (E,E)-FPP in a productive manner (van Schie et al., 2007) or even GGPP as a third possible substrate (Arimura et al., 2008; Martin et al., 2010). It has been proposed that trace amounts of GPP are present in the cytosol, and minor amounts of (E,E)-FPP are available to plastid-localised terpene synthases (Aharoni et al., 2006; Wu et al., 2006). Hence, the active site plasticity of some TPS to accommodate isoprenyl diphosphates of different chain length may be biologically relevant.
The newly identified monoterpene synthase SlTPS3 seems to be less promiscuous enzyme, as it can only accept GPP or NPP, the (Z)-isomer of GPP, as substrates. SlTPS3, expressed primarily in the trichomes, was significantly induced by treatment with JA. In vitro, recombinant protein produced β-myrcene, limonene, terpinolene, camphene, geranial and linalool. Although in tomato emission of β- myrcene and limonene have not been correlated with repellence to whiteflies (Bleeker et al., 2009) or spider mites (Ament et al., 2004), for example in Arabidopsis, myrcene is one of the (minor) constituents of AtTPS03 recombinant protein product-mix and it is shown that AtTPS03 is up-regulated in leaves as a response to mechanical wounding and treatment with JA (Fäldt et al., 2003). It is therefore possible that terpinolene or camphene, or perhaps more likely a blend of all the produced compounds, plays a role in the plant’s defenses. In Cupressus lusitanica (white cedar) cell cultures elicited with a yeast extract, β-thujaplicin was
Chapter 2 58 one of the major components of the ether extract of the cells. It was shown that the time course for terpinolene synthase activity coincided with β-thujaplicin biosynthesis, indicating that most of terpinolene is metabolized to β-thujaplicin rather than emitted (De Alwis et al., 2009). Hence it is also conceivable that the produced compounds of SlTPS3 (or other TPSs) are further metabolized in planta, and it is these final products that exhibit a role in the plant’s defense. SlTPS7, which is expressed in various tissues, catalyzes the formation of β-myrcene and limonene from GPP as shown before (Falara et al., 2011), however in this assay it also catalyzed the formation of (dihydro) carveol and linalyl-acetate. (Dihydro) carveol or its simpler counterpart, carveol, was most likely formed as a product of limonene hydroxylation (McCaskill and Croteau, 1997) and linalyl-acetate could be conversion of the precursors by the crude extracts. Purified protein was not tested. Interestingly, SlTPS7 could also use (E,E)- and (Z,Z)-FPP as substrates, producing an array of farnesenes as well as (E)-α-bergamotene. SlTPS8 is expressed mainly in the roots and produces 1,8-cineole, β-myrcene and limonene from GPP and NPP. 1,8-cineol is a compound reported before to be either formed directly by a root (-specific) terpene synthase (Chen et al., 2004) or be part of essential oil mixtures from roots (Shafaghat et al., 2006). In Arabidopsis it was shown that 1,8-cineole was released from the roots after bacterial infection or damage by a root-feeding insect (Steeghs et al., 2004). Here we focused on induction of TPSs in stem trichomes, where SlTPS8 shows minimal expression, and treatment with JA did not result in up-regulation of this gene in this tissue (Fig.6c), however it is possible that induction occurs in the roots. SlTPS8 could produce also sesquiterpene products when assayed with (Z,Z)-FPP.
Only some of the terpene products of the analyzed TPSs have been previously detected in the headspace of undamaged Moneymaker plants (Bleeker et al., 2009). Camphene, 1,8-cineole, γ-gurjunene, valencene and viridiflorene have not been previously reported to be emitted by Moneymaker plants, although for example α-
Chapter 2 59 gurjunene has been measured from the emissions of S.lycopersicum cv. Castlemart undamaged plants (Ament et al., 2004). Often the presence of many, but not all of the terpenes observed in the respective glands can be explained by detection of terpene synthase activity in the same tissue. Since some terpenes are made by more than one terpene synthases in the same species, or conversely, since one terpene synthase can make more than one product even from a single precursor, it is difficult to determine the direct contribution of each of them to the observed mixture even when the expression levels of individual terpene synthases are examined in detail, and it is also challenging to estimate the conditions and timing under which each product is synthesized and can be detected in planta.
When terpene biosynthesis is seen as a whole, it is noteworthy that despite the fact that monoterpenes appear to dominate S.lycopersicum volatile emissions (Buttery et al., 1987; Ament et al., 2004; Bleeker et al., 2009), the majority of TPS genes mined from the tomato genome are sesquiterpene synthases (Falara et al., 2011). In the S. lycopersicum cv. Moneymaker stem trichome transcriptome (see Chapter 5) several SlTPSs can be identified, most of which encode sesquiterpene synthases (Table 2). However, the majority of terpenes present in Moneymaker trichomes are monoterpenes (Table 3). Although transcript abundance need not be translated into protein abundance, another explanation for the low sesquiterpene content of S.lycopersicum might be found in low precursor biosynthesis. Quantitative RT- PCR on cDNA derived from stem trichomes of S.lycopersicum and S.habrochaites LA1777, which produces copious amounts of sesquiterpenes, showed that expression of HMG-CoA reductase, a key enzyme in the MEP pathway, is approximately 7-fold higher in S. habrochaites (Besser et al., 2009).
Chapter 2 60
Table 2. Transcripts of various terpene synthases found in S.lycopersicum cv. Moneymaker stem trichomes, fold JA-induction and the enzymes they encode. b; van Schie et al., 2007, c; Colby et al., 1998, d; Schilmiller et al., 2009, e; Falara et al., 2011, ND; not detected.
TPS nr Contig nr Transcript Reads JA induction Main product(s) of in vitro enzymatic assay length (bases) trichome (fold) 3 4235 2099 13 7 see Table 1 5 (MTS1) 19389 2186 263 2.3 R-linaloolb 7 12366 423 1 3 see Table 1 8 10432 264 442 - see Table 1 9 (SST1) 3548 2011 13286 - germacreneCc 12 17073 407 15 - a 16 10687 1868 95 - ND 17 10174 1190 20 - see Table 1 19 14448 776 211 - ND 20 (PHS1) 10263 1148 602 - d 24 (KS) 23265 854 1 - ent-kaurenee 31 4341 1991 1 4 see Table 1 39 10191 1131 32 2.2 linaloole 41 3672 2368 375 - ND
Table 3. Sesqui- and mono-terpenes present in trichomes of S.lycopersicum cv. Moneymaker (compiled from Bleeker et al., 2009).
Sesquiterpenes Monoterpenes azulene 2-carene caryophyllene 4-carene linalool limonene germacrene C germacrene D cuparene
terpinolene p-cymene pinene
*could be produced by conversion of germacrene
Jasmonic acid (JA) is a hormone known to be involved in plant defenses (Ballare, 2011). In cultivated tomato, it has been shown that expression of SlMTS1 (SlTPS5; van Schie et al., 2007) and emission of TMTT, a homoterpene derived from geranyllinalool (Ament et al., 2006), increase approximately 2-fold upon treatment with JA. Here we have tested the effect of JA by Q-RT-PCR on all the analyzed terpene synthases that showed expression in trichomes. Only two terpene synthases (SlTPS3 and SlTPS31) were induced by the treatment and one (SlTPS17) seemed to be down-regulated by JA, although not at a statistically significant level (discussed
Chapter 2 61 earlier). Furthermore, we were able to obtain an overview of the trichome- expressed SlTPSs and their regulation or not by JA (Table 2). The data were acquired by expression profiling (Illumina GA II) of JA-treated and control stem trichomes of 4-week-old Moneymaker plants and are described more analytically in Chapter 5. Interestingly, out of the 14 terpene synthases found in the stem trichome transcriptome only five are induced, out of which four produce monoterpenes.
In conclusion, the release of the tomato genomic sequence allowed the identification of (all) terpene synthases and the characterization of the respective genes and proteins. Here we focused on a subset of SlTPS that we cloned from cultivar Moneymaker, focusing mostly on those found in the trichomes. We analyzed the gene expression patterns, investigated whether they are induced by JA and made recombinant proteins to test the enzymatic activity of the TPSs in vitro. We were able to correlate the expression of the synthases with the products formed in the trichomes for a few terpenes and we showed that many could also use precursors other than the canonical ones to make various products. It goes without saying that as a community we are only starting to understand the tremendous complexity and variety of the tomato terpene synthase family.
ACKNOWLEDGEMENTS
We thank Harold Lemeris, Ludeck Tikovsky and Thijs Hendrix for the excellent care of the tomato plants.
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Chapter 2 68 Chapter 3
A yeast-one-hybrid screen identifies a putative transcription factor that interacts with a tomato terpene synthase promoter
Eleni A. Spyropoulou, Michel A. Haring and Robert C. Schuurink
Department of Plant Physiology, Swammerdam Institute for Life Sciences, University of Amsterdam, Science Park 904, 1098 XH Amsterdam, The Netherlands
ABSTRACT
Volatile terpenes are well known for their role in plant defenses. Glandular trichomes of tomato produce an array of such terpenes for some of which their involvement in plant-herbivore interactions has been established and the respective terpene synthases have been identified. In order to unravel the transcriptional regulation of the Solanum lycopersicum Monoterpene Synthase 1 (SlMTS1) gene in glandular tomato trichomes we functionally dissected its promoter. The JA- responsive region of the promoter was identified and a 207bp promoter fragment appeared to be sufficient for trichome-specific expression. With this fragment a yeast-one-hybrid screen was performed. The most abundant interactor is a nuclear- localized putative transcription we named Emission of Terpenes (SlEOT1).
INTRODUCTION
In tomato, the first monoterpene synthase identified (SlMTS1; van Schie et al., 2007) was shown to be expressed specifically in trichomes and trichome- containing tissues. Spider mite feeding, artificial wounding and jasmonic acid (JA) application induced expression of SlMTS1. Recombinant protein could produce (R)-linalool with GPP and (E)-nerolidol with FPP as substrate. Furthermore, linalool and nerolidol were shown to predominantly accumulate in trichomes but wounded or JA-treated plants emitted higher amounts only of linalool, which correlated with SlMTS1 transcript abundance. Plants overexpressing SlMTS1 under control of the CaMV 35S promoter emitted hundred-fold more linalool, whereas
Chapter 3 70 emission of other terpenes, including nerolidol, was not affected, indicating that SlMTS1 functions in planta as a true linalool synthase (van Schie et al, 2007). However nothing is known about the regulation of the tissue-specific and hormone- regulated expression of this gene. As in all eukaryotes such regulatory mechanisms involve sequences that flank the gene in question containing cis-regulatory elements (promoters) and proteins that recognize and can interact with these elements (trans-acting factors; Kristiansson et al., 2009). In general, a eukaryotic promoter consists of two parts. A basal part (core promoter) that contains the TATA box and the transcription start site (TSS) where the transcription machinery (RNA polymerase II holoenzyme complex) is assembled and a part that contains various protein binding sites that confer specificity of transcription (Lee and Young, 2000). Such motifs are usually short (typically ranging from four to eight nucleotides) and therefore abundant, as they can be found in the genomic sequence in a randomized fashion (Stone and Wray, 2001). Most of these motifs do not act as true binding sites for a variety of reasons, like absence of an active transcription factor or chromatin condensation (Wray et al., 2003), therefore validation of sites that can bind proteins requires experimentation. Such in vitro validation methods commonly involve footprinting, missing nucleoside or electrophoretic mobility shift assays. Footprinting assays entail the radioactive labeling of a DNA fragment followed by its DNase I degradation in the presence or absence of a binding protein. Electrophoresis of the complex and autoradiography in combination with Maxam-Gilbert sequencing then allows the identification of the protective “footprint” of the protein on the DNA sequence (Galas and Schmitz, 1978). In a similar manner, missing nucleoside experiments treat radioactively labeled DNA molecules with the hydroxyl radical thus randomly removing nucleosides, which affects the ability of a protein to bind to the gapped DNA (Hayes and Tullius, 1989). Although such techniques have been useful in identifying promoter motifs (e.g. Boter et al., 2008), electrophoretic mobility shift assay (EMSA) has emerged as the method of choice to detect protein-nucleic acid
Chapter 3 71 interactions as, although not without its limitations, it is a relatively simple, sensitive and fast technique (Hellman and Fried, 2007). It involves separating a radioactively labeled DNA fragment from DNA-protein complexes through electrophoresis on a non-denaturing polyacrylamide gel (Garner and Revzin, 1981). Validation of the DNA binding site can further be achieved by mutating the motif in question. However, in vitro assays fail to take into account the influence of chromatin structure on protein binding or transcription (Wray et al., 2003), nor does transcription factor-DNA interaction in vitro necessarily imply transcriptional activation in vivo. For example, Antirrhinum Myb305 transcription factor was shown by EMSA to bind to the promoter of the chalcone synthase gene involved in anthocyanin biosynthesis, however Myb305 failed to activate the same promoter in a yeast-one-hybrid assay (Moyano et al., 1996). In vivo assays provide a more relevant image of transcription factor (TF)- DNA interactions. Such validation methods commonly involve yeast-one-hybrid (Y1H) assays or fusion of the DNA fragment to a reporter gene and transiently or stably assaying its transcription in the presence of a TF of interest. One-hybrid screening in yeast entails the detection of an interaction between a transcription factor (prey) fused to an activation domain (AD) and a bait DNA fragment driving expression of a reporter gene. Y1H minimizes limitations of in vitro assays, like lack of post- transcriptional modifications or incorrect protein folding (Luo et al., 1996) and since the DNA-binding proteins are expressed as a fusion to an AD, transcriptional repressors as well as activators can be identified (Vidal and Legrain, 1999). Transient assays for transactivation of a promoter fragment by a transcription factor in planta or the more laborious reporter gene assays in stable transformants, provide the opportunity to test the effect of nucleotide substitutions in the binding site, swapping or altering orientation of binding sites and are therefore quite informative about binding site function in vivo (Wray et al., 2003). Finally, it is worth mentioning that advances in technology have enabled genome- wide identification of DNA-binding sites of a particular protein of interest in vivo, although such techniques can generate also a large number of false positives (Qin
Chapter 3 72 et al., 2011) and therefore identified binding sites would have to be validated as true targets of the TF with a method like those mentioned above. These techniques involve chromatin immunoprecipitation (ChIP): cross-linking of the protein of interest (TF) to the DNA in vivo and purifying the DNA-protein complexes using a specific antibody against the protein, followed by micro-array hybridization (ChIP- CHIP) or deep sequencing (ChIP-Seq; Kaufmann et al., 2010). Developed methodologies for characterizing TF DNA-binding specificities play a crucial role in deciphering the complexity of transcriptional regulation. Here, we isolated a part of the 5’ genomic sequence flanking the SlMTS1 gene in order to unravel its regulation at the transcriptional level. By creating 5’ sequential promoter deletion constructs driving expression of reporter genes in transgenic tomato plants we were able to identify the trichome-specific and JA-responsive regions of this promoter. Yeast-one-hybrid screens with the trichome-specific 207bp SlMTS1 promoter fragment identified a putative transcription factor.
MATERIALS AND METHODS
Constructs and Stable Plant Transformations
1254bp of the genomic sequence upstream from the start codon of SlMTS1 (AY840091) were cloned between restriction sites SacI (at the 5’ end of the sequence) and XbaI (at the 3’ end of the sequence just prior to the ATG) replacing the 35S promoter in vector pJVII, a pMON999 vector (Monsanto, St. Louis, MO) with a modified multiple cloning site, upstream of the ATG start codon of uidA (GUS) fused to yellow fluorescent protein (sYFP1) and followed by the Nos terminator (tNos). Five 5’ deletion constructs were generated by designing forward
Chapter 3 73 primers at positions -1046, -806, -612, -408 and -207 distal to the ATG and a reverse primer at position -18. These five PCR products were cloned in vector pJVII using the same restriction sites as for the full-length promoter and after verifying by sequencing, the expression cassettes were transferred to the binary vector pBINplus (van Engelen et al., 1995) by digesting with restriction enzymes SacI and SmaI and ligating at the same restriction sites in the multiple cloning site (MCS) of pBINplus. These six constructs and an empty pBINplus vector were introduced to Agrobacterium tumefaciens strain EHA105 and used to create transgenic plants using explants derived from cotyledons of sterile seedlings of Solanum lycopersicum cultivar Moneymaker, as previously reported (Cortina and Culianez-Macia, 2004).
Analysis of Transgenic Plants
One transgenic line was obtained with the empty pBINplus vector, four independent transgenic lines with the full-length (1254bp) SlMTS1 promoter construct, three with the 1046bp SlMTS1 promoter construct, five with the 806bp and 612bp SlMTS1 promoter constructs, eight with the 408bp SlMTS1 promoter construct and nine with the 207bp SlMTS1 promoter construct. Presence of the transgene was verified by PCR (target gene GUS) on genomic DNA isolated from leaves of the different T0 lines. YFP expression was checked under a fluorescence stereomicroscope. Trichomes were collected at the bottom of a 50ml tube by vortexing stem pieces frozen in liquid nitrogen. Crude extracts were prepared according to Jefferson et al. (1987) and the enzymatic GUS activity was determined spectrophotometrically using 4-methylumbelliferyl β-D-glucuronide (MUG) as a substrate. Values were corrected for protein content, measured using Bradford reagent.
Chapter 3 74 Hormone Treatment, RNA Isolation and cDNA synthesis
Tomato plants were grown in soil in a greenhouse with day/night temperatures of 23oC/18oC and a 16/8 h light/dark regime for 4 weeks. Jasmonic acid (JA; Duchefa, NL) was applied to plants by spraying 1mM solution made with tap water containing 0.05% SilwetL-77 (GE Silicones, VA, USA). Control plants were sprayed with tap water containing 0.05% SilwetL-77. Trichomes were collected 24h later at the bottom of a 50ml tube by vortexing stem pieces frozen in liquid nitrogen. The material was ground and total RNA was isolated using Trizol (Invitrogen, Paisley, UK). DNA was removed with DNAse (Ambion, Huntingdon, UK) according to the manufacturer and cDNA was synthesized from 1.5μg RNA using M-MuLV H- Reverse Transcriptase (Fermentas, St. Leon-Rot, Germany).
Quantitative Real Time PCR
For Q-RT-PCR cDNA equivalent to 100ng total RNA was used as template in 20μl volume and reactions were performed in the ABI 7500 Real-Time PCR System (Applied Biosystems) using the Platinum SYBR Green qPCR SuperMix-UDG kit (Invitrogen, Paisley, UK) with the following cycling program: 2min 50oC, 7min 95oC, 45 cycles of 15sec at 95oC and 1min at 60oC, followed by a melting curve analysis. Primer pairs were tested for amplification kinetics and linearity with a standard cDNA dilution curve and new primers were designed if necessary. Expression levels were normalized using ACTIN (SGN-U579547) mRNA levels. Effect of JA in gene expression was analyzed in three biological replicates by T- test using PASW Statistics 17.0 (http://www.spss.com). The homogeneity of variance was tested by Levene’s test and values were log transformed before the analysis if necessary.
Chapter 3 75 Yeast-One-Hybrid Screens
In order to be used as target binding sequence in yeast-one-hybrid screens, the 207bp SlMTS1 promoter fragment was cloned between restriction sites EcoRI (at the 5’ end of the sequence) and XbaI (at the 3’ end of the sequence just prior to the ATG) in the MCS of vector pHISi (Clontech, Mountain View, CA, USA) upstream of the minimal promoter of the HIS3 locus driving HIS3. The HIS3 gene of pHISi was used as selectable marker for integration into the nonfunctional his3 locus of yeast strain PJ69-4A. The vector was linearized at the XhoI restriction site before integration. Leaky HIS3 expression enabled enough colony growth, making it possible to use as a selectable marker. Background growth was controlled during library screening by the addition of 5mM 3-amino-1,2,4-triazole (3-AT) in the selective medium (synthetic dextrose, SD). For the construction of the cDNA library used, trichomes were shaken off frozen stem pieces of tomato S.lycopersicum cv Moneymaker, RNA was isolated using the RNeasy Plant kit from Qiagen (Valencia, CA, USA) and the following steps were executed according to the instructions of the HybriZAP−2.1 XR library construction kit and HybriZAP− 2.1 XR cDNA synthesis kit from Stratagene (Cedar Creek, Texas, USA). The size of the primary cDNA library was 3.0 × 106 pfu. The primary library was subsequently amplified and mass excised as described by the manufacturer. Screening of the library was performed using 20μg library plasmid according to the Clontech Matchmaker One-Hybrid System user manual. Transformation efficiency was on average 105 colony forming units (cfu) per μg DNA. Positive colonies were transferred on medium containing higher 3-AT concentration (10mM and 15mM) and plasmid was isolated and sequenced from colonies that could grow on the stringent selection medium.
Chapter 3 76 Gateway Cloning and Confocal Microscopy
Gateway technology (Invitrogen, Paisley, UK) was used for the construction of the CaMV 35S-driven SlEOT1 - fluorescent protein fusion. The SlEOT1 ORF excluding the stop codon was PCR-amplified introducing the AttB recombination sites and subsequently recombined in pDONR207 (BP reaction; Invitrogen, Paisley, UK) creating SlEOT1-pENTR, which was verified by sequencing. For the C-terminal fusion of mVenus (Nagai et al., 2002), SlEOT1-pENTR was recombined in a destination vector (LR reaction; Invitrogen, Paisley, UK) along with pGEM Box1 CaMV 35S and pGEM Box3 mVenus-tNos. The construct was transformed to Agrobacterium tumefaciens GV3101 (pMP90). Agroinfiltration of 5-week-old N.benthamiana leaves was performed as described by Verweij et al. (2008). A separate A.tumefaciens GV3101 (pMP90) culture harboring mCherry with a N-terminal nuclear localization signal (NLS) followed by tNos (Galvan- Ampudia, Xu and Testerink; unpublished) was co-infiltrated in a 1:1 (v/v) ratio. Cells were observed 48h later with a Zeiss LSM 510 inverted microscope. mVenus fluorescence was monitored with a 520-555nm band pass emission filter (514nm excitation) and the mCherry fluorescence was monitored with a 585-615nm band pass emission filter (561nm excitation). Images were taken with an open pinhole and processed by ImageJ (http://rsb.info.nih.gov/ij/).
List of primers used
Primer name Sequence 5' -> 3' SlMTS1_QF GTATCTGCATAGCGATGTTAAAAGTAG SlMTS1_QR TTCTAAATTCGACTTTATGGAGATACG GUS_QF TTACAGGCGATTAAAGAGCTGATAG GUS_QR TTCGTTGGCAATACTCCACATC SlACT_QF TCAGCACATTCCAGCAGATGT SlACT_QR AACAGACAGGACACTCGCACT SlEOT_attB1 GGGGACAAGTTTGTACAAAAAAGCAGGCTatgGCTAATTTCTTTTCATTAG SlEOT_attB2 GGGGACCACTTTGTACAAGAAAGCTGGGTAaggagatcttgaaggtggaaa
Chapter 3 77 RESULTS
Identification of the SlMTS1 promoter trichome-specific fragment
The Monoterpene Synthase 1 from Solanum lycopersicum (SlMTS1) has been previously described as a linalool synthase, expressed in tissues containing trichomes (van Schie et al., 2007). Genomic sequence of 1254 base pairs upstream of the start codon ATG, designated here as the full-length promoter, were cloned in front of β-glucuronidase (GUS) fused to yellow fluorescent protein (sYFP1). Five 5’ deletion constructs were generated by designing primers at positions -1046, -806, -612, -408 and -207 distal to the ATG (Fig.1). These six constructs plus an empty vector were stably transformed in tomato Moneymaker plants. YFP expression for the full-length SlMTS1 promoter was evaluated in T0 stem and leaf pieces and T1 seedlings under a fluorescence stereomicroscope and was specific for the four “head” cells of the type VI glandular trichomes (Fig.2). For quantification of GUS expression five plants of each independent T1 line were grown in soil for four weeks. Expression of the transgene was verified by YFP observation, one plant was chosen from each line and the enzymatic activity of GUS was determined in isolated stem trichomes spectrophotometrically using 4- methylumbelliferyl β-D-glucuronide (MUG) as a substrate and corrected for protein concentration (Fig.3). Trichome-specific expression was low in plants with the 408bp promoter:GUS fusion fragment, but was then partially restored at the 207bp fragment, indicating the existence of a repressor element. Expression directed by the 207bp fragment remained specific for the type VI glandular trichome “heads”, albeit less strong than that of the full-length promoter.
Chapter 3 78 -1254 GTTTCATTCAAAGTAGTGGTGTCCAATACCAATTATGTATTGCTAATGAA -1204 TGTAACACGTTGATGAATAGTATCCAATATATTGGAAAGTCATAGTTTGA -1154 GTTCCAACATCTAAAAGATATATTAGTTTAAAACATTATAAAAGGTGTGA -1104ATCAACAACACAGGAATGGTGAGGGTGTGTGTCTCTCAACTGCTGCCAAA -1054 ATTAAATTAGCTGAACCAAATCCCAATACTACCCGACGTTCAGGGAATTA -1004 AAAACCTACTACCTGCAATTTTCAATATTTTCACTCCATCGATAATAATT -954 ACGTGGTATATATATATATAATGGTTGAGATTTAAAATCTGTTTGGATCT -904 AGTTGTTTTAGGCATTTTTATTTAAATAACATTTAAATATTTTTATAATA -854 TCAATTAAATATTTTTAAAGTTAAAATAAATTAACTAGATTGCAAATTGT -804 CCTATTTTTTCCATATTGAAATAATGGTTGCGCAAGGAAGCGTTGAAATA -754 TTTATGAGTTCACCTGTATTCAGTGTTGAAATCGTTGTAGAAAGCAAGTA -704 GGACTCTATAGCATTGTGACTTTTTTTACCAAAACTAGGTTCATTAATTT -654 CTACTCTAACATCGAATTTGAGTCATGGAGTTACTTGAATCAATCAACAG -604 TATTAAATGTGCTTCATCAATATTACTAATAAATACTATCACCTATAGAG -554 GCATTATCAAACATGTATGTATATATGAGAAGCTTTCTAGTACAACAATT -505 TTCTTCGTAAAATTTATTCGCATCGGGTATTTATACTAAGTCAATACATG -454 CATATCATTTGTTTGAATTTATAATTTATTTTACTTCATTAAATCTAGTA -404 ATAATGAAAATCGCATCGAATTGGTATAAACATCCGATACGAGTAAGTGT -354 AAACACATATACACATGCAAAAACAGTGGTGGAGCTATAATTTGGTCCAA -304 GGGGTGTATAAATATAAAGAAATAAAATCGTGTAGAAGTCAAAGACTGTC -254 AGTACACCCTTCACTGTGGCTCCGCCACCGTGCACAAATATTTTAGCACA -204 TGTGCTATTTTTATGCTACACATAGTATATGTGCTATTTTTATGCTATAT -154 TTATACTACACATACTAGGTAGGTACATAGTTGATTTATTTACCATTCTA -104 TCCCTAGGAAGTTTCTCTTGTATTACTGATATACAATACAAAAAATTTTC -54 TTTATATTCTTTCATGTTTAACATCTTAAAAAGTAATTGAGCAGAACAAA -4 TAAAATG
Cis-element Name Position (Strand) Putative Function AACGTG T/G-box -1199 (-) MYC recognition site TACGTG T/G-box-like -955 (+) CACATG E-box -343 (+) MYC recognition site (G-box-like) -208 (+) TGAC(C/T) W-box -1166 (-) WRKY recognition site -688 (+) -633 (-) -465 (-) -267 (-) (T/C)CCGCC GCC-box-like -234 (+) ethylene response CCGAC C/DRE -1022 (+) cold, drought, ethylene response TGTCTC ARF -1075 (+) auxine response GATAA IBOX -964 (+) light regulation -551 (-) ATTAAT BOX 4 -662 (+) light responsiveness CNGTT(A/G) MYBCORE -1068 (-) MYB recognition site -610 (-) ATTTTCTTC TC-rich repeat -507 (+) defense/ stress response ACTCTA(C/A) YUC element -652 (+) auxin response
Figure 1. Sequence of the 1254bp SlMTS1 promoter fragment. The A of the start codon ATG (marked in bold) is designated as +1; 5’UTR is highlighted. Potential CAAT and TATA boxes are italicized. Primer sequences for the full-length and 5’ promoter deletion constructs are underlined. Selected regulatory motifs in the sequence analyzed by PLACE (http://www.dna.affrc.go.jp/PLACE/) are shown in the table.
Chapter 3 79
Figure 2. The SlMTS1 promoter drives specific expression in the “head” of type VI tomato glandular trichomes. (a) trichomes of tomato Moneymaker plant and (b) trichomes of tomato Moneymaker plant transformed with the full-length SlMTS1 promoter as seen under a microscope. (i) Normal light (brightfield), (ii) YFP filter (emission 525-545nm) and (iii) Merged image.
Figure 3. Quantification of GUS activity from isolated stem trichomes of transgenic 5’ SlMTS1 promoter deletion lines and control plants. Average relative GUS expression for each promoter construct (full-length (FL), 1046bp, 806bp, 612bp, 408bp, 207bp), empty vector control (EV) and untransformed Moneymaker plant (MM).
Identification of the SlMTS1 promoter JA responsive fragment
Since SlMTS1 is induced by jasmonic acid (JA) (van Schie et al., 2007) it was investigated which of the promoter:GUS constructs were responsive to JA
Chapter 3 80 treatment. To this end three plants from one independent line for each promoter deletion construct were grown in soil for four weeks. At this point equal number and size of stem pieces were removed from each plant and frozen in liquid nitrogen (control samples). The plants were left to recover for three days and were subsequently sprayed with 1mM JA in tap water containing 0.05% Silwet. Twenty- four hours later the rest of the stems from each plant were collected and frozen in liquid nitrogen (JA samples). Trichome RNA was isolated, cDNA was synthesized and expression of GUS was evaluated by Q-RT-PCR (Fig.4). All tested plants showed induction of SlMTS1 by JA (data not shown). Induction of GUS was observed with the 612bp SlMTS1 promoter fragment and fragments larger than that, indicating that a JA-responsive box was located between position -409 and - 612 upstream of the ATG. The experiment was repeated with plants from different independent lines with similar results (data not shown).
Figure 4. Identification of a JA responsive fragment in the SlMTS1 promoter. Q-RT-PCR results for the induction of GUS in control and JA-induced stem trichomes from transgenic SlMTS1p:GUSsYFP1 Moneymaker plants carrying the full-length (FL) promoter, promoter fragments of 1046bp, 806bp, 612bp, 408bp or 207bp, or an empty vector (EV). Asterisks denote significant difference (* p < 0.05) according to T-test, ns; not significant.
Chapter 3 81 Screening a stem trichome cDNA library for interaction with specific SlMTS1 promoter fragments
The 207bp trichome-specific SlMTS1 fragment was used to screen a Y1H cDNA library prepared from isolated stem trichomes of tomato Moneymaker plants in order to identify transcription factors that interact with the trichome-specific SlMTS1 promoter fragment. The screen was repeated three times yielding 71 clones (Table 1). One putative transcription factor appeared 34 times and this gene was designated as Emission of Terpenes 1 (SlEOT1). The binding was confirmed by using the library plasmid pAD-GAL4-2.1 containing the EOT1 clone in independent yeast-one-hybrid screens with the same 207bp trichome-specific SlMTS1 promoter fragment and the 326bp minimal promoter fragment of Petunia hybrida cv W115 (Mitchell) ODORANT1 (ODO1; Van Moerkercke et al., 2011) as negative control (Fig.5).
Figure 5. Specific interaction of SlEOT1 with the SlMTS1 promoter and not with PhODO1 promoter. Yeast cells with (a) the 207bp SlMTS1 promoter fragment or (b) the 326bp minimal PhODO1 promoter integrated into their genome were transformed with pAD- GAL4-2.1_SlEOT1 and grown on SD medium with 0, 5 or 10mM 3-AT.
The nucleotide and protein sequence of SlEOT1 is provided in Figure S1.
Two more putative transcription factors were identified through these screens (clone 9, Screen1 and clone 62, Screen 3; Table 1), however as they appeared only once each, they were not further investigated.
Chapter 3 82 Table 1. List of clones obtained from three Y1H screens of the 207bp trichome-specific SlMTS1 promoter fragment with a tomato stem trichome cDNA library. The same putative TF appeared 34 times (SlEOT1) and is marked by an asterisk.
Chapter 3 83 Table 1 continues from previous page.
Screening with the full-length SlMTS1 promoter (1254bp) gave an elevated number of background colonies even when higher concentrations of 3-AT, the competitive inhibitor of the yeast HIS3 protein, (up until 30mM) were used. However, several colonies of two screens were sequenced and one was identified as SlEOT1 (Table 2).
In order to identify transcription factor(s) that interact with the JA responsive fragment of the SlMTS1 promoter (Fig.4), base pairs -409 to -612 of the promoter were cloned in the pHISi vector as described for the 207bp fragment. The stem trichome cDNA library was screened in two yeast-one-hybrid assays but yielded a very limited number of colonies, none of which were transcription factors.
Chapter 3 84 Fragment -613 to -806 of the SlMTS1 promoter was then used in two more screens, as the JA induction of GUS in these plants is slightly stronger (Fig.4) and finally a combination of the two fragments (base pairs -409 to -806) was screened once but the number of colonies obtained remained low (Table 3).
Table 2. List of clones obtained from two Y1H screens of the full-length SlMTS1 promoter with a tomato stem trichome cDNA library. Putative TF SlEOT1 is marked by an asterisk.
Chapter 3 85 Table 3. List of clones obtained from five Y1H screens of the JA responsive SlMTS1 promoter fragments with a tomato stem trichome cDNA library.
SlEOT1 is localized to the nucleus
In order to determine the subcellular localization of SlEOT1, the mVenus (Nagai et al., 2002) reporter gene was fused in frame with the ORF of SlEOT1 under the control of a double CaMV 35S promoter. The translational fusion was transiently expressed in N.benthamiana leaves using agro-infiltration (Verweij et al., 2008). As illustrated in Figure 6, confocal imaging showed co-localization of this fusion protein with the nuclear marker (nuclear localization signal (NLS)-mCherry; Galvan-Ampudia, Xu and Testerink; unpublished) in epidermal cells. Co- localization was observed several times in independent experiments also in mesophyll cells (data not shown).
Chapter 3 86
Figure 6. Subcellular localization of SlEOT1. Transient expression of 35S:mVenus- SlEOT1 in N.benthamiana leaves showing nuclear localization. (a) epidermal cell bright field image, (b) NLS-mCherry image, (c) mVenus-SlEOT1 image and (d) merged image. Images were obtained with a Zeiss LSM 510 confocal laser scanning microscope.
DISCUSSION
Transcription factors (TF) are proteins that can interact with cis-regulatory sequences in promoters of genes and thus influence transcription (Grotewold, 2008). Increased emission of volatile terpenoids is known to be part of a plant’s induced defense mechanism upon herbivory, that is under control of phytohormone signaling (Alba et al., 2012). However, not much is known about the regulation of terpene biosynthesis on the transcriptional level. To identify TFs that are involved in this process, the promoter (1254bp) of a trichome-specific, jasmonic acid (JA)- inducible monoterpene synthase from tomato (SlMTS1; van Schie et al., 2007) was isolated in order to be screened for interacting proteins found in a trichome cDNA library (yeast-one-hybrid). Screening of a promoter fragment with a cDNA library in yeast is based on the fact that genes with a DNA-binding domain can be
Chapter 3 87 expressed as fusion proteins carrying a C-terminal activation domain and are therefore able to activate reporter gene expression, allowing growth of yeast cells on a selective medium (MATCHMAKER One-Hybrid System user manual; Clontech, CA, USA). Yeast-one-hybrid screens have successfully been used before for the identification of transcription factors (Liu et al., 1998; Menke et al., 1999; Lopato et al., 2006; Gaudinier et al., 2011). However, there are restrictions in the size of the DNA fragment that can be efficiently used as bait, since the majority of yeast promoters range from approximately 150bp to 400bp and their upstream activation sequences are positioned within a few hundred base pairs upstream of the TATA box (Dobi and Winston, 2007). Consequently there is an effect of distance on the activation in yeast in vivo and usually only specific cis-acting regulatory elements or other short DNA fragments are used in yeast-one-hybrid screens. Therefore, the SlMTS1 promoter was dissected by creating five 5’ deletion constructs driving a GUS-sYFP1 fusion, in order to identify the minimal promoter fragment and the fragment that confers JA inducibility. As expected (van Schie et al., 2007), expression of the full-length 1254bp SlMTS1 promoter was specific for the secreting cells of type VI trichomes (Fig.2). The 1046bp fragment showed equal level of GUS activity and the next two deletion constructs showed diminished, but still trichome-specific activity. Interestingly, the 408bp promoter fragment abolished expression in all tissues, indicating the presence of a repressor element (between -408 and -208 bases) and/or a transcriptional enhancer in the -409 to -612bp fragment. However, the 207bp fragment proximal to the coding region contained all cis-elements required for trichome-specific expression (Fig.3). Co-existence of both activating and repressing cis-elements in a promoter has been reported before (Ennajdaoui et al., 2010) and in fact, studies from various plant species reveal that the regulation of promoter-driven expression of genes can be variable and not always straightforward: for example, the promoter of the tomato sucrose transporter SlSUT1 is insufficient for proper SUT1 expression, which requires regulatory cis-elements found in the introns (Weise et al., 2008) and the organ-specificity of the Arabidopsis and tomato gene Lateral Suppressor (LAS) is
Chapter 3 88 conferred by an enhancer in the 3’UTR, which also has suppressor functions, overwriting promoter activity (Raatz et al., 2010). In the case of SlMTS1 nevertheless, we were able to identify the minimal promoter (207bp) as well as a potential JA-responsive fragment (-612 to -409bp; Fig.4) with 5’ sequential deletions. For a more detailed analysis of the cis-acting elements of the promoter, internal and/or 3’ sequential deletions would have to be made. The cDNA library used for screening with the promoter fragments was constructed from tomato stem trichomes. Unfortunately this library failed to produce candidate transcription factors interacting with the JA-responsive fragment (Table 3) either because these genes are not (highly) expressed in uninduced tissue, which would require the construction of a cDNA library from plants treated with JA, or because it is not a one-to-one interaction taking place and the presence of a co-factor is required, in which case the potential interacting TF(s) can not be identified by yeast-one-hybrid. Screening of the stem trichome cDNA library with the trichome- specific SlMTS1 fragment however yielded one putative transcription factor (Table 1; Fig.5) that localizes in the nucleus (Fig.6). This trichome-specific 207bp fragment proximal to the coding region of SlMTS1 includes the 5’ untranslated region (UTR; Fig.1). The 98bp 5’UTR was isolated by 5’RACE, however there is a possibility that low abundant, longer mRNAs exist. Alternative 5’-ends of transcripts have been reported before and in Arabidopsis 40- 50% of genes have frequently more than one start sites (Alexandrov et al., 2006). 5’UTRs can contain translation control elements and/or binding sites for regulatory proteins. Similarly, 3’UTRs can also contain a multitude of translational regulatory elements (Wilkie et al., 2003). Common roles of UTRs in the post-transcriptional regulation of gene expression involve mRNA translation efficiency, subcellular localization and stability (Mignone et al., 2002), with the latter being one of the foremost forms of post-transcriptional regulation in eukaryotic cells (Abler and Green, 1996). For example, already decades ago it was demonstrated in rice protoplasts that the wheat Em leader sequence contains, apart from an ABA response element in its promoter, an element in the 5’UTR that quantitatively
Chapter 3 89 increases the ABA response and it was postulated that it plays a role in stabilizing or increasing the efficiency of translation of the mRNA (Marcotte et al., 1989). An example of a protein found to interact with such an element is the tobacco E2F factor that is involved in the cell cycle progression and was shown to bind the respective motif in the 5’UTR of the Ribonucleotide reductase 1b (RNR1b) gene and thus repress expression in differentiated tissues, whereas confering S phase- specific expression in meristems (Chaboute et al., 2002). Since the 207bp fragment contained the (canonical) 5’UTR, it was not clear whether SlEOT1 binds to the 109bp promoter region or the 98bp 5’UTR. To this end electrophoretic mobility shift assays (EMSAs) using purified recombinant GST-tagged SlEOT1 were performed with the 207bp or deletion fragments. However, no binding was observed with any of the tested DNA fragments in vitro. This could be due to the lack of a co-factor or additional TF necessary to facilitate the interaction (present in yeast) or because the protein fusion was not functional. The assay was not repeated with purified protein after the GST-tag had been cleaved off. Furthermore, in silico searches for consensus sequences in promoters of tomato trichome-specific genes failed to reveal a conserved motif, therefore at the moment the exact binding site of SlEOT1 remains unknown. The 98bp 5’UTR of SlMTS1 and the 109bp promoter fragment have been cloned behind the minimal promoter of the HIS3 locus in yeast in order to investigate the binding specificity of SlEOT1. SlEOT1 could either be involved in the post-transcriptional (in case it binds to the 5’UTR) or transcriptional (in case it binds to the promoter sequence) regulation of SlMTS1, which remains to be further investigated. However, based on homology with other proteins and further experimental data (Chapter 4) SlEOT1 is in all likelihood a transcription factor, in which case its binding site is most likely in the promoter sequence.
Chapter 3 90 ACKNOWLEDGEMENTS
We thank Mattijs Bliek (Vrije Universiteit, Amsterdam) for kindly providing the yeast strain PJ69-4A, Carlos Galvan-Ampudia and Ronald Breedijk for help with confocal imaging constructs and microscopy, Kai Ament for constructing the trichome cDNA library, Petra Bleeker for initially isolating the SlMTS1 promoter by genome walking and Harold Lemeris, Ludeck Tikovsky and Thijs Hendrix for the excellent care of the tomato plants.
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Chapter 3 94 SUPPLEMENTAL DATA
Figure S1. Nucleotide and protein sequence of SlEOT1. Coding sequence shown in capital letters, non-coding sequence in small letter type. N-terminal and C-terminal conserved domains identified by blasting in NCBI (http://www.ncbi.nlm.nih.gov/Structure/cdd/ wrpsb.cgi; discussed in Chapter 4) are underlined.
ATGGCTAATTTCTTTTCATTAGGTGGGAATCAAGAACAACAACATCAAGAAATTAGCAGCAGCCAAG CATTAGTACCCACAGAGAGTAATAATTGGTTTTTGTACAGAAATGAACATCATCATCATCATCATAA TCAAGAAATACCCAACACTTACAAAGGTTTTGAGTTATGGCAAAGTGGTAACACTCCACAACACCAA CACCAACACCACCAACAACAACAACAGTTTCGTCATCCGATTTATCCTTTGCAAGATCTTTATTCCAC TGATGTTGGATTAGGGGTTGGGCCAAGCAGAAGTGGCTTTGATATATCtgcaggtgatcatcagaaacagatttag aatttaaactttatctattcagtactttctaaagtacttatagatctataatttaagtttgataaatttaatatttatgttctaaacaattcacaacattttgcaatta gggatttcgaaacgtatttactgaagcatgttagaattcccagcctcgaaaaggcatgggaatttggtctatggacttgggaaattctccattcatgag ctaacttttgaggttaaattaggttcatatgtcatatctttacatgatatcagagtaagattcatctcaattctttgttcaccaatattggccccccatattattg tgtccacaatctagttaacctacgctggcccctccatattacagtgtccacgttctagttaacgagatctgggcttgcagaagagtgtaaagaattcag aaaaaggatgagtatttggtctccttgtataaacttgagcaatccttccttcatgagctagctagttttggaattaagttagactagatgtcatatcttttaat atttatgttctcactgtagaaccatatagcaacgaaactatagtactatttgttgcaccgctctctctatatatatcgtgcatattaagttcaattgaatctgtt gctaaaaggcgggatggggattattattgTGCAGGTGATCATGAGGCGTCGAGGTCGGGATTCGTGATGATGAGGA GTGGTGGAGGAGGAATAAGTTGCCAAGATTGTGGGAACCAAGCTAAGAAAGATTGTCAACATATGA GGTGTAGGACTTGTTGTAAGAGTAGAGGGTTTCAGTGTCAAACTCATGTGAAAAGTACTTGGGTTCC AGCAGCTAAAAGGAGAGAAAGGCAACAACAACTTGCTGCTTTGCAACAACAACAACAAGGACATAA TAATAATAATAATAATCATAAGAATAAAAGGCAAAGGGAGGATCCAAGTGCTTCTTCTCTTGTGTCT ACTCGTTTGCCTTCAAACACTAATGgtaaagtacttcatgtttttcttaccttttcattgctacgtctgttttaatttaaaggtcttagtttga ctgaacatgaatataagatgttgaaattgaaaaacgtagataaatatttaaattgaaacgagggaataatattaattttttttgtatcacacaaagacatag agtcttgagatccatcatgtaaagaagattaatttgatcattgcctaaatgaattctatataaagtaagtctatagagaaaagagaccctatagtaaattc gtcagctttttctttttctatttgtcattctcttcttccatcatcactcttcttttttattactctacaaaagattgacaaaaattcgtaatgagatatattcaaattttt gagttaattatgaatttttaattctagttaatagaaagtgtgaataaattatttatatgtattactaacaaaatagcaaaactaaaactttacttgtacccttgc gcgtgtgtatgcacaatttctttctcttagacctacacatgatatttatctcgaccctaaaaagatcaccattattcttaatttcaattttcgtcaatttttttttaa gataataactattatttgagtaataatatatgtgacttacccaaaaaactgttagtggagtgagtatttgagaaaccaactctctaattcatgtataataatt ggtgttatcatatattgtcattagtattggaattaacttatatatctattagtaaatgtacttttgaaataataactattatttgagtaataatatatgttgcttacc aaaaaaataactattagttgagtggctattaactctccaaatatgtataataattggtgttatcattttcattagtattggaattaacttatatatatagtaaatg cacttgcatttcaaatttttttacctgcttttccttttagttcgattaaaataaattgactatttttcaagcaagtgtttattctaaacttttcagatgaaatgtttaa aaaaaccacaagattaaatagtgttttgatacatttgacatatttttagttttagaccataaaattcaaattgctttactaaatttcgtgtcaagtgatactagg taaaaaaaaatatttatttgcaatacattagtccaaataaacctaattttgtattatggaatttcatgtgttatttttagGGTTAGAAGTGGGAAAA TTTCCATCAAAAGTACGTACAAGTGCTGTATTTCAGTGTATTCAAATGAGTTCAATTGAGGATGATGA AGATCAATTAGCATATCAAGCTGCTGTGAGCATTGGTGGACATGTTTTCAAAGGAATTTTATATGATC AAGGTCATGAAAGTCAGTACAATAACATGGTTGCAGCCGGAGGCGATACGTCTTCCGGTGGTAGTGC TGGCGGAGTTCAGCACCACCACCATAATTCCGCTGCAGTAGCTACCGCCACCACTACAAGTGGTGGC GATGCTACTGCAGCGGGTCCATCGAATTTTCTAGATCCTTCTTTATTTCCAGCTCCCCTTAGCACTTTT ATGGTAGCTGGTACGCAATTTTTTCCACCTTCAAGATCTCCTTGA
MANFFSLGGNQEQQHQEISSSQALVPTESNNWFLYRNEHHHHHHNQEIPNTYKGFELWQSGNTPQHQHQ HHQQQQQFRHPIYPLQDLYSTDVGLGVGPSRSGFDISAGDHEASRSGFVMMRSGGGGISCQDCGNQAKK DCQHMRCRTCCKSRGFQCQTHVKSTWVPAAKRRERQQQLAALQQQQQGHNNNNNNHKNKRQREDPS ASSLVSTRLPSNTNGLEVGKFPSKVRTSAVFQCIQMSSIEDDEDQLAYQAAVSIGGHVFKGILYDQGHESQ YNNMVAAGGDTSSGGSAGGVQHHHHNSAAVATATTTSGGDATAAGPSNFLDPSLFPAPLSTFMVAGTQ FFPPSRFP*
Chapter 3 95 Chapter 4
Characterization of EMISSION OF TERPENES, a trichome-specific transcription factor from Solanum lycopersicum involved in terpene biosynthesis
Eleni A. Spyropoulou, Michel A. Haring and Robert C. Schuurink
Department of Plant Physiology, Swammerdam Institute for Life Sciences, University of Amsterdam, Science Park 904, 1098 XH Amsterdam, The Netherlands
ABSTRACT
Terpene biosynthesis in tomato glandular trichomes has been well studied with the role of several terpenes in defense responses of the plant already established. Stress-induced production of terpenes is known to be under regulatory control of the pathway genes by transcription factors (TFs) and for some plant species TFs involved in this process have been identified. In tomato, however, nothing is known about the regulation of the tissue- and/or hormone-specific expression of the terpene synthases. Here we report the characterization of the trichome-specific Emission of Terpenes 1 (SlEOT1), the first Solanum lycopersicum transcription factor identified involved in (mono)terpene biosynthesis that was previously discovered in a yeast-one-hybrid screen of the SlMTS1 promoter. SlEOT1 can transactivate the SlMTS1 promoter in Nicotiana benthamiana leaves.
INTRODUCTION
Secondary metabolite production is under control of transcriptional regulation of the pathway genes, which is of special relevance for the terpenoids as some of these compounds accumulate upon a stress stimulus (Broun et al., 2006). Such regulatory mechanisms involve transcription factors (TF)- proteins that can recognize and bind specific elements in the promoters of the genes they regulate (Grotewold, 2008). TFs are usually classified as activators or repressors, or general transcription factors when they are involved in the RNA polymerase II transcription-initiation complex, or yet other transcriptional regulators may be
Chapter 4 97 involved in DNA conformation (architectural proteins), by e.g. facilitating bending of the DNA and thereby binding or interacting with other proteins, or act as cofactors mediating effects of activators or repressors (Singh, 1998). The last two categories usually lack DNA binding domains and do not fall into the typical transcription factor definition, which generally describes a DNA binding domain, a transcription regulation domain, an oligomerization site and a nuclear localization signal (Liu et al., 1999). Transcription factors can also be classified according to their DNA binding domain, that usually gives the name to the TF families (Yamasaki et al., 2008). Big plant TF families include the MYB, bHLH, AP2/ERF, bZIP and WRKY families, some of which are found also in other eukaryotes, while others are specific for plant lineages, such as the AP2/ERF and WRKY proteins (Riechmann et al., 2000). DNA binding domains are usually short motifs, and binding to the double-stranded DNA groove commonly involves an α-helix or a β-sheet (Wray et al., 2003), with many plant-specific binding domains utilizing β-sheet regions, whereas prokaryotes or other eukaryotes recognize DNA through α-helices (Yamasaki et al., 2008). For example, the WRKYGQK residues that gave the name to the WRKY family proteins correspond to the most N-terminal β-strand that exhibits the DNA binding activity. The WRKY domain consists of a four-stranded β-sheet, with a zinc-binding pocket formed by conserved cysteine and histidine residues (Rushton et al., 2010). The WRKY family is a class of zinc finger (ZnF) transcription factors that all involve a sequence motif in which a zinc atom is bound by cysteine and histidine residues stabilizing the secondary structure of the finger. Other ZnF classes include the Dof and RING finger families (Takatsuji, 1998). Transcription factors commonly also contain transcription regulation domain(s), which influence the effect that the TF will have once bound to DNA- activating or repressing (Mejia-Guerra et al., 2012). Furthermore, many transcription factors are able to form homo- or hetero-dimers through their oligomerization sites, which are
Chapter 4 98 usually highly conserved domains, and this can thus have an effect on DNA binding specificity, affinity for the promoter(s) in question or nuclear localization (Liu et al., 1999). Binding specificity, interaction with other proteins or subcellular localization can be regulated further by posttranslational modification of the TF itself, most commonly involving phosphorylation (Vom Endt et al., 2002). It is also worth pointing out that the ability of transcription factors to regulate gene expression can rest on their own regulation, therefore they are usually part of a transcriptional network (Hobert, 2008) that can involve upstream master- regulators influencing TFs found in the middle of the hierarchy, which in turn can regulate TFs or other genes at the lower levels (Yu and Gerstein, 2006) or that entails less hierarchical structures with hubs of genes involving TFs mutually regulating each other and/or downstream genes or finally that combines these two types of network structures (Jothi et al., 2009). Terpenoid biosynthesis can involve a signaling cascade that starts with the perception of a stress signal, followed by de novo synthesis or activation of transcription factor(s), which in turn regulate expression of genes, leading to the production of enzymes that catalyze the biosynthesis of terpenoid metabolites (Zhao et al., 2005). In Catharanthus roseus, such a signaling cascade involves a MeJA-inducible transcription factor of the MYC family (CrMYC2) that regulates at least two identified TFs of the AP2 family (ORCA2 and ORCA3) that in turn regulate expression of the strictosidine synthase (STR) involved in the terpene indole alkaloid biosynthesis (Zhang et al., 2011). Other examples of transcription factors involved in the stress-induced biosynthesis of terpenoids come from the WRKY proteins of the zinc finger family from various plant species (Suttipanta et al., 2011; Ma et al., 2009; Xu et al., 2004). Here we report the characterization of a novel zinc finger transcription factor from Solanum lycopersicum that contains a RING finger-like conserved N-terminal domain and a less conserved C-terminal IGGH domain found in proteins of many plant species, but which appear to have acquired different functions. In tomato it concerns a trichome-specific transcription factor involved in (mono)terpene
Chapter 4 99 biosynthesis, which is a member of a small gene family, also potentially involved in regulating terpene production.
MATERIALS AND METHODS
Hormone Treatment, RNA Isolation and cDNA synthesis
Tomato plants were grown in soil in a greenhouse with day/night temperatures of 23oC/18oC and a 16/8 h light/dark regime for 4 weeks. Jasmonic acid (JA; Duchefa, NL) was applied to plants by spraying 1mM solution made with tap water containing 0.05% SilwetL-77 (GE Silicones, VA, USA). Control plants were sprayed with tap water containing 0.05% SilwetL-77. Trichomes were collected 24h later at the bottom of a 50ml tube by vortexing stem pieces frozen in liquid nitrogen. The rest of the tissue-types analyzed were also frozen in liquid N2 and then the material was ground and total RNA was isolated using Trizol (Invitrogen, Paisley, UK). DNA was removed with DNAse (Ambion, Huntingdon, UK) according to the manufacturer and cDNA was synthesized from 1.5μg RNA using M-MuLV H- Reverse Transcriptase (Fermentas, St. Leon-Rot, Germany). Petunia plants (Petunia hybrida cv W115 Mitchell) were grown under the same conditions for two months and RNA isolation, DNAse treatment and cDNA synthesis was carried out as described for the tomato plants.
Chapter 4 100 Quantitative Real Time PCR
For Q-RT-PCR cDNA equivalent to 100ng total RNA was used as template in 20μl volume and reactions were performed in the ABI 7500 Real-Time PCR System (Applied Biosystems) using the Platinum SYBR Green qPCR SuperMix-UDG kit (Invitrogen, Paisley, UK) with the following cycling program: 2min 50oC, 7min 95oC, 45 cycles of 15sec at 95oC and 1min at 60oC, followed by a melting curve analysis. Primer pairs were tested for amplification kinetics and linearity with a standard cDNA dilution curve and new primers were designed if necessary. Expression levels were normalized using ACTIN (SGN-U579547) mRNA levels. Three biological replicates (unless otherwise mentioned) were analyzed. In Figure 10 expression of SlEOT1 in transgenic (M) lines compared to untransformed plants was tested by nested ANOVA followed by a Dunnett posthoc test. Similarly, significance for differences in transgene expression between the M lines was tested by nested ANOVA followed by Tuckey’s B posthoc test (tomato line was used as fixed factor and technical replica as nested factor in all cases). The homogeneity of variance was tested by Levene’s test and values were log transformed before the analysis if necessary. Statistics were performed using PASW Statistics 17.0 (http://www.spss.com). For Petunia hybrida, ACTIN was used for normalization of expression levels in one biological replica.
Cloning and construct design
The coding sequence of SlEOT1 (Chapter 3) was cloned in the pKG1662 vector (KeyGene, Wageningen, NL; for a map of the vector see patent nr US2011/0113512A1) between restriction sites NcoI (at the ATG) and SacI (after the stop codon) followed by the Nos terminator (tNos) and driven by the CaMV 35S promoter. 1961bp, 1236bp and 1398bp genomic sequence upstream of the start codon of SlTPS3, SlTPS7 and SlTPS8 (Chapter 2) respectively, were cloned in
Chapter 4 101 pKG1662 between restriction sites HindIII (at the 5’ end of the sequence) and NcoI (at the 3’ end of the sequence just prior to the ATG) replacing the 35S promoter upstream of uidA (GUS) followed by tNos. Similarly, 1557bp and 1270bp of the genomic sequence upstream from the start codon of SlTPS9 (Chapter 2) and SlEOT1, respectively, were cloned in vector pJVII, a pMON999 vector (Monsanto, St. Louis, MO) with a modified multiple cloning site (MCS). The first was cloned between restriction sites SacI (at the 5’ end of the sequence) and NheI (at the 3’ end of the sequence just prior to the ATG) and the second between HindIII (at the 5’ end of the sequence) and XbaI (at the 3’ end of the sequence just prior to the ATG) replacing the 35S promoter, upstream of the ATG start codon of GUS fused to yellow fluorescent protein (sYFP1) followed by the tNos. All constructs were verified by sequencing and then the expression cassettes were transferred to the MCS of the binary vector pBINplus (van Engelen et al., 1995) between HindIII and SmaI restriction sites for SlTPS3p, SlTPS7p, SlTPS8p and SlEOT1p and SacI and SmaI restriction sites for SlTPS9p. The final constructs were transformed to Agrobacterium tumefaciens GV3101 (pMP90).
Transient transactivation assay in Nicotiana benthamiana leaves
A.tumefaciens GV3101 (pMP90) cultures were grown overnight from a single colony and diluted in infiltration buffer (50mM MES pH 5.8, 0.5% glucose, 2mM
NaH2PO4, 100μM acetosyringone; Sigma-Aldrich) to OD600 of 0.3. Leaves from five week old Nicotiana benthamiana plants were then infiltrated with mixtures carrying various promoter:GUS or promoter:GUSsYFP1 reporter constructs and the 35S:EOT1 effector construct in a 1:1 ratio. In order to normalize for transformation efficiency and protein extraction efficiency, a construct containing Photinus pyralis (firefly) luciferase (LUC) driven by the CaMV 35S promoter was co-infiltrated with the reporter/effector mix in a 2:5 ratio (LUC: reporter/effector mix). Two leaves of three plants were infiltrated for each combination. Two days
Chapter 4 102 later leaf disks from the infiltrated areas were collected, frozen in liquid nitrogen and crude extracts were prepared in extraction buffer containing 25mM Tris phosphate pH 7.8, 2mM DTT, 2mM CDTA pH 7.8, 10% glycerol and 1% Triton X-100. The enzymatic GUS activity was determined spectrophotometrically using 4-methylumbelliferyl-β-D-glucuronide (MUG) as a substrate according to Jefferson et al. (1987). The luciferase assay was performed using the same extraction buffer, according to van Leeuwen et al. (2000) and measured in a FluoroCount Microplate Fluorometer (Packerd BioScience Company) using a 560nm emission filter. Enzymatic GUS activity was normalized for luciferase activity for each sample. Significant differences between samples were tested by ANOVA followed by Tuckey’s B posthoc test using software PASW Statistics 17.0 (http://www.spss.com). The homogeneity of variance was tested by Levene’s test and values were log transformed before the analysis if necessary. The experiments were repeated at least twice with similar results.
Stable Plant Transformations
The open reading frame (ORF) of SlEOT1 was cloned between restriction sites NcoI at the ATG and SacI after the stop codon in the pKG1662 vector (KeyGene, Wageningen, NL) downstream of the Solanum habrochaites MKS1 (AY701574; Fridman et al., 2005) promoter and after verifying by sequencing, the expression cassette was transferred to the binary vector pBINplus (van Engelen et al., 1995) by digesting with restriction enzymes HindIII and SmaI and ligating in the MCS of pBINplus at the same restriction sites. The construct was introduced to Agrobacterium tumefaciens strain GV3101 (pMP90) and used to create transgenic plants using explants derived from cotyledons of sterile seedlings of Solanum lycopersicum cultivar Moneymaker, as previously reported (Cortina and Culianez- Macia, 2004).
Chapter 4 103 The pBINplus SlEOT1p:GUSsYFP1 construct described in the previous section was transformed to tomato Moneymaker plants as explained for the pBINplus ShMKS1p:EOT1 construct.
Analysis of Transgenic Plants
Fifteen independent lines were obtained for the pBINplus ShMKS1p:EOT1 construct and presence of the transgene was verified by PCR (using primers for NPTII) on genomic DNA isolated from leaves of the different T0 lines. Two cuttings were made from each of the T0 lines and after four weeks trichomes were isolated and cDNA synthesized as described above. Specific Q-RT-PCR primers were designed for checking the expression of the endogenous SlEOT1 (primers were designed in the 3’ UTR), the expression of the transgene (a forward primer was designed in the 5’ UTR of ShMKS1 and a reverse in the ORF of SlEOT1) and finally a set of primers was designed in the SlEOT1 ORF for checking the overall expression of this gene. Five independent lines were obtained for the pBINplus SlEOT1p:GUSsYFP1 construct and expression of the yellow fluorescent protein was observed under a fluorescence stereomicroscope.
Yeast-One-Hybrid Screen
The full-length SlMTS1 promoter or promoter fragments -1254 to -1047bp, -1046 to -807bp, -806 to -613bp, -612 to -409bp, -408 to -208bp and -207 to -1bp were cloned between restriction sites EcoRI (at the 5’ end of the sequence) and XbaI (at the 3’ end of the sequence) in the MCS of vector pHISi (Clontech, Mountain View, CA, USA) upstream of the minimal promoter of the HIS3 locus driving HIS3. The HIS3 gene of pHISi was used as selectable marker for integration into the
Chapter 4 104 nonfunctional his3 locus of yeast strain PJ69-4A. The vector was linearized at the XhoI restriction site before integration. Leaky HIS3 expression enabled enough colony growth, making it possible to use as a selectable marker. Background growth was controlled during library screening by the addition of 5mM 3-amino- 1,2,4-triazole (3-AT; Sigma-Aldrich) in the selective medium (synthetic dextrose, SD). Transformations were performed using 1μg of the library plasmid pAD-GAL 4-2.1 containing clone SlEOT1 (Chapter 3).
List of primers used
Primer name Sequence 5' -> 3' PhEOT_atgF ATGGCTAATTTCTTTTCACTAGGTG PhEOT_tgaR TCAAGGAGATTTTGGAGATGGAAA PhEOT_QF CAGCAGAAGCCTCAAATTATCTTG PhEOT_QR CAAACGAACATAACTGTATCACACAG PhACT_QF TGCTGATCGTATGAGCAAGGAA PhACT_QR GGTGGAGCAACAACCTTAATCTTC SlACT_QF TCAGCACATTCCAGCAGATGT SlACT_QR AACAGACAGGACACTCGCACT SlEOT_ORF_QF TACAAGTGGTGGCGATGCTAC SlEOT_3'UTR_QR ACCTCAATATTATCAATGTGGACAATC SlEOT_ORF_QF2 AATTAGCAGCAGCCAAGCATTAG SlEOT_ORF_QR3 GTGTTGGTGTTGTGGAGTGTTAC ShMKS1_5'UTR_QF CATATATTGTGCACTAAATCCCAAC SlEOT_ORF_QR2 CTAATGCTTGGCTGCTGCTAATT
RESULTS
SlEOT1 is specifically expressed in tomato trichomes and not induced by jasmonic acid
SlEOT1 was isolated as an interactor of the 207bp trichome-specific fragment of the SlMTS1 promoter (Chapter 3) by a Y1H screen. In order to investigate the expression pattern of this gene, Q-RT-PCR was performed on cDNA from different
Chapter 4 105 S.lycopersicum cv. Moneymaker tissues; namely leaves, stems, isolated stem trichomes and roots from 4-week-old plants, as well as flowers and fruit of mature plants. As shown in Figure 1a, expression of SlEOT1 was specific for trichomes. This was confirmed by creating stably transformed tomato Moneymaker plants expressing a GUSsYFP1 fusion under the control of the 1270bp SlEOT1 promoter. In five independent transformants the expression of SlEOT1 was similar to that of SlMTS1 in the four “head” cells of the type VI glandular trichomes (Fig.2).
Figure 1. Tissue specific expression and JA induction of SlEOT1. Relative transcript levels for SlEOT1 as determined by Q-RT-PCR. Mean values (+SE) of 3 biological replicas are shown, normalized for Actin expression. L; leaf, WS; whole stem, BS; bald stem, T; stem trichomes, R; root, Fr; fruit, Fl; flower; C; control and JA; jasmonic acid-induced stem trichomes. ns; not significant according to T-test.
Since SlEOT1 interacted with SlMTS1, a JA-inducible gene, the subsequent step was to investigate whether expression of this gene could also be regulated by application of JA. To this end, 4-week-old Moneymaker plants were sprayed either with control solution or with solution containing JA and 24h later stem pieces were collected and Q-RT-PCR was performed on cDNA from isolated stem trichomes. Induction of SlMTS1 was confirmed in this material (data not shown) but SlEOT1 was not induced by JA (Fig.1b).
Chapter 4 106
Figure 2. The SlEOT1 promoter is expressed in the “head” of type VI tomato glandular trichomes. (a) trichomes of tomato Moneymaker plant and (b) trichomes of tomato Moneymaker plant transformed with the full-length SlEOT1 promoter driving GUSsYFP1 as seen under an EVOS inverted microscope. (i) Normal light (brightfield), (ii) YFP filter (emission 525-545nm) and (iii) Merged image.
SlEOT1 is a zinc finger-like protein that has a conserved RING finger-type motif and a less conserved IGGH domain
Homology search of the SlEOT1 ORF after translation (BLASTX) against the National Center for Biotechnology Information (NCBI) non-redundant database returned sequences of putative transcription factors from various plant species such as Ricinus communis, Vitis vinifera and Populus trichocarpa as best hits. The best hits from Arabidopsis thaliana were a Short Internode (SHI)- related protein (Stylish 1; STY1, At3g51060) with which it had 42,4% identity, SHI (At5g66350) with which it had 40,8% identity and a Lateral Root Primordium (LRP, At5g12330) protein with which it had 25,6% identity. Searches for homologous sequences of the SlEOT1 ORF (BLASTN) in the potato CDS sequences (PGSC DM v3.4) available at the Solanum Genomics Network (SGN; http://solgenomics.net/) database returned a cDNA that after translation had 83,5% identity with the SlEOT1 protein. Searches for homologous sequences of the SlEOT1 ORF (BLASTN) in the Petunia inflata genomic scaffolds available from
Chapter 4 107 the Petunia Consortium (released through the Sol Genomics Network) returned a sequence from which the ORF was predicted and subsequently cloned full-length from Petunia hybrida cDNA. The two sequences shared 56% identity on protein level. Interestingly, in Petunia plants the SlEOT1 homolog appeared to be also specifically expressed in the trichomes (Fig.3).
Figure 3. Tissue specific expression of Petunia hybrida EOT1. Relative transcript levels for PhEOT1 as determined by Q-RT-PCR, normalized for Actin. L; leaf, WS; whole stem, BS; bald stem, T; stem trichomes, R; root, Fl; flower.
STY1 and SHI of Arabidopsis belong to a gene family that consists of 10 members (Fridborg et al., 2001; Kuusk et al., 2002). Similarly in tomato, homology searches on nucleotide level against the SGN database revealed that SlEOT1 belongs to a small family that consists of at least five more genes (Table 1). Three of these genes are also expressed in stem trichomes (transcripts were identified in a stem trichome transcriptomic database; see Chapter 5). For one that is positioned on chromosome two (SGN, Genomic Release 2.41) no EST had been previously identified and one (SGN-U563829) was up-regulated in trichomes by treatment of the plant with jasmonic acid (transcripts were identified in a stem trichome quantitative database comparing trichomes from control and JA-treated plants; see Chapter 5). The EOT1/SHI family appears to be specific for plants, as homology searches in human, insect, fungus, nematode, microbe and protozoa genomes (http://www.ncbi.nlm.nih.gov/sutils/genom_table.cgi?organism) returned no hits. However, homologues have been found in the moss Physcomitrella patens (Eklund
Chapter 4 108 et al., 2010a). A phylogenetic tree with the SlEOT1 protein and homologues from other plant species is shown in Figure 4.
Table 1. SlEOT1 family members in the tomato genome. When not available, putative full- length sequence was predicted by Genscan (http://genes.mit.edu/GENSCAN.html) using sequences obtained a from SGN genomic sequence or b also contig sequences from a stem trichome database (see Chapter 5). SGN nr Chromosome Trichome DB Reads C Reads JA Fold AA lenght Comments Contig nr JA-induction SGN-U566013 1 - - - - 226 a SGN-U587292 2 4489 5 7 1,4 351 SlEOT1 - 2 8844 39 40 1,02 345 b SGN-U563829 3 12542 26 53 2,04 319 b SGN-U569648 4 26035 10 5 0,5 372 b SGN-U586059 11 - - - - 350 b
Figure 4. Phylogenetic tree of SlEOT1 with homologues from other plant species (Arabidopsis thaliana, Ricinus communis, Physcomitrella patens, Populus trichocarpa, Solanum tuberosum, Petunia hybrida and Nicotiana benthamiana). Sequences were aligned using ClustalW. The phylogenetic tree was constructed after bootstrap analysis (n=1000) using Lasergene DNAstar Megalign software (DNASTAR, Madison, USA). Tomato family members are in bold.
What all the above-mentioned proteins had in common was a N-terminal zinc finger-like conserved domain with a less conserved C-terminal companion domain (Fig.5; NCBI: http://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi). The zinc
Chapter 4 109 finger-like domain was first described in Arabidopsis (Smith and Fedoroff, 1995). The root-specific gene carrying this domain was identified by insertional mutagenesis with a gene trap transposon and it was designated as lateral root primordium 1 (LRP1). LRP1 was activated during the early stages of root primordium development and switched off prior to the emergence of lateral roots (Smith and Fedoroff, 1995). It was also shown to positively regulate root elongation that is normally repressed via histone deacetylation of LRP1 chromatin through the action of SWP1 (Krichevsky et al., 2008). Smith and Fedoroff (1995) observed that the cysteine arrangement in LRP1 can be shown as C-X2-C-X12-C-
X2-C-X7-C (X being a variable amino acid), which is similar to the arrangement of cysteins and histidines in the conserved domain of the zinc binding sites of the protein kinase C family of proteins: H-X12-C-X2-C-X10-14-C-X2-C-X4-H-X2-C-X7-C (Hubbard et al., 1991). LRP1 was later shown to belong, together with STY1, to the SHI gene family (Fridborg et al., 2001; Kuusk et al., 2002). The sequence arrangement of the conserved N-terminal motif seen as C-X2-C-X7-C-X-H-X2-C-
X2-C-X7-C-X2-H was named C3HC3H RING domain (Fridborg et al., 2001) due to the similarity with the zinc binding RING finger motif identified in the human
RING1 gene (C-X2-C-X9-27-C-X1-3-H-X2-C-X2-C-X4-48-C-X2-C (C3HC4); Freemont, 1993; Lovering et al., 1993). The C3HC3H RING domain of SlEOT1 and of the other homologous proteins mainly differs from the classic C3HC4 RING motif in that the last zinc binding cysteine (C) residue has been replaced with a histidine (H) and that the first and second loops (amino acids between second and third C and between fifth and sixth C) have different length (Saurin et al., 1996). A typical structure of a zinc finger involves an antiparallel β-sheet followed by an α- helix with the zinc ion stabilizing the structure that is bound tetrahedrally by the conserved cysteines and histidines, and the DNA binding site residing in the helical part of the protein (Klug, 2010). RING fingers associate two zinc atoms with each atom bound tetrahedrally either by four cysteines or by three cysteines and one histidine in a “cross-brace” motif thus forming one integrated structural unit instead of two zinc finger units (Borden and Freemont, 1996).
Chapter 4 110 The second (C-terminal) domain of the SHI family proteins with high sequence identity (although less conserved than the N-terminal domain) was named IGGH domain due to these four highly conserved residues of the region (Fridborg et al., 2001) and is unique for this family proteins.
Figure 5. Protein alignment of SlEOT1 with related proteins from other plant species. Putative zinc finger domain (C3HC3H RING type) and C-terminal companion domain (IGGH type) of SlEOT1 are shown highlighted, in lowercase. The alignment was generated in CLC Workbench (www.clcbio.com) using ClustalW. Putative nuclear localization (NLS) signal as well as regions typically found in this protein family (glutamine (Q)-rich regions and acid residues cluster; ARC) are shown (see Discussion).
Chapter 4 111 Amino acid sequence similarities in the N- and C-terminal domains between the tomato family members and the Arabidopsis STY1, SHI and LRP1 are shown in Figure 6.
Figure 6. Amino acid sequence alignment between the SlEOT1 tomato family members and Arabidopsis STY1, SHI and LRP1. (a) N-terminal domain and (b) C-terminal domain. The alignment was generated in CLC Workbench (www.clcbio.com) using ClustalW. C3HC3H RING motif and IGGH motif are boxed.
SlEOT1 specifically transactivates the SlMTS1 promoter in Nicotiana benthamiana leaves
In order to investigate whether SlEOT1 can interact with the promoters of other terpene synthases besides SlMTS1, a transient assay was used in Nicotiana benthamiana leaves, which has been previously used successfully to investigate transcription factor interactions with plant promoters (Van Moerkercke et al., 2011). In the reporter constructs, expression of β-glucuronidase (GUS) was driven by trichome specific promoters (SlMTS1, SlTPS9, SlTPS3), the SlTPS7 promoter (expressed in various tissues) or the root specific SlTPS8 promoter. Co-infiltration
Chapter 4 112 with the 35S:SlEOT1 effector construct, now expressed in leaves, was predicted to transactivate the promoters with which it could interact, leading to GUS expression in this heterologous system. Initially, in order to confirm that none of the promoter:GUS constructs used gave background expression in N.benthamiana leaves (especially SlTPS7 that is expressed also in tomato leaves; Chapter 2), the assay was performed excluding the 35S:SlEOT1 effector construct. No GUS activity was detected for any of the mixtures infiltrated, except for the positive control SlMTS1p:GUS /35S:SlEOT1 reporter /effector mix (data not shown). Subsequently, the assay was performed including the 35S:SlEOT1 effector construct. SlEOT1-enhanced SlMTS1 reporter activity up to 5-fold compared to the CaMV 35S:RFP-infiltrated plants was detected, but not for any of the other terpene synthase promoters tested (Fig.7), indicating that SlEOT1 can specifically regulate SlMTS1. Furthermore, from the SlEOT1p:GUSsYFP1 /35S:SlEOT1 reporter /effector mix it became clear that SlEOT1 can’t bind and transactivate its own promoter (Fig.7).
Figure 7. Specific transactivation of SlMTS1 promoter by SlEOT1 in N.benthamiana leaves. Normalized GUS activity after co-infiltration with A. tumefaciens harboring the 35S:SlEOT1 effector construct and various promoter:GUS reporter constructs. The 35S:RFP effector construct was used as negative control. The bars represent obtained mean values and the error bars the standard error (n=3). RFP; red fluorescent protein. Bars with the same letters represent groups with the same mean according to ANOVA followed by Tuckey’s B posthoc test. Representative results from three experiments are shown.
Chapter 4 113 Interactions of SlEOT1 in planta and in yeast with the full-length or SlMTS1 promoter deletion fragments
SlEOT1 was identified as a protein binding to the 207bp trichome-specific fragment of SlMTS1 promoter in yeast (Chapter 3). As shown in Figure 8, SlEOT1 could interact also in planta with the 207bp SlMTS1 promoter fragment. Furthermore it was tested if in yeast it could also bind to the full-length or shorter SlMTS1 promoter fragments. To this end yeast cells with the full-length or various short promoter fragments integrated into their genome were transformed with pAD-GAL4-2.1_EOT1 and grown on selective medium. As shown in Figure 9, SlEOT1 could interact with the full-length promoter as well as the 207bp SlMTS1 promoter fragment, but none of the other promoter parts.
Figure 8. Transactivation of the full-length and 207bp SlMTS1 promoter by SlEOT1 in N.benthamiana leaves. Normalized GUS activity after co-infiltration with A.tumefaciens harboring the 35S:SlEOT1 effector construct and either the full-length (FL) or the 207bp MTS1 promoter:GUS reporter construct. The 35S:RFP effector construct was used as negative control. The bars represent obtained mean values and the error bars the standard error (n=3). RFP; red fluorescent protein. Bars with the same letters represent groups with the same mean according to ANOVA followed by Tuckey’s B posthoc test. Representative results from two experiments are shown.
Chapter 4 114
Figure 9. Interaction of SlEOT1 with the SlMTS1 promoter. Yeast cells with the full-length (FL) or shorter SlMTS1 promoter fragments A (-1254 to -1047bp), B (-1046 to -807bp), C (-806 to -613bp), D (-612 to -409bp), E (-408 to -208bp) and F (-207 to -1bp) integrated into their genome were transformed with pAD-GAL4-2.1_EOT1 and grown on SD medium with 5mM 3-AT.
Analysis of transgenic SlEOT1-overexpressing lines
The promoter of ShMKS1, a trichome-specific gene (Fridman et al., 2005) was used to overexpress SlEOT1 in stably transformed tomato plants. Fifteen independent lines were created and the SlEOT1 expression compared to untransformed Moneymaker (MM) plants was evaluated in isolated stem trichomes from T0 or MM cuttings. Initially, a forward primer was designed in the 5’UTR of ShMKS1 and a reverse primer in the coding sequence of SlEOT1. Almost all lines showed expression of the transgene, with lines 15, 7 and 11 having the highest levels. No product was formed, as expected, for the MM plants (black bars, Fig.10). Since only the open reading frame (ORF) of SlEOT1 (and not the UTRs) was used for the stable transformations’ construct, the endogenous levels of SlEOT1 were evaluated by designing a reverse primer in the 3’UTR of the gene (white bars, Fig.10). Since the efficiency was not identical between primers detecting the endogenous gene and the transgene however, the significance of the observed differences between transgene expression and endogenous levels in each line was not statistically tested but a new set of primers was designed in the ORF of SlEOT1 in order to compare the overall expression between transgenic lines and
Chapter 4 115 untransformed plants. However it appeared that the levels of SlEOT1 were not significantly altered, even in line 15 which showed the highest transgene expression (grey bars, Fig.10).
Figure 10. Expression of SlEOT1 in stably transformed overexpressing lines. Relative transcript levels in isolated stem trichomes for the overall expression of SlEOT1 (“ALL”, grey bars), the endogenous SlEOT1 expression (white bars) and the transgene expression driven by the ShMKS1 promoter (black bars), as determined by Q-RT-PCR in cuttings of 15 independent T0 lines and untransformed Moneymaker control. Mean values (+SD) of 2 biological replicas and 2 technical replicas are shown for the transgenic lines and of 3 biological and 3 technical replicas are shown for the Moneymaker plants, normalized for Actin expression. Bars with the same letters represent groups with the same mean according to ANOVA followed by Tuckey’s B posthoc test.
DISCUSSION
Secondary metabolite production is known to be under regulatory control of the pathway genes by transcription factors (Vom Endt et al., 2002). Well-studied examples come from experiments with the flavonoid pathway in various plant species (Broun et al., 2006), but our knowledge about transcription factors regulating the terpenoid pathway is somewhat limited, with the first direct evidence
Chapter 4 116 coming from the field of terpene indole alkaloid biosynthesis in Catharanthus roseus (Menke et al., 1999; van der Fits and Memelink, 2000). In Chapter 3 the identification of a putative transcription factor (TF) involved in the tomato (mono)terpene biosynthesis was described that was discovered as the predominant interactor in a screen of a trichome cDNA library with the SlMTS1 promoter in yeast. This gene was designated Emission of Terpenes 1 (SlEOT1) and was further characterized here. SlEOT1 and SlMTS1 were both expressed in the secreting cells of glandular trichomes (Fig.1 and Chapter 3; Fig.2), but unlike SlMTS1, SlEOT1 was not induced by jasmonic acid (Fig.2, Table 1). Gene regulation that leads to the coordinate secondary metabolite production being a complex process, the involvement of more than one TF is not uncommon or unlikely. For example, regulation of the strictosidine synthase (STR) involved in the terpene indole alkaloid biosynthesis in Catharanthus roseus, entails at least two transcription factors. Yeast-one-hybrid screen of the STR promoter detected two TFs, octadecanoid-derivative responsive Catharanthus AP2-domain (ORCA) 1 and 2 (Menke et al., 1999). ORCA1, in contrast with ORCA2, was not induced by MeJA and elicitor treatments and it was therefore speculated that this gene was not involved in JA- and elicitor-induced STR gene expression like ORCA2 but, based on the homology of the C-terminal region of this protein with the Arabidopsis DREB2A/B proteins, it was reasoned that it might be involved in drought- responsive gene expression. However it should be mentioned that ORCA1 failed to significantly transactivate STR promoter:GUS activity in C.roseus cells perhaps because it might require phosphorylation to exhibit enhanced binding (Menke et al., 1999). ORCA3, another MeJA-inducible AP2/ERF-domain TF, was isolated by T-DNA activation tagging and was shown to also bind to the STR promoter as well as to the tryptophan decarboxylase (TDC) and cytochrome P450 reductase (CPR) promoters (van der Fits and Memelink, 2000). It was speculated that ORCA2 and ORCA3 might have different functions but that are both required for the full-scale jasmonate-induced responses (van der Fits and Memelink, 2001). In the case of SlEOT1, as it is not induced by JA, it is possible that it is involved in the steady-
Chapter 4 117 state transcription of SlMTS1. Alternatively, SlEOT1 might be part of a transcriptional cascade involving an (upstream) jasmonate-inducible TF or another regulator that alters SlEOT1 protein levels and subsequently that of SlMTS1. Some TFs, although capable of binding DNA alone, are known to alter their specificity and/or association kinetics when bound with a cofactor and others are known to produce a much stronger effect on transcription when bound with interacting proteins (Wray et al., 2003). Although binding of SlEOT1 to the SlMTS1 promoter fragment was not confirmed in vitro by EMSA (Chapter 3), it was shown that it could interact with the 207bp and full-length SlMTS1 promoter in yeast (Fig.9). More importantly it could interact with the same promoter fragment and the full-length promoter also in planta (Fig.8), which indicates that SlEOT1 functions as a transcriptional activator. As discussed in Chapter 3, in yeast-one-hybrid screens also repressors can be identified, since the library proteins are all fused to an activation domain. However the fact that SlEOT1, when transiently expressed in N.benthamiana leaves, could induce the SlMTS1 promoter suggests that it functions as an activator. Its mode of action seems to be specific for SlMTS1, as it could not transactivate other terpene synthase (TPS) promoters tested (Fig.7), which however does not exclude that SlEOT1 can regulate other TPSs too, that were not investigated here. A more definitive conclusion about the role of SlEOT1 in terpene biosynthesis should be drawn from overexpressing or silencing it in stably transformed tomato plants. An initial attempt to overexpress this TF under the control of the trichome- specific ShMKS1 promoter failed to produce plants with elevated SlEOT1 expression (Fig.10). Transcript levels of SlMTS1 in the lines with the highest transgene expression (M15, M7, M11) were tested by Q-RT-PCR and were found not altered, also in JA-treated plants (data not shown). The most likely explanation for these observations is that the ShMKS1 promoter was not strong enough to significantly overexpress SlEOT1. Therefore, transgenic overexpressing and silencing lines have been created under the control of the strong, trichome-specific SlTPS9 (Chapter 2) promoter and are awaiting further characterization.
Chapter 4 118 The exact role of SlEOT1 in terpene biosynthesis as proven by stably transformed plants remains elusive, however its function as a transcription factor is supported by domains found in its sequence as well as by the experimental data presented so far. Transcription factors possess a DNA-binding domain that attaches to cis- elements in the promoters of the genes they regulate and a second domain, which can be either an activation or repressor domain (Wray et al., 2003). SlEOT1 was shown to function as a transcriptional activator (Fig.7 and 8). It contains three potential activation domains: two glutamine (Q)-rich regions and one acidic region cluster (Fig.5), both of which are known to function as such (Giniger et al., 1985; Courey and Tjian, 1988). Furthermore it was shown to localize in the nucleus (Chapter 3; Fig.6) and a putative nuclear localization signal (NLS) typically found in transcription factor proteins (Boulikas, 1994) is present in its N-terminal conserved domain (Fig.5). Most importantly, it was shown to bind DNA in yeast (Fig.9) and in planta (Fig.7 and 8) with the interaction probably taking place through its conserved zinc finger RING-like domain (Fig.5 and 6). It should be mentioned here however that the DNA binding activity of the RING finger domain is not conserved among proteins that contain this motif and such proteins are also known for example to act as E3 ubiquitin ligases like the mammalian Cb1 or have functions involving protein-protein interactions like the Arabidopsis COP1. However they exert such functions in combination with other structural modules found in their sequence (SH2 domain for Cb1; Freemont, 2000 and Coil region for COP1; Torii et al., 1998). SlEOT1 contains also a C-terminal IGGH domain (Fig.5 and 6) only found in the SHI family members (Fridborg et al., 2001) and in Arabidopsis, STY1 was also shown to be a DNA-binding transcriptional activator that is involved in auxin-mediated leaf and flower development (Sohlberg et al., 2006; Kuusk et al., 2006). The IGGH domain was shown to be responsible for intrafamily homo- or hetero-dimerization, however the activation domain (ARC; Fig.5) also found in this C-terminal region was required for the full transcriptional activation effect (Eklund et al., 2010b). Interestingly, neither a truncated STY1 protein lacking the putative DNA-binding domain (N-terminal) nor the one lacking
Chapter 4 119 the dimerization domain (C-terminal) could interact with the promoter of the flavin monooxygenase YUCCA (YUC4) gene, unlike the full-length protein (Eklund et al., 2010b) suggesting that several regions of STY1 are responsible for this interaction. Furthermore, STY1 was shown to be unable to bind to the 5’UTR of YUC4 but specifically interacted with an element found (upstream) in close proximity to the TATA box. This element or a variation of it, ACTCTA(C/A), was found in five of eleven YUC gene’s promoters but STY1 could only activate two of them (Eklund et al., 2010b), suggesting that there are more elements or factors involved. Based on the above, SlEOT1 probably exerts its function in a similar way. In the promoter fragment of SlMTS1 a YUC element can be found (Chapter 3; Fig.1), however it is in a part of the promoter, which SlEOT1 does not bind in yeast (Fragment C; Fig.9) but could potentially serve as a binding site for another family member. A similar element (ATTCTAT) is found in the promoter region of the 207bp minimal SlMTS1p fragment (position -110bp distal to the ATG) and could be the binding site of SlEOT1, which remains to be tested. Remarkably, although other SlEOT1 family members are also present in tomato trichomes (Table 1), the Y1H screens of the 207bp minimal SlMTS1p fragment with a trichome cDNA library identified only SlEOT1 (Table 1; Chapter 3) indicating a very specific interaction of SlEOT1 with the SlMTS1 promoter binding site. The SlEOT1 conserved DNA binding, dimerizing and activation domains in all likelihood function in a similar way as those of AtSTY1. However, the suggested function for the Arabidopsis and other plant species SHI family proteins in development and auxin biosynthesis is unlikely to hold true for SlEOT1, and perhaps the other tomato family members, also expressed in the trichomes (like the JA-inducible SGN-U563829), could be involved in terpene biosynthesis regulation too. The Arabidopsis SHI family members have been shown to have partially redundant functions in promoting gynoecium, stamen and leaf development (Kuusk et al., 2006) through regulation of auxin homeostasis (Sohlberg et al., 2006). SlEOT1 however was not expressed in those tissues but in the glandular trichomes of
Chapter 4 120 tomato. Only STY2 was reported to be expressed in the Arabidopsis (non- glandular) trichomes (Kuusk et al., 2002). Analysis of Genevestigator (https://www.genevestigator.com/gv/) data revealed that most of the SHI genes have a broad expression pattern (Topp and Rasmussen, 2012) and since sty1-1 sty2-1 shi3 lrp1 quadruple mutant develops leaf-like structures on which intact trichomes are found (Kuusk et al., 2006) these genes do not seem to play a role in regulating processes that take place in Arabidopsis trichomes. Moreover, although LRP1 is expressed and has a function in the root (Smith and Fedoroff, 1995; Krichevsky et al., 2008), its expression has been also detected in flowers where it appears to serve an additional role, since sty1-1 lrp1 double mutants enhanced the style phenotype of the sty1-1 single mutant (Kuusk et al., 2006). This indicates that Arabidopsis SHI family members can have distinct roles in various tissues, which could hold true for the tomato SHI family members as well. Furthermore, two Physcomitrella patens members of the SHI gene family have been also identified and overexpression or knockout of these moss genes led to changes in auxin homeostasis (Eklund et al., 2010a). Finally, also in Populus trichocarpa two SHI homologues were identified, and reduction of their expression through RNAi lines led to enhanced shoot and root growth, however the auxin levels remained unaltered (Zawaski et al., 2011). Interestingly, in petunia, which like tomato belongs to the Solanaceae family, PhEOT1 appeared to be expressed specifically in the trichomes (Fig.3). The Solanaceae members seem to form a separate clade in the phylogenetic tree (Fig.4) and it could be that PhEOT1 also regulates terpene biosynthesis, as petunia leaf washes and extracts contained terpenes (data not shown), possibly produced in the trichomes. However in general, sequence conservation between plants need not imply functional conservation, even between plants of the same family. Taken together, all the above-presented lines of evidence support that SlEOT1 functions as a transcription factor and analysis of the SlEOT1 silenced and overexpressing lines will shed more light into the role of this gene in the terpene biosynthesis.
Chapter 4 121 ACKNOWLEDGEMENTS
We thank Marjolijn Kelder and Esther van Mullekom (KeyGene N.V., Wageningen, NL) for the stable transformations, Mattijs Bliek (Vrije Universiteit, Amsterdam) for kindly providing the yeast strain PJ69-4A, Juan M. Alba Cano for help with the statistical analyses and Harold Lemeris, Ludeck Tikovsky and Thijs Hendrix for the excellent care of the tomato plants.
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Chapter 4 125 Chapter 5
RNA sequencing of Solanum lycopersicum trichomes identifies transcription factors that interact with terpene synthase promoters
Eleni A. Spyropoulou1, Raymond Hulzink2, Michel A. Haring1 and Robert C. Schuurink1
1Department of Plant Physiology, Swammerdam Institute for Life Sciences, University of Amsterdam, Science Park 904, 1098 XH Amsterdam, The Netherlands 2KeyGene N.V., Agro Business Park 90, 6708 PW Wageningen, The Netherlands
ABSTRACT
Glandular trichomes are production and storage organs of specialized metabolites such as terpenes, which play a role in the plant’s defense. The present study aimed to shed light on the regulation of terpene biosynthesis in Solanum lycopersicum by identification of transcription factors (TFs) that control the expression level of biosynthetic genes. A trichome transcriptome database was created with a total of 27,195 contigs, which contained 743 annotated transcription factors. Sixteen TFs were selected for further analysis, out of which two were shown to interact with terpene synthase promoters. For one, a MYC TF, binding to a monoterpene synthase promoter was confirmed in yeast and stably transformed overexpressing lines have been constructed in order to shed further light into the role of this TF in the regulation of terpene biosynthesis.
INTRODUCTION
Trichomes have interested scientists initially for the taxonomic classification of plant species since the seventeenth century. Over the decades the interest in these structures expanded to involve their biochemistry, differentiation and development as well as their physiological and ecological roles (Hallahan and Gray, 2000). Specialized glandular trichomes, produce and accumulate large quantities of terpenoids, phenylpropanoids, flavonoids and alkaloids, which they can also secrete (Schilmiller et al., 2008). Sequencing techniques in combination with metabolite profile analysis of glandular trichomes and proteomics techniques have shed light onto the biosynthesis of these specialized metabolites in the trichomes of various plant species (Tissier, 2012).
Chapter 5 127 Functional evaluation of expressed sequence tags (ESTs) from oil gland secretory cells of peppermint (Mentha x piperita; Lamiaceae) revealed that 25% of the ESTs code for enzymes involved in secondary metabolism. Since in mint oil glands the mevalonate (MVA) pathway is blocked, only enzymes of the methylerythritol (MEP) pathway were identified, that lead to the production of terpenoids- a highly represented class of compounds in these glands. Lipid transfer protein (LTP) homologues were also very abundant, whereas enzymes involved in the flavonoid metabolism formed a quantitatively less significant group in mint trichomes (Lange et al., 2000). In basil (Ocimum basilicum; Lamiaceae), 13% of the ESTs of a leaf peltate gland database belonged to genes of the phenylpropanoid pathway. Genes of the terpenoid pathways as well as lipid transfer proteins represented only 1% of the ESTs each (Gang et al., 2001). EST and micro-array analysis of alfalfa (Medicago truncatula; Fabaceae) glandular trichomes revealed that these glands primarily produce flavonoid compounds, with the second largest group being proteins of lipid metabolism or transport. Transcripts from the terpenoid biosynthetic pathway were underrepresented, in agreement with the fact that no terpenes have been detected in alfalfa trichomes (Aziz et al., 2005). In hop (Humulus lupulus; Cannabaceae) lupulin glands, three major classes of secondary metabolites have been found: essential oils, prenylated polyketides and prenylflavonoids. In a hop gland cDNA library enzymes from all biosynthetic pathways were identified in high abundance. LTPs were also highly expressed (Wang et al., 2008). Finally in a tobacco (Nicotiana tabacum; Solanaceae) trichome cDNA library, secondary metabolism accounted for only ~3% of the total metabolism-related ESTs, which seemed much lower than in other plant species and was attributed to primary metabolism-related genes being much more abundant and thus limiting the number of selected clones from the secondary metabolism (Cui et al., 2011). This however is not in accordance with other plant species where primary metabolism enzymes accounted also for a large number of the EST clones (e.g. 48% in mint oil gland secretory cDNA library; Lange et al., 2000).
Chapter 5 128 Although EST sequencing has been instrumental in the discovery of enzymes of trichome-specialized metabolism so far (Schilmiller et al., 2010), next generation sequencing (NGS), which is becoming widely available and cost-effective, can give a more in-depth picture of (trichome) transcriptomes. Apart from the large volume of data obtained, a major advantage of NGS technologies is perhaps that the DNA fragments do not need to be subcloned in a suitable vector, such as was necessary for Sanger sequencing, but can be used directly (Wilhelm and Landry, 2009) in one of the major, commercially available sequencing platforms to date: Genome Sequencer (454 Life Sciences, Roche), Genome Analyzer (Illumina) and Sequencing by Oligo Ligation and Detection (SOLiD; Applied Biosystems). Template in all three technologies is fragmented double-stranded DNA (genomic or cDNA). Roche’s 454 GS FLX Titanium system is based on emulsion PCR and pyrosequencing, producing reads of about 400bp with a sequencing accuracy of ~99% (http://www.454.com/index.asp). The Illumina Genome Analyzer (GA) system is based on solid-phase bridge PCR and a “sequencing by synthesis” approach and makes use of fluorescent dye-labelled reversible terminator nucleotides for imaging. Depending on the sequencing chemistry it can produce reads of 35-150 bases (http://www.illumina.com). Finally, the Applied Biosystems SOLiD technology is based on emulsion PCR in combination with sequencing by ligation with fluorescently labeled oligonucleotides, producing reads of 50-75 bases (http://www.appliedbiosystems.com). Each of these technologies has its advantages and disadvantages (Metzker, 2010), but since 454 sequencing produces the longest reads, it has been opted for creating transcriptomic databases by researchers (Brautigam and Gowik, 2010). Contigs are assembled based on the overlapping sequence of the reads and the identity within the overlapping region and this task becomes easier and more accurate with longer reads. Depending on whether a reference transcriptome (or genome) is available (and if this is desired) the reads can be mapped to the reference database and then the overlapping reads assembled into contigs. Alternatively, the reads can be
Chapter 5 129 directly assembled into contigs (de novo assembly; Garber et al., 2011). In quantification of transcripts and expression profiling, next generation sequencing overcomes limitations of micro-array experiments, such as probe cross- hybridization (Lister et al., 2009) and for such applications, technologies like that of Illumina or Applied Biosystems are perhaps better suited, as they produce a larger sequence output per run compared to the 454 Roche technology (18-30Gb compared to 0.45Gb per run; Metzker, 2010). Deep sequencing of cDNA using NGS technologies (termed RNA sequencing) is already being used for characterization of trichome transcriptomes- for example from plants of medical importance like Artemisia annua (Asteraceae; Wang et al., 2009) or Huperzia serrata and Phlegmariurus carinatus (Huperziaceae; Luo et al., 2010) and/or for gene discovery: 454 sequencing in combination with shotgun proteomics and metabolite analysis of tomato (Solanum lycopersicum; Solanaceae) trichomes led to the discovery of the leaf-trichome-specific β-charyophellene/α- humulene synthase (CAHS; Schilmiller et al., 2010) and 454 sequencing of trichomes deriving from wild and cultivated tomato varieties led to the discovery and characterization of various sesquiterpene synthases, providing insight into the evolution of terpene synthases (Bleeker et al., 2011). Terpene biosynthesis in tomato plants is of major interest as these compounds are believed to play a role in the plant’s defense (van Schie et al., 2007; Bleeker et al., 2009; Schilmiller et al., 2009; Kang et al., 2010) in this agronomically important crop. The sequencing of the cultivated tomato genome has enabled the characterization of its terpene synthase (TPS) gene family (Falara et al., 2011) but not much is known about the regulation of the terpenoid pathway. Transcriptional control of biosynthetic genes is a major mechanism by which secondary metabolite production is regulated (Vom Endt et al., 2002). Well-studied cases of transcription factors regulating plant secondary metabolic pathways involve the biosynthesis of flavonoids and terpenoid indole alkaloids (Broun et al., 2006). Here, we used 454 pyrosequencing of tomato stem trichomes as a tool for gene discovery. First, a transcriptome database was created from normalized cDNA,
Chapter 5 130 which was mined for transcription factors (TF). Then, in order to narrow down the number of TFs potentially involved in terpene biosynthesis, an expression profiling database was created using Illumina sequencing of trichome RNAs from plants treated with or without jasmonic acid (JA), since JA is known to regulate plant’s indirect defenses in response to herbivore feeding and wounding (Ballare, 2011). Two transcription factors were shown to transiently transactivate terpene synthase promoters in Nicotiana benthamiana leaves.
MATERIALS AND METHODS
Hormone Treatment and RNA Isolation
Tomato plants (Solanum lycopersicum cultivar Moneymaker) were grown in soil in a greenhouse with day/night temperatures of 23oC/18oC and a 16/8h light/dark regime for four weeks. They were then sprayed either with JA solution (1mM JA; Duchefa, NL, in tap water + 0,05% SilwetL-77; GE Silicones, VA, USA) or with control solution (0,05% SilwetL-77 in tap water). Stem pieces were collected 30min, 2h, 8h and 24h later for pyrosequencing or 24h later for expression analyses and trichomes were isolated by shaking the stems in liquid nitrogen. Total RNA was isolated using TRIzol (Invitrogen, Paisley, UK) according to the manufacturer’s instructions. Equal amount of trichome RNA from the different time points was pooled creating the control (C) and JA samples. RNA used for pyrosequencing was then purified on a RNeasy Plant column (Qiagen, Valencia, CA, USA).
Chapter 5 131 Transcriptome Database Construction
RNA quality was determined with the Agilent RNA pico chip (Agilent Technologies, Waldbronn, Germany). Synthesis and amplification of cDNA was performed using the SMART PCR cDNA Synthesis and Advantage 2 PCR kits (Clonthec Inc., CA, USA) according to the manufacturer’s instructions with some modifications of adapters to eliminate 3’ poly(A)-stretches prior to sequencing. cDNA quality was determined with the Agilent DNA 7500 chip (Agilent Technologies, Waldbronn, Germany) or on an 1% agarose/EtBr gel. Normalization of the cDNA was carried out using the Evrogen TRIMMER kit (Evrogen, Moscow, Russia) according to the manufacturer’s protocol with some modifications of the adapters. The normalization efficiency was determined both on an agarose/EtBr gel (1%) and with an Agilent DNA 7500 chip (Agilent Technologies, Waldbronn, Germany). Amplified cDNA was purified and concentrated using the Qiaquick PCR purification kit (Qiagen, Valencia, CA, USA). cDNA shearing and FLX Titanium library preparation was carried out using the Roche GS FLX Titanium General Library Preparation Method kit (Roche Diagnostics, Mannheim, Germany) according to the manufacturer’s protocol. The size range of the fragments was determined with an Agilent DNA 1000 chip (Agilent Technologies, Waldbronn, Germany). Exclusion of smaller-sized fragments was performed using the double SPRI method as described in the Roche GS FLX Titanium General Library Preparation protocol (Roche Diagnostics, Mannheim, Germany). End-polishing, small fragment removal, library immobilization, fill-in reaction and single-stranded library isolation was performed using the GS FLX Titanium General Library Preparation Method kit (454 Life Sciences, Roche Diagnostics, Mannheim, Germany) according to manufacturer’s instructions.
Chapter 5 132 Expression Profiling Database Construction
Starting from the same total RNA samples (C and JA, see above), mRNA was amplified and purified using the MessageAmp II aRNA Amplification kit (Applied Biosystems/Ambion, CA, USA) according to manufacturer’s instructions. RNA quality was determined with the Agilent RNA pico chip (Agilent Technologies, Waldbronn, Germany). Synthesis of cDNA was performed using the MessageAmp II aRNA Amplification kit (Applied Biosystems/Ambion, CA, USA) according to manufacturer’s instructions with modifications of the adapters to enable sequencing of 3’ cDNA ends. cDNA was purified with the Qiaquick PCR purification kit (Qiagen, Valencia, CA, USA). cDNA quality was determined with the Agilent DNA 7500 chip (Agilent Technologies, Waldbronn, Germany) or on an 1% agarose/EtBr gel. Shearing and ligation was carried out using standard Illumina PE adapters containing a specific sample ID tag. Adapter-ligated cDNA fragments were column purified with the Qiaquick PCR purification kit (Qiagen, Valencia, CA, USA). The size range of the fragments was determined with an Agilent DNA 1000 chip (Agilent Technologies, Waldbronn, Germany). Exclusion of smaller- sized fragments was performed using a single SPRI procedure as described in the Agencourt Ampure PCR Purification protocol (Agencourt Bioscience Corporation, MA, USA). The size range of single-stranded fragments was determined with an Agilent RNA pico 6000 chip (Agilent Technologies, Waldbronn, Germany). Expression profiling was performed using the Illumina GA II System (Illumina, USA).
Databases Assembly, EST Annotation and Homology Searches
The 454 sequencing reads (Control and JA combined) were assembled into contigs de novo by Vertis Biotechnologie AG, Germany using the CLCbio software (http://www.clcbio.com). Nucleotide sequences of the contigs were then blasted
Chapter 5 133 against the SGN tomato unigenes v2 database (ftp.solgenomics.net/unigene_builds/ combined_species_assemblies/tomato_species) for annotation, using a local Eblast tool (E value 1e–9). The GA II reads (Control and JA separately) were mapped to the annotated contigs of the 454 sequencing trichome database by Vertis Biotechnologie AG, Germany. The resulting contigs were also imported in the bioinformatics tool Blast2GO v.2.5.0 (Conesa et al., 2005) and were compared against the National Center for Biotechnology Information (NCBI) non-redundant protein database BLASTX (E value 1e–3). Further analyses with this tool included functional annotation by Gene Ontology (GO) terms and Enzyme Commission numbers (EC code), InterPro terms (InterProScan; Quevillon et al., 2005) and metabolic pathways (Kyoto Encyclopedia of Genes and Genomes, KEGG; Ogata et al., 1998).
cDNA Synthesis and Quantitative Real Time PCR
DNA was removed from RNA with DNAse (Ambion, Huntingdon, UK) according to the manufacturer and cDNA was synthesized from 1.5μg RNA using M-MuLV H- Reverse Transcriptase (Fermentas, St. Leon-Rot, Germany) in 20μl volume. For Q-RT-PCR, cDNA equivalent to 100ng total RNA was used as template in 20μl volume and reactions were performed in the ABI 7500 Real-Time PCR System (Applied Biosystems) using the Platinum SYBR Green qPCR SuperMix-UDG kit (Invitrogen, Paisley, UK) with the following cycling program: 2min 50oC, 7min 95oC, 45 cycles of 15sec at 95oC and 1min at 60oC, followed by a melting curve analysis. Primer pairs were tested for amplification kinetics and linearity with a standard cDNA dilution curve and new primers were designed if necessary. Expression levels were normalized using ACTIN (SGN-U579547) mRNA levels. Effect of JA in gene expression was analyzed in three biological replicates by T- test using PASW Statistics 17.0 (http://www.spss.com). The homogeneity of variance was tested by Levene’s test.
Chapter 5 134 Cloning and construct design
TFs SlMYC1, SlWRKY3 and SlWRKY4 (sequence of the full-length ORFs obtained from the 454 trichome database) were cloned between restriction sites NcoI (at the ATG) and SacI (at the 3’ end of the sequence) in vector pKG1662 (KeyGene, Wageningen, NL; for a map of the vector see patent nr US2011/0113512A1) driven by the CaMV 35S promoter. TF SlWRKY1 (sequence of the full-length ORF obtained from the 454 trichome database) was cloned downstream of the CaMV 35S promoter in vector pJVII, a pMON999 vector (Monsanto, St. Louis, MO) with a modified multiple cloning site, between restriction sites XbaI (at the ATG) and BsrGI (at the 3’ end of the sequence). The sequences of these four TFs are provided in Figure S1. All constructs were verified by sequencing and then the expression cassettes containing 35S promoter, cDNA of interest and nos terminator were transferred to the MCS of the binary vector pBINplus (van Engelen et al., 1995) between HindIII and SmaI restriction sites. The final constructs were transformed to Agrobacterium tumefaciens GV3101 (pMP90). The promoter:GUS constructs used in the transient transactivation assay have been described earlier (Chapter 4).
Transient transactivation assay in Nicotiana benthamiana leaves
A.tumefaciens GV3101 (pMP90) cultures were grown overnight from a single colony and diluted in infiltration buffer (50mM MES pH 5.8, 0.5% glucose, 2mM
NaH2PO4, 100μM acetosyringone; Sigma-Aldrich) to OD600 of 0.3. Leaves of five week old Nicotiana benthamiana plants were then infiltrated with mixtures carrying various promoter:GUS reporter constructs and the 35S:EOT1 effector construct in a 1:1 ratio. In order to normalize for transformation efficiency and protein extraction efficiency, a construct containing Photinus pyralis (firefly) luciferase (LUC) driven by the CaMV 35S promoter was co-infiltrated with the
Chapter 5 135 reporter/effector mix in a 2:5 ratio (LUC: reporter/effector mix). Two leaves of three plants were infiltrated for each combination. Two days later leaf disks from the infiltrated areas were collected, frozen in liquid nitrogen and crude extracts were prepared in extraction buffer containing 25mM Tris phosphate pH 7.8, 2mM DTT, 2mM CDTA pH 7.8, 10% glycerol and 1% Triton X-100. The enzymatic GUS activity was determined spectrophotometrically using 4-methylumbelliferyl β-D-glucuronide (MUG) as a substrate according to Jefferson et al. (1987). The luciferase assay was performed using the same extraction buffer, according to van Leeuwen et al. (2000) and measured in a FluoroCount Microplate Fluorometer (Packerd BioScience Company) using a 560nm emission filter. Enzymatic GUS activity was normalized for luciferase activity for each sample. Significant differences between samples were tested using PASW Statistics 17.0 (http://www.spss.com) by ANOVA followed by a Tuckey’s B posthoc test. The homogeneity of variance was tested by Levene’s test and values were log transformed before the analysis if necessary. The experiments were repeated at least twice with similar results.
Yeast-One-Hybrid Screen
The full-length SlMTS1 promoter or promoter fragments -1254 to -1047bp, -1046 to -807bp, -806 to -613bp, -612 to -409bp, -408 to -208bp and -207 to -1bp were cloned between restriction sites EcoRI (at the 5’ end of the sequence) and XbaI (at the 3’ end of the sequence) in the MCS of vector pHISi (Clontech, Mountain View, CA, USA) upstream of the minimal promoter of the HIS3 locus driving HIS3. The HIS3 gene of pHISi was used as selectable marker for integration into the nonfunctional his3 locus of yeast strain PJ69-4A. The vector was linearized at the XhoI restriction site before integration. Leaky HIS3 expression enabled enough colony growth, making it possible to use as a selectable marker. Background growth was controlled during library screening by the addition of 5mM 3-amino-
Chapter 5 136 1,2,4-triazole (3-AT) in the selective medium (synthetic dextrose, SD). Transformations were performed using 1μg of the library plasmid pAD-GAL 4-2.1 carrying the ORF of SlMYC1 cloned between restriction sites NheI (at the ATG) and XhoI (after the stop codon) in the MCS of the vector.
List of primers used
Primer name Sequence 5' -> 3' SlMYC1_QF2 TGCTGAATCGCGATGAAATTATGTC SlMYC1_QR2 GCCTCAACTCGAGATCTCTAGTA SlWRKY1_QF GGATCAGTCGTTGTACGGTG SlWRKY1_QR CTTCCTGACTTTTGGTTCCACG SlWRKY3_QF CAACAACATCAAGGTTCAGAATATAC SlWRKY3_QR TAGTCAATGCTACAAGAAACTTGTAC SlWRKY4_QF GAAGGTTACTGAGCCATTTCCAG SlWRKY4_QR AACCTGATTAATAACGCTGATACCAG SlACT_QF TCAGCACATTCCAGCAGATGT SlACT_QR AACAGACAGGACACTCGCACT
RESULTS
Assembly of 454 sequencing data and Genome Analyzer II transcript profiling
We have created a tomato trichome EST database by sequencing a mixture of glandular and non-glandular trichome RNAs, deriving from stems of Solanum lycopersicum cv. Moneymaker plants. The resulting cDNA was normalized before being used as input for 454 GS FLX Titanium pyrosequencing. A full plate was sequenced consisting of two halves: one with cDNAs originating from control plants and the other half with cDNAs originating from plants treated with JA. In total we obtained 979,076 high-quality reads with an average length of 337bp. The reads from control and JA samples combined were assembled de novo resulting in
Chapter 5 137 27,195 contigs with an average length of 931bp, leaving 24,187 reads unmatched (singletons), with an average length of 241bp. Nucleotide sequences of the contigs were blasted against the Solanaceae Genomics Network (SGN) tomato database for annotation, using a local E-Blast tool. 3,295 contigs were not annotated. For creating the transcript profiling databases with Genome Analyzer II, the same RNA material as for the 454 sequencing was used, but this time the cDNA derived from Control and JA-treated stem trichomes was not normalized before being used as input. From the Control sample 5,631,975 3’ sequences were obtained and from the JA sample 5,882,547, corresponding to one plate lane. 4,840,738 and 5,169,891 reads from the Control and JA-samples, respectively, were mapped uniquely to the 27,195 contigs of the trichome database. In addition, 38,699 (C) and 45,375 (JA) reads were mapped non-specifically and 791,237 (C) and 712,656 (JA) remained unmapped.
Homology searches, gene ontology and protein function
In order to dissect the S. lycopersicum stem trichome transcriptome the unique ESTs (27,195 contigs) were submitted to homology searches after translation (BLASTX) and were compared against the National Center for Biotechnology Information (NCBI) non-redundant protein database using Blast2GO (www.blast2go.com). 4,733 sequences did not return a BLASTX hit. The majority of the top hits were to protein sequences of Vitis vinifera, followed by Populus trichocarpa, Ricinus communis and Solanum lycopersicum. Next, gene ontology (GO) and enzyme classifications (EC) were performed in order to classify the ESTs. It must be noted that one sequence could be assigned to more than one GO term. An overview of the GO annotations obtained (at level 2 of 5) is presented in Figure 1 (level 1 giving a general description whereas level 5 a more specific description of a given gene therefore list coverage decreases at higher levels, whereas term specificity increases). The highest percentage of
Chapter 5 138 molecular function GO terms were involved in binding and catalytic activity (Fig.1c). In the biological processes, the majority of the GO terms was grouped into two categories- those of metabolic and cellular process (Fig.1b). Furthermore, for the cellular component class the assignments were mostly given to cell and organelle (Fig.1a). Finally, within the predicted ECs, the prevailing categories of enzymes were transferases and oxidoreductases (Fig.1d).
Figure 1. Gene ontology (GO) and enzyme classifications (EC) for S.lycopersicum stem trichome transcriptome at level 2. (a) Cellular component GO terms, (b) biological process GO terms, (c) molecular function GO terms and (d) general EC terms.
Chapter 5 139 The search of additional databases for protein families, domains, regions and sites was performed remotely from Blast2GO via the InterPro EBI web server. The 30 top InterPro entries obtained are presented in Table 1. The most dominant class of enzymes was protein kinases. Abundantly represented were also cytochrome P450s. Finally, within Blast2GO, the EC numbers were classified in KEGG pathways, enabling the presentation of enzymatic functions in the context of the metabolic pathways in which they are part of (Blast2GO Tutorial; Conesa and Goetz, 2009). Among the pathways identified, the ones related to secondary metabolism are shown in Table 2. Lipid transfer proteins represented 0.19% of the tomato stem trichome transcripts.
Table 1. Summary of the 30 most common InterPro entries found in the S.lycopersicum stem trichome transcriptome.
InterPro Frequency Description IPR011009 571 Protein kinase-like domain IPR000719 521 Protein kinase, catalytic domain IPR002290 336 Serine/threonine- / dual-specificity protein kinase, catalytic domain IPR008271 286 Serine/threonine-protein kinase, active site IPR020635 283 Tyrosine-protein kinase, catalytic domain IPR016040 263 NAD(P)-binding domain IPR013083 239 Zinc finger, RING/FYVE/PHD-type IPR017441 187 Protein kinase, ATP binding site IPR002885 181 Pentatricopeptide repeat IPR015943 175 WD40/YVTN repeat-like-containing domain IPR001841 172 Zinc finger, RING-type IPR012677 172 Nucleotide-binding, alpha-beta plait IPR016024 166 Armadillo-type fold IPR001245 164 Serine-threonine/tyrosine-protein kinase catalytic domain IPR000504 158 RNA recognition motif domain IPR001680 153 WD40 repeat IPR011046 152 WD40 repeat-like-containing domain IPR011990 147 Tetratricopeptide-like helical IPR011989 145 Armadillo-like helical IPR001128 141 Cytochrome P450 IPR017986 133 WD40-repeat-containing domain IPR017853 130 Glycoside hydrolase, superfamily IPR012287 119 Homeodomain-related IPR001611 115 Leucine-rich repeat IPR009057 115 Homeodomain-like IPR016196 112 Major facilitator superfamily domain, general substrate transporter IPR012336 108 Thioredoxin-like fold IPR013781 107 Glycoside hydrolase, subgroup, catalytic domain IPR002213 102 UDP-glucuronosyl/UDP-glucosyltransferase IPR002401 95 Cytochrome P450, E-class
Chapter 5 140 Table 2. KEGG pathways related to biosynthesis of secondary metabolites found in the S.lycopersicum stem trichome transcriptome.
KEGG pathway EC nr Enzyme name Nr of Sequences Terpenoid biosynthesis ec:1.1.1.208 (+)-neomenthol dehydrogenase 1 ec:4.1.1.33 diphosphomevalonate decarboxylase 3 ec:2.2.1.7 1-deoxy-D-xylulose-5-phosphate synthase 2 ec:1.17.1.2 4-hydroxy-3-methylbut-2-enyl diphosphate reductase 1 ec:2.7.7.60 2-C-methyl-D-erythritol 4-phosphate cytidylyltransferase 1 ec:2.5.1.1 dimethylallyltranstransferase 1 ec:1.17.7.1 (E)-4-hydroxy-3-methylbut-2-enyl-diphosphate synthase 1 ec:2.7.1.148 4-(cytidine 5'-diphospho)-2-C-methyl-D-erythritol kinase 1 ec:1.1.1.267 1-deoxy-D-xylulose-5-phosphate reductoisomerase 1 ec:1.1.1.34 hydroxymethylglutaryl-CoA reductase (NADPH) 6 ec:2.5.1.31 ditrans,polycis-undecaprenyl-diphosphate synthase 4 ec:2.3.3.10 hydroxymethylglutaryl-CoA synthase 6 ec:5.3.3.2 isopentenyl-diphosphate Delta-isomerase 2 ec:2.5.1.32 phytoene synthase 2 Phenylpropanoid biosynthesis ec:2.1.1.104 caffeoyl-CoA O-methyltransferase 4 ec:1.11.1.7 peroxidases 56 ec:3.2.1.21 beta-glucosidase 7 ec:2.1.1.68 caffeate O-methyltransferase 1 ec:1.14.13.11 trans-cinnamate 4-monooxygenase 1 ec:6.2.1.12 4-coumarate---CoA ligase 4 Flavonoid biosynthesis ec:2.1.1.104 caffeoyl-CoA O-methyltransferase 4 ec:2.3.1.74 naringenin-chalcone synthase 2 ec:1.14.11.23 flavonol synthase 7 ec:1.14.13.88 flavonoid 3',5'-hydroxylase 3 ec:1.14.11.9 flavanone 3-dioxygenase 1 ec:1.1.1.219 dihydrokaempferol 4-reductase 2 ec:1.14.13.21 flavonoid 3'-monooxygenase 3 ec:1.14.13.11 trans-cinnamate 4-monooxygenase 1 ec:5.5.1.6 chalcone isomerase 1 Alkaloid biosynthesis ec:4.3.3.2 strictosidine synthase 2 ec:4.1.1.28 aromatic-L-amino-acid decarboxylase 4 ec:1.14.11.20 deacetoxyvindoline 4-hydroxylase 1 ec:2.6.1.42 branched-chain-amino-acid transaminase 3 Steroid biosynthesis ec:1.14.21.6 lathosterol oxidase 1 ec:2.1.1.41 sterol 24-C-methyltransferase 1 ec:2.5.1.21 squalene synthase 2 ec:5.3.3.5 cholestenol Delta-isomerase 1 ec:2.1.1.6 catechol O-methyltransferase 1 ec:1.1.1.145 3beta-hydroxy-Delta5-steroid dehydrogenase 2 ec:1.14.14.1 unspecific monooxygenase 1 ec:1.3.99.5 3-oxo-5alpha-steroid 4-dehydrogenase 4
A closer look was taken at the terpene biosynthesis pathway in order to see if the precursor pathways were up-regulated by JA treatment. As shown in Figure 2a, expression of some precursor genes in tomato was induced by JA although not strongly (max induction ~2.5-fold for HDS). Like in other plants (Tholl and Lee, 2011), genes encoding enzymes of the precursor pathways are not always single copy and it appears that expression levels and JA inducibility can vary. Transcript abundance of precursor genes is presented in Figure 2b for comparison reasons with the expression levels of the terpene synthases (TPSs) found in stem trichomes. Transcripts for enzymes involved in the jasmonic acid biosynthesis and signaling pathway were also identified in the database. Obtained data for some of these enzymes are presented in Table 3.
Chapter 5 141 (a)
Abbreviation Name Chr SGN nr Reads Reads Fold JA C JA-induction AACT acetoacetyl-coenzyme A thiolase 5 SGN-U566720 2065 1550 1,33 7 SGN-U566719 743 752 0,99 HMGS 3-hydroxy-3-methylglutaryl-CoA synthase 8 SGN-U579858 88 90 0,98 8 SGN-U578388 1208 696 1,74 HMGR 3-hydroxy-3-methylglutaryl-CoA reductase 1 2 SGN-U580675 13 6 2,17 3-hydroxy-3-methylglutaryl-CoA reductase 2 2 SGN-U578017 113 125 0,90 3-hydroxy-3-methylglutaryl-CoA reductase 3 3 SGN-U579319 255 138 1,85 MVK mevalonate kinase 1 SGN-U567385 1170 704 1,66 pMVK phosphomevalonate kinase 8 SGN-U583971 280 309 0,91 MDC mevalonate diphosphate decarboxylase 4 SGN-U587221 20 18 1,11 11 SGN-U581971 157 200 0,79 DXS1 1-deoxy-D-xylulose 5-phosphate synthase 1 1 SGN-U567647 142 199 0,71 DXS2 1-deoxy-D-xylulose 5-phosphate synthase 2 11 SGN-U582996 219 128 1,71 DXR 1-deoxy-D-xylulose 5-phosphate reductoisomerase 3 SGN-U585813 3869 2801 1,38 MCT 4-diphosphocytidyl-2-C-methyl-D-erythritol synthase 1 SGN-U566797 635 684 0,93 CMK 4-diphosphocytidyl-2-C-methyl-D-erythritol kinase 1 SGN-U583224 1623 1338 1,21 MDS 2C-methyl-D-erythritol 2,4-cyclodiphosphate synthase 8 SGN-U568497 216 235 0,92 HDS 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate synthase 11 SGN-U567167 179 69 2,59 HDR 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate reductase 1 SGN-U580658 17159 15322 1,12 IDI isopentenyl diphosphate isomerase 5 SGN-U569721 20 11 1,82 4 SGN-U577516 5632 4219 1,33 GPS geranyl diphosphate synthase 8 SGN-U573523 16 19 0,84 NDPS neryl diphosphate synthase 8 SGN-U583641 16533 26450 0,63 FPS farnesyl diphosphate synthase 12 SGN-U580757 65 55 1,18 10 SGN-U578686 4 7 0,57 10 SGN-U581576 94 142 0,66 GGPS geranyl geranyl diphosphate synthase 4 SGN-U571085 36 42 0,86 9 SGN-U575882 374 372 1,01 2 SGN-U573348 101 196 0,52
(b)
TPS nr Contig nr Transcript Reads Reads Fold length(bases) JA C JA-induction 3 4235 2099 93 13 7,15 5 19389 2186 607 263 2,31 7 7522 1069 3 1 3,00 9 3548 2011 18005 13286 1,36 12 17073 407 24 15 1,60 16 10687 1868 108 95 1,14 17 10174 1190 25 20 1,25 19 14448 776 263 211 1,25 20 10263 1148 840 602 1,40 24 23265 854 1 1 1,00 31 4341 1991 4 1 4,00 39 10191 1131 71 32 2,22 41 3672 2368 421 375 1,12 Figure 2. (a) Enzymes involved in the biosynthesis of mono-(C10), sesqui-(C15) and di- (C20) terpenes and (b) terpene synthases found in stem trichomes. An explanation of the abbreviations used in the pathways and the GAII reads for each enzyme are shown in the tables. Genes upregulated (> 1.7x) and downregulated (< 0.6x) by JA treatment are shown in red and green respectively. Chr; chromosome.
Chapter 5 142 Table 3. List of selected enzymes involved in the jasmonic acid biosynthesis and signaling pathway (see references). Abbreviation Contig nr SGN Nr Annotation Transcript Reads Reads Fold length(bases) JA C JA-induction LOXAa 6402 SGN-U592535 Lipoxygenase A 2837 207 9 23,00 AOCa 24817 SGN-U562649 Allene oxide cyclase 1645 11170 2352 4,75 JAZ1b 6863 SGN-U579837 Jasmonate ZIM-domain 1 1156 72 4 18,00 JAZ3c 20751 SGN-U564446 Jasmonate ZIM-domain 3 986 111 13 8,54 COI1a 2260 SGN-U568988 Coronatine-insensitive 1 2260 65 48 1,35 a; Li et al., 2004: LOXA (U09026), AOC (AW624058), COI1 (NM_001247535), b; Thines et al., 2007: JAZ1 (EF591123), c; Katsir et al., 2008: JAZ3 (EU194561)
Selection of candidate transcription factors potentially involved in terpene biosynthesis
Based on the annotated contigs 743 transcription factors of different classes were found: 69 WRKY, 151 MYB, 8 MYC, 52 bZIP, 9 ARF, 71 ERF, 17 ZnF, 28 bHLH, 12 MADS, 1 NAC and 325 of unknown function/class. Out of those, 325 were up-regulated (>1.1x) by the treatment with JA, 208 were down-regulated (<0.9x) and expression of 210 TFs remained unaltered (1.1x - 0.9x). Since jasmonic acid is known to play a role in the plant’s direct and indirect defenses (Ballare, 2011) we were interested in those transcription factors that are induced by JA and could therefore potentially be involved in up-regulating terpene biosynthesis. 56 of the TFs that were up-regulated by JA showed an induction higher than 2-fold. The sequence of these 56 TFs was blasted against the tomato genomic sequence (Solanaceae Genomics Network, SGN) and complete ORFs were constructed when possible (if not provided by the 454 sequencing). These sequences were submitted to homology search after translation (BLASTX) against the National Center for Biotechnology Information (NCBI) database for identifying conserved domains. Out of this analysis 16 TFs were selected according to their class to be further investigated (Table 4) based on the published data presented below. There are only few transcription factors that are known to be involved in regulation of the terpenoid pathway. The first evidence came from Catharanthus roseus cells overexpressing ORCA3, a jasmonate-responsive APETALA2 (AP2)-domain
Chapter 5 143 transcription factor, that showed increased accumulation of terpenoid indole alkaloids (van der Fits and Memelink, 2000). In 2004 Xu et al. identified a methyl- jasmonate-inducible WRKY transcription factor from Gossypium arboreum that regulates the sesquiterpene synthase (+)-δ-cadiene synthase A in cotton fibers. In 2009 Ma et al. demonstrated that a methyl-jasmonate-inducible WRKY transcription factor from Artemisia annua is involved in the regulation of artemisinin biosynthesis. Finally, CrWRKY1 was recently identified as being involved in the root-specific accumulation of serpentine in C.roseus plants and as being induced by phytohormones including JA (Suttipanta et al., 2011). Based on this knowledge eleven transcription factors of the AP2 class and four of the WRKY class were selected for further investigation of their potential involvement in terpene biosynthesis. Lastly, a sixteenth gene was added to the list, a MYC transcription factor, since in Arabidopsis, AtMYC2, the first transcription factor shown to be involved in the transcriptional regulation of the JA signaling pathway, is known to regulate pathogen and wound response genes (Dombrecht et al., 2007).
Table 4. List of selected transcription factors (TF) potentially involved in terpene biosynthesis.
Name Contig nr SGN nr Annotation Transcript Reads Reads Fold length(bases) JA C JA-induction SlAP2_9 83 SGN-U572361 ERF (ethylene response factor) subfamily B-3 of ERF/AP2 TF family 371 57 23 2,5 SlAP2_2 1719 SGN-U596590 DREB subfamily A-1 of ERF/AP2 TF family 422 80 11 7,3 SlAP2_6 5289 SGN-U563871 AP2 domain-containing TF 933 267 72 3,7 SlAP2_3 7031 SGN-U563215 DREB subfamily A-1 of ERF/AP2 TF family 981 35 8 4,4 SlAP2_7 7865 SGN-U587768 DREB subfamily A-4 of ERF/AP2 TF family 775 13 4 3,3 SlAP2_4 10714 SGN-U586437 ERF (ethylene response factor) subfamily B-3 of ERF/AP2 TF family 611 16 4 4,0 SlAP2_10 14672 SGN-U585539 AP2 domain-containing TF 297 111 45 2,5 SlAP2_5 16204 SGN-U577088 ERF (ethylene response factor) subfamily B-4 of ERF/AP2 TF family 523 4 1 4,0 SlAP2_11 25582 SGN-U584756 ERF (ethylene response factor) subfamily B-2 of ERF/AP2 TF 660 7 3 2,3 SlAP2_1 25985 SGN-U586438 ERF (ethylene response factor) subfamily B-3 of ERF/AP2 TF family 942 96 3 32,0 SlAP2_8 26482 SGN-U581852 Ethylene-responsive element-binding factor 4 homolog 788 346 137 2,5 SlWRKY2 9827 SGN-U565154 WRKY family TF 1366 4 1 4,0 SlWRKY3 10561 SGN-U584367 WRKY family TF 1326 3 1 3,0 SlWRKY1 13200 SGN-U565157 WRKY family TF 1099 7 1 7,0 SlWRKY4 20918 SGN-U571278 WRKY family TF 1453 3 1 3,0 SlMYC1 24332 SGN-U576396 MYC TF 2174 191 128 1,5
Chapter 5 144
Tissue specificity and JA responsiveness of selected transcription factors
The candidate transcription factors (TFs) were selected from a sequencing trichome database as potential regulators of terpene biosynthesis. Ideally these TFs would be trichome-specific genes, induced by jasmonic acid. In order to investigate the expression pattern of these genes, cDNA was synthesized from different S.lycopersicum cv. Moneymaker tissues: leaves, stems, isolated stem trichomes and roots from 4-week-old plants, as well as flowers and fruit of mature plants. In Figure 3 expression data are presented for four of the sixteen selected transcription factors. For the other candidate TFs expression in the trichomes was much lower than that in other tissues (data not shown) and these were thus discarded from further analysis. TF SlMYC1 was predominately expressed in trichomes, but also in leaves and flowers (Fig.3a). SlWRKY1 was expressed in leaves, trichomes, roots and flowers (Fig.3b). SlWRKY3 was a trichome-specific gene (Fig.3c) and SlWRKY4 was expressed in trichomes, roots and fruit (Fig.3d). None of the selected transcription factors was significantly induced by JA according to the Q- RT-PCR data (Fig.3). SlWRKY4 expression appeared to be roughly 1.7-fold reduced in JA treated plants (p=0.07).
Chapter 5 145
Figure 3. Tissue specific expression and JA induction of selected TFs. Relative transcript levels for (a) SlMYC1 (b) SlWRKY1, (c) SlWRKY3 and (d) SlWRKY4 as determined by Q- RT-PCR. Mean values (+SE) of 3 biological replicas are shown, normalized for Actin expression. L; leaf, WS; whole stem, BS; bald stem, T; stem trichomes, R; root, Fr; fruit, Fl; flower; C; control and JA; jasmonic acid induced stem trichomes. ns; not significant according to T-test.
SlMYC1 and SlWRKY4 can transactivate terpene synthase promoters in Nicotiana benthamiana leaves
In order to investigate whether these TFs can interact with terpene synthase promoters, a transient assay was used in Nicotiana benthamiana leaves, which has
Chapter 5 146 been previously shown to work for the interaction between SlEOT1 and the SlMTS1 promoter (Chapter 4). In the reporter construct, expression of β- glucuronidase (GUS) was driven by the trichome specific promoter of SlMTS1 and therefore GUS activity was not detected in the leaf. However, co-infiltration with the 35S:SlEOT1 effector construct, which was expressed in leaves, transactivated the SlMTS1 promoter, leading to GUS expression in this heterologous system. As negative control a 35S:RFP construct was used. Various other reporter constructs with promoters of terpene synthases SlTPS3, SlTPS7 and SlTPS8 driving expression of β-glucuronidase (GUS) or GUSsYFP1 fusion (SlTPS9; Chapter 2) were used as well. As shown in Figure 4, SlWRKY4 could transactivate the SlMTS1 promoter albeit to a lower extent than SlEOT1. The SlTPS3 and SlTPS7 promoters were weakly transactivated by SlWRKY4. Transactivation of all tested promoters by SlWRKY1 and SlWRKY3 was similar to that of the negative control (35S:RFP) (data not shown).
Figure 4. Transactivation of terpene synthase promoters by SlWRKY4 in N. benthamiana leaves. Normalized GUS activity after co-infiltration with A. tumefaciens harboring the 35S:SlEOT1 (positive control) or the 35S:SlWRKY4 effector construct and various promoter:GUS reporter constructs. The 35S:RFP effector construct was used as negative control. The bars represent the obtained mean values and the error bars the standard error (n=3). RFP; red fluorescent protein. Bars with the same letters represent groups with the same mean according to Tuckey’s B posthoc test. Representative results from two experiments are shown.
Chapter 5 147 The other TF investigated, SlMYC1, could transactivate all terpene synthase promoters tested except SlTPS8 and interaction with the trichome specific SlMTS1 and SlTPS3 promoters was the strongest (Fig.5). However it should be noted that GUS activity of a promoter driving the GUSsYFP1 fusion was lower than when the same promoter driving GUS alone was transactivated by an effector construct (data not shown), possibly because the fusion protein was less stable or because it was more difficult to be synthesized. Therefore, interaction of SlMYC1 with the trichome-specific SlTPS9 promoter is potentially stronger than that shown here.
Figure 5. Transactivation of terpene synthase promoters by SlMYC1 in N. benthamiana leaves. Normalized GUS activity after co-infiltration with A. tumefaciens harboring the 35S:SlEOT1 (positive control) or the 35S:SlMYC1 effector construct and various promoter:GUS reporter constructs. The 35S:RFP effector construct was used as negative control. The bars represent the obtained mean values and the error bars the standard error (n=3). RFP; red fluorescent protein. Bars with the same letters represent groups with the same mean according to Tuckey’s B posthoc test. Representative results from three experiments are shown.
Furthermore it was investigated whether this transcription factor could interact with individual promoter fragments of the model SlMTS1 promoter in yeast. As shown in Figure 6, SlMYC1 could interact only with the full-length promoter and not with the various promoter fragments. These data suggest either the existence of two
Chapter 5 148 binding sites necessary for activity or that the binding site was interrupted in the promoter deletion fragments.
Figure 6. Interaction of SlMYC1 with the SlMTS1 promoter. Yeast cells with the full-length (FL) or shorter SlMTS1 promoter fragments A (-1254 to -1047bp), B (-1046 to -807bp), C (-806 to -613bp), D (-612 to -409bp), E (-408 to -208bp) and F (-207 to -1bp) integrated into their genome were transformed with pAD-GAL4-2.1_SlMYC1 and grown on SD medium with 5mM 3-AT.
Since SlEOT1, SlMYC1 and SlWRKY4 were shown in independent experiments to be able to transactivate the SlMTS1 promoter, we investigated what effect a combination of these transcription factors would have on the transactivation of SlMTS1 promoter. To this end, Agrobacterium cultures carrying the CaMV 35S- driven effector constructs were mixed in pairs or all three together and combined with the SlMTS1p:GUS reporter construct in N. benthamiana leaves (Fig.7). Interestingly, co-expression of SlEOT1 and SlMYC1 almost tripled the transactivation of SlMTS1 promoter compared to the effect of each TF alone. Adding SlWRKY4 did not have an additional effect, but rather seemed to have a negative effect on the combinatorial action of the other two TFs exerted on the SlMTS1 promoter, although not at a statistically significant level.
Chapter 5 149
Figure 7. Transactivation of SlMTS1 promoter by SlEOT1, SlMYC1, SlWRKY4 or combination thereof in N. benthamiana leaves. Normalized GUS activity after co- infiltration with A. tumefaciens harboring the 35S:SlEOT1 (positive control), 35S:MYC1, 35S:SlWRKY4 effector constructs or combination thereof and the SlMTS1p:GUS reporter construct. The 35S:RFP effector construct was used as negative control. The bars represent the obtained mean values and the error bars the standard error (n=4). RFP; red fluorescent protein. Bars with the same letters represent groups with the same mean according to Tuckey’s B posthoc test. Representative results from two experiments are shown.
DISCUSSION
Although trichomes constitute a small fraction of a plant’s total tissue, they received distinctive attention for their ability to synthesize, store and secrete specialized metabolites. Through the production of mainly EST libraries, as well as micro-arrays and more recently high-throughput sequencing of (glandular) trichomes, research has focused on the expression of genes involved in the terpenoid, phenylpropanoid, alkaloid and flavonoid biosynthesis in various plant species, including tomato (McDowell et al., 2011; Schilmiller et al., 2010; Besser et al., 2009), sweet basil (Xie et al., 2008; Gang et al., 2001), tobacco (Cui et al., 2011; Harada et al., 2010), mint (Lange et al., 2000), alfalfa (Aziz et al., 2005), Artemisia annua (Wang et al., 2009) and hop (Wang et al., 2008).
Chapter 5 150 Here we used massive parallel pyrosequencing on the 454 GS FLX Titanium platform to sequence S.lycopersicum stem trichome RNAs with the ultimate goal to identify transcription factors that are involved in terpene biosynthesis. In order to obtain a broad transcriptomic database optimal for gene discovery, it was deemed necessary to use normalized cDNA, so as to maximize representation of low abundant transcripts and reduce representation of highly abundant transcripts. Initial attempts to map the obtained reads to publicly available EST databases led to a high percentage of unmapped reads and assignment of the same reads to multiple unigenes and therefore the reads were in the end assembled de novo. 2.47% of the reads could not be matched and were not used in further analysis. 87.9% of the resulting contigs were subsequently annotated after blasting against the SGN tomato database using a local E-Blast tool. First, in order to obtain a general overview of the transcripts that can be found in the tomato stem trichomes we used the functional annotation workstation Blast2GO. When blasting against the NCBI database through this automated software 82,6% of the contigs were annotated. Blast2GO provides a collection of tools that enable the assignment of Gene Ontology terms as well as Enzyme Classification codes with KEGG maps and InterPro motifs to the submitted sequences. However, for the data mining and the interpretation of the statistical results it must be kept in mind that one sequence can be assigned to more than one GO term. According to the InterPro entries (Table 1) by far the highest represented in the S.lycopersicum stem trichomes are protein kinases, which is not surprising since protein phosphorylation is a process implicated in responses to various signals and many regulatory enzymes are controlled by reversible phosphorylation (Stone and Walker, 1995). In addition, there are many proteins that contain WD40 repeats. Such proteins could have various roles in the trichomes as they are known to regulate diverse cellular functions, like cell division and trans-membrane signaling (Neer et al., 1994) and some could participate in the trichome formation, as in Arabidopsis, TRANSPARENT TESTA GLABRA1 (TTG1), a WD40-repeat protein, is one of the partners in the complex responsible for trichome initiation
Chapter 5 151 (Ishida et al., 2008). Moreover, several tomato stem trichome proteins appear to have zinc-finger domains. Since zinc finger-containing proteins are involved in a plethora of processes such as DNA recognition, transcriptional activation, protein folding and assembly and lipid binding (Laity et al., 2001) it is hard to speculate the particular function of these proteins in the trichomes. Furthermore, proteins with Armadillo-type repeats are also highly represented in the tomato stem trichomes. The Armadillo proteins are known to be involved in intra-cellular signaling and in Arabidopsis they have been shown to promote lateral root development (Coates et al., 2006). Finally, among the highly represented proteins are also cytochrome P450s, enzymes that are important for the biosynthesis of several compounds, such as hormones and defensive compounds involved in plant- insect interaction (Schuler, 2010). In order to investigate whether in stem trichomes of tomato Moneymaker plants, jasmonic acid (JA) regulation of terpene biosynthesis is also on the precursor level except on the level of individual TPSs (Falara et al., 2011; Chapter 2), the quantitative database was mined for the genes encoding enzymes of the precursor pathways. The copy number of these genes varies between different plant species (Tholl and Lee, 2011) and as shown in Figure 2 different family members can vary in their expression levels and/or JA-inducibility. For example, although the methylerythritol (MEP) pathway enzymes are generally single copy (Phillips et al., 2008), 1-deoxy-d-xylulose 5-phosphate synthase (DXS) in contrast with Arabidopsis, which contains a single functional gene, has diversified into two isogenes in other plant species among which is tomato (Walter et al., 2002). Interestingly, while SlDXS1 is ubiquitously expressed, SlDXS2 is expressed only in a few tissues and in leaf trichomes its transcript abundance is much higher than that of SlDXS1 (Paetzold et al., 2010). Furthermore, SlDXS2 is moderately induced by wounding in cultivar Moneymaker (Paetzold et al., 2010), which correlates with the observed moderate induction of SlDXS2 by JA (1.7-fold, Fig.2). However, in cultivar Castlemart Manduca sexta larvae feeding up-regulates SlDXS2 expression approximately threefold (Sanchez-Hernandez et al., 2006). The regulation of
Chapter 5 152 precursor enzymes of the MEP pathway by wounding, hormones or elicitors has been demonstrated in various plant species (Sanchez-Hernandez et al., 2006; Okada et al., 2007; Oudin et al., 2007; Arimura et al., 2008; Kim et al., 2009; Paetzold et al., 2010). Evidence for the regulation of precursor enzymes of the mevalonate (MVA) pathway is also abundant (Yang et al., 1991; Choi et al., 1994; Ha et al., 2003; Hui et al., 2003; Kondo et al., 2003; Bede et al., 2006). For example, HMGR enzyme activity and protein level was shown to be increased by fungal infection in potato tubers and sweet potato root (Kondo et al., 2003). Furthermore, HMGR1 transcripts were induced by treatment with methyl jasmonate also in potato, whereas HMGR2 transcripts were reduced (Choi et al., 1994). However, in response to caterpillar herbivory, transcripts of alfalfa HMGR1 were reduced (Bede et al., 2006). In tomato stem trichomes HMGR1 and HMGR3 were induced by JA treatment approximately 2-fold, whereas expression of HMGR2 remained unaltered (Fig.2). None of the prenyl diphosphate synthases was induced in tomato trichomes by JA treatment, whereas two seemed to be downregulated (SGN-U578686; FPS and SGN-U573348; GGPS; Fig.2). However, expression of GGPS1 (SGN-U574849; transcripts not found in the stem trichome database) in tomato cv. Castlemart leaves was induced by spider mite feeding and JA (Ament et al., 2006) and in pepper roots FPS was induced by fungal infection (Ha et al., 2003). The emerging picture indicates that intermediates of the MEP and MVA pathways are subject to differential regulation between species and it would be interesting to confirm the obtained data for the Moneymaker stem trichomes by Q-RT-PCR.
As mentioned above, the primary goal of this sequencing run was to identify transcription factors that regulate terpene biosynthesis. Based on the annotated contigs (blasted against the SGN database) 2.7% of the transcripts in the tomato stem trichomes encode transcription factors. For comparison, in Arabidopsis thaliana ~6% of the genes in all tissues encode TFs (TAIR10 genome release, http://arabidopsis.org). In order to narrow down the number of transcription factors
Chapter 5 153 to those that could potentially be involved in plant defense, a quantitative transcript profiling database was created by sequencing trichome RNAs from plants treated with JA or control solution. Terpene production and/or release are part of the plant’s indirect defenses, which involve emission of herbivore-induced volatiles that can be recognized by carnivorous and parasitoid insects. These defense responses to chewing arthropods are coordinated mainly by JA (Ballare, 2011) and therefore it was hypothesized that TFs involved in the regulation of terpene biosynthesis could also be JA-inducible genes. The transcription factors known to be involved in regulation of the terpenoid pathway (CrORCA3; van der Fits and Memelink, 2000; GaWRKY1; Xu et al., 2004; AaWRKY1; Ma et al., 2009; CrWRKY1; Suttipanta et al., 2011) are all jasmonate-inducible genes. However, from the field of JA signaling research in Arabidopsis, it was recently discovered that two previously unidentified MYC transcription factors (AtMYC3 and AtMYC4) are also direct targets of JAZ repressors and act additively with AtMYC2 in the activation of JA responses. Interestingly, in contrast to AtMYC2, AtMYC3 and AtMYC4 are only marginally induced by JA treatment (Fernandez- Calvo et al., 2011). Based on all the above, the initial selection of transcription factors to be analyzed from our quantitative stem trichome database was limited to TFs of the WRKY and AP2 class that showed a 2-fold or higher induction by JA treatment (2.3-fold was the induction rate of control gene SlMTS1). None of the MYC transcription factors of our database showed induction higher than 2, so for further analysis the closest homolog of AtMYC2 was selected. After discarding TFs that were not trichome-specific or at least showed highest expression in trichomes, the list was narrowed down to four candidate transcription factors. According to the Q-RT-PCR data however, none of these TFs was significantly induced by JA treatment (Fig.3). This could be explained in two ways. First, since the read count of these genes is very low both in the Control and JA samples (Table 4), perhaps the fold induction is over-estimated as 1 read versus 3 or 7 need not necessarily translate to a significant induction. Alternatively, since the RNA material used in the GAII sequencing run originated from pooled trichomes collected 30min, 2h, 8h
Chapter 5 154 and 24h after JA treatment whereas the material for Q-RT-PCR was collected 24h after treatment, it is possible that these TFs show higher induction at an earlier time point, which remains to be tested. A further and more specific indication of whether these TFs are involved in the terpene biosynthesis pathway would be to observe an interaction between the transcription factor and a terpene synthase. In transient transactivation assays in N.benthamiana leaves two of the four selected transcription factors were able to transactivate at least one terpene synthase promoter. SlWRKY4 showed strongest interaction with SlMTS1 and in lesser extent with SlTPS3 and SlTPS7. Although this TF is expressed highly in roots (Fig.3), it could not interact with the root- specific SlTPS8. It could also not interact with the trichome-specific sesquiterpene synthase SlTPS9 so it is possible that SlWRKY4 can interact only with monoterpene synthase promoters or at least not the sesquiterpene synthase tested here (Fig.4). As shown in Table 5 this TF and the respective TPSs that it can transactivate are co-expressed in various tissues where the regulation could take place in the plant. SlMYC1 showed strongest interaction with SlMTS1 and SlTPS3 and to a lesser extent with SlTPS7 and SlTPS9 but no interaction with SlTPS8 (Fig.5), although this TF is also expressed in the root albeit not strongly (Fig.3). As shown in Table 5 SlMYC1 is expressed (at different levels) in every plant tissue and is able to transactivate all the terpene synthase promoters tested except one, so it seems to be a regulator of multiple TPSs, in contrast to SlEOT1 that is only expressed in the trichomes and can specifically interact with the SlMTS1 promoter and none of the other TPS promoters tested.
Chapter 5 155 Table 5. Expression patterns and interaction overview of TFs and TPSs. Positive interaction of the transcription factors with the respective SlTPS promoters in N.benthamiana leaves is indicated by a colored box that represents the tissue in which they are co-expressed. Expression in the various tissues is indicated by +++, ++, +, +/- and -- according to Q-RT-PCR values. Darker shaded boxes indicate a stronger transient interaction between the TF and TPS.
Leaf Stem Trichomes Root Fruit Flower
SlWRKY4 -- -- ++ ++ + +/-
SlMTS1 -- -- ++ ------
SlTPS3 -- -- ++ +/- -- +/-
SlTPS7 + +/- -- +/- -- ++
SlTPS8 ------++ -- --
SlTPS9 -- -- +++ ------Leaf Stem Trichomes Root Fruit Flower
SlMYC1 + +/- ++ +/- +/- +
SlMTS1 -- -- ++ ------
SlTPS3 -- -- ++ +/- -- +/-
SlTPS7 + +/- -- +/- -- ++
SlTPS8 ------++ -- --
SlTPS9 -- -- +++ ------Leaf Stem Trichomes Root Fruit Flower
SlEOT1 -- -- + ------
SlMTS1 -- -- ++ ------
SlTPS3 -- -- ++ +/- -- +/-
SlTPS7 + +/- -- +/- -- ++
SlTPS8 ------++ -- --
SlTPS9 -- -- +++ ------
The interaction of SlMYC1 with the promoter of SlMTS1 in planta was confirmed also in yeast (Fig.5 and 6), however the binding region of this TF could not be identified. In the promoter sequence of SlMTS1 (Fig.1, Chapter 3) there are two G- box-like elements (CACATG instead of the canonical CACGTG), one T/G-box element (AACGTG) and one T/G-box-like element (TACGTG), which could potentially be the binding site(s) of SlMYC1. However even though the promoter deletion fragments used in yeast to test binding of SlMYC1 did not interrupt any of
Chapter 5 156 these motifs (Fig.6) no interaction was observed, suggesting that perhaps this TF has a binding site that has not been described before. The promoter of SlTPS3, with which SlMYC1 interacts less strongly, contains one G-box-like element at position -1315 bases upstream of the ATG start codon and one T/G-box element at position -147 bases. The SlTPS7 promoter, with which it can also interact, albeit even less strongly, contains one T/G-box at position -260 bases upstream of the ATG start codon. The SlTPS9 promoter however, does not contain any of these elements, further indicating the existence of an uncharacterized motif on which SlMYC1 binds. When using the naïve motif search program MEME (http://meme.sdsc.edu/meme/intro.html) for DNA sequences that are present in all four promoters which SlMYC1 can transactivate transiently in N.benthamiana leaves, one 8bp motif was identified in the plus or minus (for SlTPS9) orientation: CTAGG(T/A)(A/G)G. The validation of this putative regulatory element or of the G-box-like or T/G-box motifs as the binding site(s) for SlMYC1 in the respective promoters would require further experimentation. Most strikingly, it was shown that SlEOT1 and SlMYC1 act synergistically in the activation of the SlMTS1 promoter (Fig.7). Combinatorial control of transcriptional regulation is commonly found in plants and other eukaryotes (Singh, 1998). For example, in abscisic acid (ABA) signaling, the 67bp promoter region of the dehydration-responsive gene rd22 contains a MYC and a MYB recognition site, where AtMYC2 and AtMYB2 bind. In Arabidopsis leaf protoplasts it was shown that these TFs could individually activate transcription of β-glucuronidase driven by this 67bp promoter region of rd22 and that the transient transactivation was stronger when AtMYC2 and AtMYB2 were combined (Abe et al., 1997). Transgenic plants overexpressing these TFs each showed ABA hypersensitivity but the effect was more profound in plants overexpressing both cDNAs (Abe et al., 2003). In order to investigate in-depth the role of SlMYC1 in terpene biosynthesis, transgenic overexpressing plants have been created under the control of the strong, trichome-specific SlTPS9 (Chapter 2) promoter and are awaiting further analysis. T0 plants will be crossed with T0 individuals from the SlEOT1 overexpressing
Chapter 5 157 plants (Chapter 4) so as to explore the combinatorial effect of these transcription factors in tomato plants.
ACKNOWLEDGEMENTS
We thank Mattijs Bliek (Vrije Universiteit, Amsterdam) for kindly providing the yeast strain PJ69-4A, Carlos Villarroel Figueroa for help with the bioinformatics, Juan M. Alba Cano for help with the statistical analyses and Harold Lemeris, Ludeck Tikovsky and Thijs Hendrix for the excellent care of the tomato plants.
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Chapter 5 163 Yang Z, Park H, Lacy GH, Cramer CL (1991) Differential activation of potato 3-hydroxy- 3-methylglutaryl coenzyme A reductase genes by wounding and pathogen challenge. Plant Cell 3 (4):397-405
SUPPLEMENTAL DATA
Figure S1. Nucleotide sequence of transcription factors SlWRKY1 (SGN-U565157), SlWRKY3 (SGN-U584367), SlWRKY4 (SGN-U571278) and SlMYC1 (SGN-U576396).
SlWRKY1 atgGAAGATAGTTCATACAAAAATCTATTTTTTCATAAACAAGAAGATTCCACCGGAACTCCACCGGA TAATGCTGCTGATTCTTGTTTTTCCGGTGATGAAGCGGCGGAAGTTAGCATGCCATCACCTAGAAAA AGTAGGAGAGGAGCAAAAAAGAAAGTAATTTCAGTGCCAATAATTGAAGGTGATGGATCAAGAAGT AAAGGGGAAGTTTATCCACCACAAGATTCTTGGTCTTGAGAAAATATGGACAAAAACCAATTAAAG GATCACCTTATCCCAGGGGATATTATCGATGCAGTAGCTCCAAAGGCTGTCCCGCCAGAAAACAAGT CGAGCGTAGCCGCCTGGACCCCACCATGCTTCTCATTACCTATTGCTCCGAACACAATCACCAAATCC CGGCCGCCGCCGCCGCCAAACACCACCATCACAACCACCCTACTACCACCACCAGTTCACCCACTAC CTCTACCGGCACCGCCGGAGACAATAATGCTACTGCGGCTGTAGCCACGGACTCTACCGTAGTAGAC AAATCATCCCCCGAAGAACCAGATCTATTTGCTTATCAACACGATAATGGATTTTCAGAGCTCGCCG GTGAGTTAGGTTGGTTTTCCGATATGGGAACAACAACTACGTTTATGGAGAGTACGTCGTCGTCCAT GGCGGGATCCACGTGGAACGACAGTGACGTGGCGTTAATGTTGCCGATTCGAGAAGAGGATCAGTC GTTGTACGGTGATCTCGGTGAATTACCGGAATGTTCAGTTGTTTTCCGGCGGTACAGTGTGGAAACTC CCTGCTGCGGAGGTACAGGAtaa
SlWRKY3 atgTCTGATAATCCTTTTTATCATGATTACATGGGAATTGGTGGAGGGATCAATAATACATTCCCTTTTT TGGTTGAAAATCCTTCAAATTATAATAACCAACCAATAACTCCAAATATTCAAAATCAACAATTTGTT CCTTCTTCTTATATGACTCTCACTGAGTGTTTACATGGCTCTATGGACTACAACACACTATCAAATGC TTTTGGTATGTCTTGTTCATCATCATCTGAAGTTGTTTGTCCACATATCGATAATCAAGACTCTACTGA AAAATATAACGTCTCTACTACTGCAGAAGAACCTGTCTTAGATTCACCTCTAGGAGATAATCAAACG AGCGTTGAAGTTCCACCAACGCCAAATTCTTCGATATCTTCTACTTCTAATGAGGTTGGAGAGCAAG AAGATTCTTCCAAAATCAAGAAACATGTTCAAGTAAAAGGGAGCCAAGAAGAAGGAGAAGACAAGT CTAAGAAAGAGTGCATAGCAAAAAAGAAAGGAGAAAAAAAGATAAAAGAACCAAGATTTGCCTTC ATGACAAAGAGTGAGATTGATAATCTTGAAGATGGTTATCGATGGAGAAAATATGGACAAAAGGCA GTGAAGAACAGCCCTTTTCCTAGGAACTATTATAAATGCACAACTCAAAAGTGCAATGTGAAGAAAC GTGTTGAAAGGTCATATGAAGATTCATCAATTGTGATAACAACATATGAAGGCCAACACAATCATCA TTGTCCAGCAACTCTTAGGGGAAATGCATCTTTTTTATCTTCATCACATTTTATGCCTAATTTTCCTCC ACAACTATTTTCCCAAATGCTAAATATTCCACCAAACAACCAAAATCTCCTCATTACTTCTTCGACCT ATAATTATAATAATAATAATAACAATTATTATTATCAACAACAACAACATCAAGGTTCAGAATATAC CCTATTTGGTGGAGGCACAAATAATGGTGATGCTTCATGGGTCCAAAAACAAGAGCCATCTtag
SlWRKY4 atgGAAGAAGTAATGGAAGAAAATCAAAGGCTAAAGAAACACTTAGATAAAATCATGAAGGATTATC GGAATCTTCAAATGCAATTCCATGAAGTTGCACAAAGAGATGCTGAAAAAACTAATACTGATGTTAA ACATGACGAAGCTGAACTTGTTTCCCTTAGCCTAGGAAGGACTTCAAGTGACACAAAAAAGGAGTTA
Chapter 5 164 TCCAAATTAATTTTGAGCAAAAAAGAGAATGATGAGAAGGAAGAAGATAACCTAACCCTAGCATTA GATTGCAAGTTTCAATCATCGACCAAATCTTCACCATCAAATCTCAGTCCAGAGAATAGTTTAGGAG AAGTGAAGGACGACGAAAAAGGAACTGATCAAACATGGCCACCTCACAAAGTTCTCAAGACGATAA GGAATGAAGAAGATGATGTGACACAACAAAACCCTACTAAAAGAGCAAAGGTTTCCGTCAGAGTTA GATGTGACACCCCAACGATGAACGATGGATGTCAATGGAGAAAATATGGACAAAAAATTGCAAAAG GGAACCCATGTCCTAGAGCTTACTACCGTTGCACGGTAGCACCAAATTGCCCCGTAAGAAAACAGGT ACAAAGATGCATTCAAGACATGTCTATATTGATCACAACATACGAAGGAACACATAACCATCCACTT CCTCATTCAGCCACATCAATGGCTTTCACCACTTCAGCAGCCGCTTCCATGCTATTATCCGGTTCATC CAGCTCCGGATCGGGCCCCACAAGTAGTACAGCCTCTGCCACAACCTCTGCGCTCAACTATTGCTTCT CTGATAACTCAAAACCAAACCCATTTTATAACCTTCCACATTCATCCATTTCTTCATCATCACATTCTC AGTACCCTACAATCACTCTTGATTTAACATCAAACTCGTCCACTTCCTCATTTCCTGGCCAAAATTAT AGAACAATCGCGAATAGTAATAATTATCCCCCGCGATATAATAATAATAATTCATCCACAAATATTC TCAATTTCAGTTCCTTTGAATCTAATCATCTTCTTCCTATGTCTTGGAGTAACAGAAATAACCAAGAC ACCCATTCCCAGTCTTATCTACAAAACAATATAAAAAGCGCGGCATCTACACAGACTTTATTACCAC AAGACACAATTGCAGCTGCAACAAAAGCAATAACATCAGATCCGAAGTTTCAATCTGCATTGGCTGT TGCTCTTACATCTATCATTGGCTCACGCAGCGGAAATCATCATATTGACGAAAAATCTGGGCAGAAT ATGAAGGTTACTGAGCCATTTCCAGTGCTTTGTAGCTTCCCATCCACCTCTCCTGGTGATCACAAAGA TTACACACTGtga
SlMYC1 atgACGGACTATAGATTATGGAGTAATACCAATACTACTAATACATGTGATGATACTATGATGATGGA TTCTTTTTTATCTTCCGATCCATCCTCTTTTTGGCCTGCTTCCACTCCCAATCGTCCGACTCCGGTGAA CGGAGTCGGAGAAACGATGCCGTTTTTCAATCAAGAATCACTACAGCAAAGGCTTCAGGCTTTAATT GACGGTGCTCGTGAATCATGGGCATATGCTATTTTCTGGCAATCGTCAGTTGTTGATTTTGCGAGCCA AACTGTATTGGGTTGGGGAGATGGGTATTATAAAGGAGAAGAAGATAAGAATAAACGGAGAGGGTC GTCTAGTTCAGCAGCTAATTTTGTTGCTGAGCAAGAGCATAGAAAGAAGGTGCTTCGGGAGCTGAAT TCATTAATATCCGGTGTACAAGCTTCCGCCGGAAACGGAACTGATGATGCAGTGGATGAGGAAGTGA CGGATACTGAATGGTTTTTTCTGATTTCAATGACCCAATCGTTTGTTAACGGTAACGGGCTTCCGGGC TTGGCGATGTACAGTTCAAGCCCAATTTGGGTTACTGGAACAGAGAAATTAGCTGCTTCTCAATGTG AACGGGCCAGGCAAGCCCAAGGTTTCGGGCTTCAGACGATTGTGTGTATTCCTTCAGCTAACGGTGT AGTGGAGCTTGGTTCGACTGAGCTGATATTCCAAAGCTCGGATTTGATGAACAAGGTTAAGTATTTG TTTAACTTCAATATTGATATGGGGTCTGTTACAGGCTCAGGTTCGGGCTCAGGCTCTTGTGCTGTGCA TCCTGAGCCCGATCCTTCGGCCCTTTGGCTTACGGATCCATCTTCCTCGGTTGTGGAACCTAAGGATT CGTTAATTCATAGTAGTAGTAGGGATGTTCAACTTGTGTATGGAAATGAGAATTCTGAAAATCAGCA GCAGCATTGTCAAGGATTTTTCACAAAGGAGTTGAATTTTTCGGGTTATGGATTTGATGGAAGTAGT AATAGGAATAAAACTGGAATTTCTTGTAAGCCGGAGTCCAGGGAGATATTGAATTTTGGTGATAGTA GTAAGAGATTTTCAGGGCAATCACAGTTGGGTCCTGGGCCTGGGCTCATGGAGGAGAACAAGAACA AGAACAAGAACAAGAAAAGGTCACTTGGATCAAGGGGAAACAATGAAGAAGGAATGCTTTCGTTTG TTTCGGGTGTGATCTTGCCAACTTCAACAATGGGGAAGTCCGGGGATTCTGATCACTCAGATCTCGA AGCCTCAGTGGTGAAGGAGGCCGTTGTAGAACCTGAAAAGAAGCCGAGGAAGCGAGGGAGGAAAC CAGCCAATGGAAGGGAGGAGCCATTGAATCACGTGGAAGCGGAGAGACAGAGGAGGGAGAAATTG AATCAAAGATTCTACGCGCTCAGAGCCGTAGTCCCAAATGTGTCTAAAATGGATAAGGCATCACTTC TTGGAGATGCAATTGCATACATCAATGAGTTGAAATCAAAAGTTCAAAATTCAGATTTAGATAAAGA GGAGTTGAGGAGCCAAATTGAATGTTTAAGGAAGGAATTAACCAACAAGGGATCATCAAACTATTC CGCCTCCCCTCCATTGAATCAAGATGTCAAGATTGTCGATATGGACATTGACGTTAAGGTGATTGGA TGGGATGCTATGATTCGTATACAATGTAGTAAAAAGAACCATCCAGCTGCCAGGCTAATGGCAGCCC TCAAGGACTTGGACCTAGACGTGCACCACGCTAGTGTTTCCGTGGTGAATGATTTGATGATCCAACA AGCCACAGTCAAAATGGGGAGCCGGCTTTATGCTCAAGAACAGCTTAGGATAGCATTGACATCAAA AATTGCTGAATCGCGAtga
Chapter 5 165 Chapter 6
General Discussion
Gene clusters in plant specialized metabolite pathways
Genes involved in different steps of specialized metabolite production pathways are often found in a cluster. Such a feature is quite common in bacterial genomes. In eukaryotes, functionally related genes are thought to be typically dispersed throughout the genome, however cases of gene clusters involved in the synthesis of specialized metabolites in plants have been reported, all of which were implicated in synthesis of defense compounds (Chu et al., 2011). For example, in oat (Avena strigosa) the biosynthesis of the antibacterial triterpenoid avenacin involves the β- amyrin synthase (AsbAS1) that is clustered together with the genes required for 2,3- oxidosqualene cyclization, β-amyrin oxidation, glycosylation and acylation in a region of the genome not conserved in other cereals (Qi et al., 2004). In Arabidopsis the synthesis and modification of the triterpenes thalianol and marneral is determined by two gene clusters found on chromosome 5 (Field and Osbourn, 2008; Field et al., 2011). In rice (Oryza sativa) five genes required for the biosynthesis of momilactones, diterpenoid phytoalexins, are clustered on chromosome 4 and their expression is regulated by a bZIP transcription factor (OsTGAP1) also located on chromosome 4 (Okada et al., 2009). It was therefore intriguing to determine whether such clustering occurs in tomato. With very few exceptions (SlTPS40; Chr 6, SlTPS17 and SlTPS24; Chr 7, SlTPS41; Chr 8), terpene synthases found on the same chromosome cluster together in a phylogenetic tree (Fig.1 and Chapter 2; Fig.1), which suggests that these clusters have been derived from tandem duplications and functional divergence and could
Chapter 6 166 imply co-ordinate regulation of their production by other genes found in close proximity.
+ + + + TPS3 TPS4 TPS5 TPS7 TPS8 TPS31 TPS32 TPS33 TPS35 Chromosome 1 M MMMMS S UU
TPS25 TPS27 TPS38 Chromosome 2 UUS
TPS28 Chromosome 4 U
TPS9 TPS10 TPS12 TPS36 TPS40 Chromosome 6 SSDUS
TPS16 TPS24 Chromosome 7 UD
TPS18 TPS19 TPS20 TPS21 TPS41 Chromosome 8 UUUUM
TPS14 Chromosome 9 S
+ TPS37 TPS39 Chromosome 10 MM
TPS17 Chromosome 12 S Figure 1. Functional or potentially functional TPS genes organized per chromosome. TPSs expressed in trichomes are in bold. For those genes expressed in trichomes, + denotes induction by jasmonic acid treatment. M/S/D or U refers to the biochemical function of the TPSs as (M) mono- / (S) sesqui-/ (D) di-terpene synthases or (U) unknown function when no enzymatic data are available. Compiled from Falara et al., 2011 and data from the stem trichome transcriptomic databases (Chapter 5).
The TPS-e/f subclade SlTPS18, SlTPS19, SlTPS20 (PHS1; Schilmiller et al., 2009) and SlTPS21 genes that group together (Chapter 2; Fig.1) and the TPS-c subclade SlTPS41 gene are found on chromosome 8 together with the neryl diphosphate synthase (NDPS1). NDPS1 catalyzes the formation of NPP- the substrate used by PHS1 to produce monoterpene products found in type VI leaf trichomes of S. lycopersicum (Schilmiller et al., 2009). On the same chromosome, genes encoding
Chapter 6 167 terpene-modifying enzymes (one cytochrome P450, one putative alcohol oxidase and two putative acyltransferaces) are also found (Falara et al., 2011). Since the enzymatic activities of SlTPS18, 19, 21 and 41 are not known (SlTPS41 is most homologous with the copalyl diphosphate synthase CPS1 (SlTPS40) that converts GGPP to CPP as part of the gibberellin biosynthesis; Rebers et al., 1999), it was suggested that NDPS1 and SlTPS41 could encode enzymes that provide substrates for these TPSs, whose products could be further modified by the terpene-modifying enzymes present on chromosome 8 (Falara et al., 2011). However, there is also evidence that genes involved in the synthesis of some tomato terpene products are not confined to gene clusters. The chromosomal substitution line IL10-3 contains a segment from chromosome 10 of S. pennellii in a S. lycopersicum M82 background that results in a reduced level of sesquiterpenes (Schilmiller et al., 2010). SlTPS9 and SlTPS12 are located on chromosome 6 (Fig.1) but their expression is reduced in the isogenic line IL10-3 (Falara et al., 2011). Therefore it is likely that genes involved in the regulation of these two TPSs are located on this segment from chromosome 10. Most monoterpene synthases (TPS-b clade) are found on chromosome 1 and some (TPS-g clade) are found on chromosome 10 (Fig.1), but the plastidial pathway precursor genes are not all located on one of these chromosomes. However, half of the genes for these precursor enzymes are indeed located on chromosome 1 (Chapter 5; Fig.2). Sesquiterpene synthases (most of which belong to the TPS-a clade) are found on various chromosomes (Fig.1) and, furthermore, the cytosolic pathway precursor genes are also scattered across almost all chromosomes, including chromosomes 3 and 5, which have not yet been shown to contain TPS genes (Chapter 5; Fig.2). In conclusion from the above-mentioned cases from the various plant species, there seems to be clustering of genes coding for specialized metabolite synthases and downstream modifying enzymes, but not with genes coding for precursor enzymes. Chromosome 1 contains the most TPSs, including the monoterpene synthases studied here (SlTPS3, SlTPS5 (SlMTS1), SlTPS7 and SlTPS8) that group together,
Chapter 6 168 whereas the sesquiterpene synthase we investigated, SlTPS9, is located on chromosome 6 (Fig.1). The identified transcription factors SlEOT1, SlMYC1 and SlWRKY4 that can interact with the promoters of some of these terpene synthases are located on chromosomes 2, 8 and 3 respectively and are not part of a gene cluster containing these TPSs. Finally, it is worth mentioning that although SlTPS3, SlTPS4, SlTPS5, SlTPS7 and SlTPS8 are quite homologous and therefore cluster together in the TPS phylogenetic tree (Chapter 2; Fig.1), their promoter regions do not share any significant identity (nucleotide identity ranging from 3,5% between SlTPS5p and SlTPS7p to 16,7% between SlTPS3p and SlTPS8p when comparing sequences of 1200bp upstream of the ATG start codons). This means that there is less sequence conservation in the promoters than in the coding sequences of these genes.
Trichome-specific promoters and cis-elements
Relatively few trichome-specific promoters of genes involved in the terpenoid biosynthesis pathway have been described to date (Tissier, 2012) and even fewer have been extensively characterized. SlMTS1 and SlEOT1 promoters are specifically expressed in the four secreting cells of tomato glandular trichomes (Chapter 3; Fig.2 and Chapter 4; Fig.1) and, to the best of our knowledge, are the first trichome-specific promoters characterized from Solanum lycopersicum. For the promoter of SlMTS1 5’ sequential deletions were created and fused to two different reporter genes, which allowed the study of the fragments’ expression patterns (Chapter 3; Fig.3). Interestingly, all promoter deletion fragments (except the -408bp fragment) retained the trichome-specific expression pattern. A similar case has been reported before for the promoter of the tobacco trichome-specific P450 gene (CYP71D16), that catalyzes the hydroxylation of cembratrienol to form cemratriendiol (Wang et al., 2002). In that instance all promoter deletion constructs had a trichome-specific expression and the full-length promoter was required for
Chapter 6 169 strong gland expression (Wang et al., 2002). In the case of the SlMTS1 promoter however, the -408bp fragment was unable to drive expression in any tissue. Deletion of a 110bp fragment close to the 5’ end of the tobacco trichome-specific cembratriendiol synthase (NtCBTS2a) promoter resulted in complete loss of reporter gene expression (β-glucuronidase (GUS); Ennajdaoui et al., 2010). In an elegant study of this promoter, involving sequential and internal deletions, it was also shown that deleting a 360bp promoter fragment (directly downstream of the 110bp fragment) resulted in significant ectopic expression of GUS (Ennajdaoui et al., 2010). Ectopic expression was not observed with the SlMTS1 promoter, for which however internal or 3’ sequential deletions were not made. Finally, it is worth pointing out that the 194bp fragment proximal to the start codon of NtCBTS2a (which includes the 75bp 5’UTR) did not produce GUS activity (Ennajdaoui et al., 2010), unlike the 207bp fragment proximal to the start codon of SlMTS1 (which includes the 98bp 5’UTR). This fragment not only contained all cis-elements necessary for trichome-specific expression, but also contained the binding site of the SlEOT1 transcription factor, possibly found on the 109bp of promoter sequence (Chapter 4). In this 109bp minimal promoter fragment the predicted TATA and CAAT boxes are located (Chapter 3; Fig.1). In comparison, as little as 250bp of the 5'-flanking regions of the Nicotiana sylvestris putrescine N- methyltransferase (PMT) 1, 2 and 3 genes were able to confer root-specific expression and MeJA responsiveness (Shoji et al., 2000). The 111 base pairs upstream of the transcriptional start site of NsPMT1 in particular, contained three cis-elements (G box, GCC-motif and a TA-rich region separating them) that were all required for MeJA-inducible transcription, indicating some level of interaction between these elements and the transcription factors (TFs) that bind to them (Xu and Timko, 2004). The SlMTS1 promoter contains several regulatory motifs (Chapter 3; Fig.1), including binding sites for MYC, WRKY and MYB transcription factors, which indicates regulation by multiple TFs. Given that SlMTS1 is a stress-inducible gene (by wounding, spider mite feeding and jasmonate
Chapter 6 170 treatment; van Schie et al., 2007), its expression could be liable to strict, coordinate regulation involving multiple transcription factors.
Transcription factors regulating tomato terpene biosynthesis
Three transcription factors (SlEOT1; Chapter 4, SlMYC1 and SlWRKY4; Chapter 5) were shown to interact with the SlMTS1 promoter, although the exact binding sites were not identified. Interaction of the three identified TFs could take place with the native SlMTS1 promoter in a heterologous system in planta. This contrasts with the finding for MYC2 from Catharanthus roseus, which only interacts with a tetramerized G box from the STR promoter in yeast and in C. roseus cells but not with a native STR promoter fragment containing the same G box (Zhang et al., 2011). SlEOT1 (Chapter 4; Fig.7) and SlMYC1 (Chapter 5; Fig.5) could transactivate SlMTS1 driven reporter gene expression stronger than SlWRKY4 (Chapter 5; Fig.4). SlWRKY4 seemed to have a negative effect on the combinatorial action of the other two TFs exerted on the SlMTS1 promoter (Chapter 5; Fig.7). WRKY transcription factors are known to function as activators, but also as repressors of transcription (Rushton et al., 2010). Nevertheless since SlWRKY4 could transactivate the SlMTS1 promoter, it most likely functions as an activator and if it exerts any negative effect on the combinatorial action of SlEOT1 and SlMYC1, it might do so by physically blocking one or more binding site(s). However the interaction of SlWRKY4 with SlMTS1 promoter (fragments) remains to be confirmed in yeast. Unlike SlEOT1, SlMYC1 and SlWRKY4 were not specifically expressed in trichomes (Chapter 4; Fig.1&2 and Chapter 5; Fig.3). Nicotine biosynthesis in Nicotiana tabacum and artemisinin biosynthesis in Artemisia annua takes place in the roots and glandular trichomes respectively, however none of the TFs identified so far involved in these pathways was expressed specifically in these tissues (De Geyter et al., 2012). Similarly, the two tomato TFs could both be involved in
Chapter 6 171 regulating terpene biosynthesis in trichomes and have other, yet unidentified roles, in other tissues where they are expressed. Since some of the terpene synthases (with whose promoters the TFs were shown to interact) were expressed in various tissues, SlMYC1 and SlWRKY4 could play a role in regulating those TPSs in the tissues in which they are co-expressed (see for overview Chapter 5; Table 5). SlMYC1 was not significantly induced by JA (Fig.3 and Chapter 5; Table 4). In Arabidopsis, AtMYC3 and AtMYC4, which act additively with AtMYC2 in the activation of JA responses, are only marginally induced by MeJA treatment, unlike AtMYC2 (Fernandez-Calvo et al., 2011). Therefore, SlMYC1 could be involved in the regulation of induced terpene biosynthesis in combination with other, unidentified tomato MYC transcription factors (Fig.2). It should be mentioned nevertheless that SlMYC1 shares higher homology with AtMYC2 than with AtMYC3 and 4. Furthermore, what is worthy of note is that, as indicated in Chapter 5, none of the annotated MYC transcription factors in the transcriptome database showed strong induction by JA (higher at least than 2-fold). Tomato JAMYC2 and JAMYC10 (shown to be induced by JA; Boter et al., 2004) were not present in our transcriptomic database nor in the SGN database (http://solgenomics.net/tools/blast/index.pl), as the sequence provided in Genbank (http://www.ncbi.nlm.nih.gov/genbank/) by the authors comes from potato. Nevertheless, it could be that other JA-inducible genes, annotated as bHLH transcription factors, that do not share high homology with the AtMYCs are involved in the process and were not investigated here. Alternatively, it could be that, in tomato, regulation of SlMTS1 activity does not involve a master-regulator MYC transcription factor, unlike N. tabacum (Shoji and Hashimoto, 2011) and C. roseus (Zhang et al., 2011). Still, the MYC-JAZ conserved system could also be present in tomato. S. lycopersicum COI-JAZ complex formation, stimulated by JA- Ile has already been shown (Thines et al., 2007; Katsir et al., 2008), but no targets of SlJAZ proteins have been identified to date. In tomato trichomes transcripts for COI1 and JAZ are present (Chapter 5; Table 3).
Chapter 6 172 SlMYC1 interacted with two trichome-specific JA-inducible terpene synthases (SlMTS1 and SlTPS3), one trichome-specific but not JA-inducible terpene synthase (SlTPS9) and one terpene synthase that is very lowly expressed in trichomes and not induced by JA in that tissue (SlTPS7; Chapter 2; Fig.2&6 and Chapter 5; Fig.5). It therefore seems to be a regulator of multiple terpene synthases. SlEOT1 on the other hand, could interact only with SlMTS1 (Chapter 4; Fig.7), which however does not exclude that it could interact with other terpene synthases not investigated here. Furthermore, it is possible that SlMTS1 (and/or other TPSs) are additionally regulated by some of the other SlEOT1 family members (Chapter 4; Table 1). In Arabidopsis, SHI family proteins were shown to act as dimers or multimers (Kuusk et al., 2002; Eklund et al., 2010). SlEOT1 does not need an interacting family member for binding to the SlMTS1 promoter (Y1H; Chapter 3&4), however it is possible that interaction with one of the other proteins has an effect on its activity (Fig.2).
Figure 2. A model for the transcriptional regulation of SlMTS1. (A) In an uninduced situation SlEOT1 could be involved in the steady-state transcription of SlMTS1. Additional binding of SlMYC1 on the SlMTS1 promoter is enough to increase the expression level. SlEOT1 and SlMYC1 could potentially be interacting with each other. (B) In an induced situation SlEOT1 in combination with SlMYC1 and a JA-inducible SlMYC2 or in
Chapter 6 173 combination with the JA-inducible EOT1/SHI family member (SlEOT2) could enhance expression of SlMTS1. Assuming binding site similarity between the Arabidopsis STY1 and SlEOT proteins, the existence of a YUC and a YUC-like element in the SlMTS1 promoter indicates interaction with at least two proteins of the EOT1/SHI class. Alternatively, it could be SlMYC1 in combination with a JA-inducible SlMYC2 that regulates induced SlMTS1 expression. Potential binding sites are shown (YUC, YUC-like, G box, G box- like). This model could be extended to other TPSs, possibly involving additional SlEOT1 family members. SlWRKY4 is not included in the model as its interaction with SlMTS1 promoter remains to be confirmed in yeast.
A role for EOT1/SHI family proteins in plant defense responses?
The SHI family proteins from various plant species are undoubtedly involved in auxin responses and plant growth (discussed in Chapter 4). AtSTY1 in particular, interacts with the promoter of at least one member of the YUCCA (YUC) family of flavin monooxygenases involved in the tryptophan-dependent auxin biosynthesis pathway (Fig.3A; Sohlberg et al., 2006; Eklund et al., 2010). Interestingly however, the Arabidopsis AP2/ERF transcription factor ORA59 (Pre et al., 2008) is also a STY1 target, possibly through a YUC element found in the ORA59 promoter (Eklund et al., 2010). ORA59 is involved in resistance against necrotrophic pathogens, integrating JA and ethylene signals to regulate expression of defense genes like Plant defensin 1.2 (PDF1.2) (Fig.3B; Pre et al., 2008). ORA59 was also shown to regulate three genes involved in tryptophan biosynthesis (Fig.3A&B; Pre et al., 2008). Therefore it is possible that STY1 regulates tryptophan availability and thus could promote either auxin biosynthesis or defense responses. Intriguingly, a yeast-one-hybrid (Y1H) screen with a region from the ORCA3 promoter with a library of MeJA-treated Catharanthus roseus cells identified a clone encoding a protein of the SHI family (Vom Endt et al., 2007). The C. roseus SHI ortholog (clone 2D81) shares highest sequence identity with SRS5 (http://www.arabidopsis.org/Blast/) and potentially binds in the YUC element found in the ORCA3 promoter region (Eklund et al., 2010). 2D81 could interact with this region also in vitro and its binding was not affected by mutations
Chapter 6 174 in the jasmonate-responsive sequences (Vom Endt et al., 2007). Binding to a mutated YUC element was not tested.
A Chorismate B
IGPS Necrotrophic Pathogen Indole-3-glycerol phosphate TSA JA ET Indole
TSB EIN3 EIL1 Tryptophan AtSTY1 TAA ERF1 ORA59 Indole-3-pyruvate AtSTY1 YUC PDF1.2 Indole-3-acetic acid (IAA)
auxin biosynthesis/ defense response? growth regulation
C D 2D81 Shikimate pathway ORCA3 MVA MEP pathway pathway Tryptophan MEP pathway TDC DXS IPP/ DMAPP Tryptamine STR Secologanin SlEOT1 TPS
Terpenes Strictosidine
TIA
TIA biosynthesis/ terpene biosynthesis/ defense response? defense response? Figure 3. Interactions and (suggested) roles of EOT1/SHI family members in various plant species. (A), (B) Arabidopsis thaliana (Eklund et al., 2010; Pre et al., 2008), (C) Catharanthus roseus (Vom Endt et al., 2007) and (D) Solanum lycopersicum (this work). Dashed lines indicate more than one enzymatic step. Pathway enzymes regulated by EOT1/SHI proteins are in boxes, transcription factors are in ovals, EOT1/SHI proteins are in shaded ovals. In panel A, enzymes involved in tryptophan metabolism that are regulated by ORA59 (Pre et al., 2008) are in bold. The AtSTY1-YUC interaction (panel A) has been
Chapter 6 175 experimentally proven for YUC4 and YUC8 (Eklund et al., 2010), the SlEOT1-TPS interaction (panel D) has been experimentally proven for SlMTS1 (this work). The 2D81- ORCA3 interaction (panel C) was detected in a Y1H screen and has been confirmed only in vitro (Vom Endt et al., 2007; see also text). IGPS; indole-3-glycerol phosphate synthase, TSA/B; tryptophan synthase subunit A/B, TAA; tryptophan aminotransferase, YUC; YUCCA flavin monooxygenases, JA; jasmonic acid, ET; ethylene, EIN3; ethylene insensitive 3; EIL1; EIN3-like 1, ERF1; ethylene response factor 1, ORA59; octadecanoid- responsive Arabidopsis AP2/ERF59, PDF1.2; plant defensin 1.2, TDC; tryptophan decarboxylase, STR; strictocidine synthase, DXS; deoxy-xylulose phosphate synthase, ORCA3; octadecanoid-derivative responsive Catharanthus AP2-domain 3, TIA; terpenoid indole alkaloid, MEP; methylerythritol, MVA; mevalonate, IPP; isopentenyl diphosphate, DMAPP; dimethallyl diphosphate, EOT1; emission of terpenes 1, TPS; terpene synthase.
ORCA3 regulates JA-induced terpenoid indole alkaloid (TIA) biosynthesis by controlling expression of tryptophan decarboxylase (TDC) that converts tryptophan to tryptamin, as well as expression of strictocidine synthase (STR) and deoxy- xylulose phosphate synthase (DXS) (Fig.3C; van der Fits and Memelink, 2000). Therefore 2D81, by regulating ORCA3, could also be controlling tryptophan availability or TIA production. Another potential link with the JA pathway comes from analysis of AtSRS7, which is mainly expressed in flower filaments and the shoot apex. In the srs7-1 mutant expression of the Defective in Anther Dehiscence 1 (DAD1) gene was increased and, similarly to JA-deficient mutants, anther dehiscence was inhibited (Kim et al., 2010). DAD1 encodes the lipolytic enzyme that catalyzes the release of linolenic acid from cellular lipids in the JA biosynthesis pathway (Ishiguro et al., 2001). It was suggested that through a feedback regulation, induction of DAD1 promotes JA biosynthesis in the mutant. However, it must be noted that exogenously applied JA did not rescue the srs7-1 floral phenotype, nor was SRS7 expression affected by JA (Kim et al., 2010). From the above-presented links of SHI family proteins with the jasmonate pathway and production of defense compounds it is already intriguing to speculate an additional role (for at least some of) these proteins in the plant defense system. An extra line of evidence comes from SlEOT1 (Fig. 3D; and possibly other family
Chapter 6 176 members) that interacts with the JA-inducible SlMTS1 (and potentially other TPSs), strengthening the case that EOT1/SHI proteins could be involved in (in)directly regulating JA-mediated defense responses in various plant species including tomato.
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Chapter 6 179 Summary
Tomatoes, like many other plants, produce and store secondary metabolites in glandular trichomes. Glandular trichomes are protrusions of epidermal origin, found on the vegetative tissue of plants. Among the metabolites that are produced in these specialized cells are volatile terpenes, which are emitted by plants under attack by herbivores. Such volatile cues are perceived by herbivore enemies (predators and parasitoids) that indirectly benefit the plant by feeding from or laying their eggs into herbivores. All terpenes are synthesized from the same building blocks (the five carbon-unit isopentenyl diphosphate and its isomere dimethallyl diphosphate) through the action of prenyl diphosphates and the downstream enzymes Terpene Synthases. Production of terpenes is tightly regulated in a temporal (and spatial) manner and such regulation involves Transcription Factors that bind to the promoters of terpene synthases, thus exerting an effect on their expression. The aim of this project was to identify transcription factors that regulate terpene synthases in tomato trichomes. For this, two approaches were taken. First, the promoter of a monoterpene synthase (SlMTS1) was used in a yeast-one-hybrid screen with a trichome cDNA library, in order to identify proteins that can bind to it. One transcription factor was identified and designated Emission of Terpenes 1 (SlEOT1). Second, a sequencing database obtained from tomato trichome cDNA pyrosequencing (GS FLX, 454 Life Sciences) and a transcript-profiling database (originating from trichomes of Control and Jasmonic acid-treated plants; Genome Analyzer II, Illumina) were mined, in order to discover transcription factors potentially involved in terpene biosynthesis. Two transcription factors were selected (SlMYC1 and SlWRKY4) and shown to interact with terpene synthase promoters. Interestingly, SlEOT1 and SlMYC1 have an additive effect in the regulation of SlMTS1.
180 Samenvatting
Net zoals vele andere planten produceren en bewaren tomaten secundaire metabolieten in haarkliertjes. Haarkliertjes bevinden zich op het vegetatieve weefsel van planten en zijn van epidermale afkomst. Een groep van metabolieten geproduceerd door deze gespecialiseerde cellen zijn vluchtige terpenen, die uitgestoten worden wanneer de plant aangevallen wordt door herbivoren. De vluchtige stoffen worden waargenomen door vijanden van de herbivoren (predatoren en parasieten) en een indirect voordeel voor de plant is dat deze natuurlijke vijanden voeden van of eieren leggen in de herbivoren. Alle terpenen worden door prenyl difosfaten en terpeen syntheses gesynthetiseerd van dezelfde bouwblokken (de vijf koolstof atoom eenheden isopentenyl difosfaat en zijn isomeer dimethylallyl difosfaat). Productie van terpenen is strikt gereguleerd in tijd (en ruimte). Deze regulatie omvat transcriptie factoren die binden aan de promoter van terpeen synthases en waarmee de expressie van terpeen syntheses gereguleerd wordt. Het doel van dit project was om transcriptie factoren te identificeren die terpeen biosynthese in haarkliertjes van tomaat reguleren. Twee methoden werden hiervoor toegepast. De eerste methode was een yeast-one-hybrid screen met een haarklier specifieke cDNA bibliotheek om de eiwitten die binden aan de promoter van een monoterpeen synthese (SlMTS1) te identificeren. In de yeast-one-hybrid screen werd een transcriptie factor ontdekt, genoemd Emission of Terpenes (SlEOT1). De tweede methode was het analyseren van een sequentie database gebaseerd op pyrosequencen van tomaten haarklier cDNA (GS FLX, 454 Life Science) en een transcript-profiling database gebaseerd op haarkliertjes van controle en jasmonzuur behandelde tomaten (Genome Analyzer II, Illumina). Het doel van deze analyse was transcriptie factoren ontdekken die potentieel betrokken zijn bij terpeen biosynthese. Twee transcriptie factoren werden geselecteerd (SlMYC1 en SlWRKY4) en aangetoond is dat deze interacteren met de promoter van terpeen syntheses. Opvallend is dat SlEOT1 en SlMYC1 een toegevoegd effect hebben op de regulatie van SlMTS1.
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