Chalcone synthase (CHS) is the key enzyme in the first committed step of the flavonoid biosynthetic pathway and catalyzes the stepwise condensation of 4-coumaroyl-CoA and malonyl-CoA to naringenin chalcone. In plants, CHS is often encoded by a small family of genes that are temporally and spatially regulated. Our earlier studies have shown that GCHS4 is highly activated by ectopic expression of an MYB-type regulator GMYB10 in gerbera (Gerbera hybrida).
The tissue- and development-specific expression patterns of three gerbera CHS genes were examined. Virus-induced gene silencing (VIGS) was used to knock down GCHS1 and GCHS4 separately in gerbera inflorescences.
Our data show that GCHS4 is the only CHS encoding gene that is expressed in the cyanidin-pigmented vegetative tissues of gerbera cv Terraregina. GCHS3 expression is pronounced in the pappus bristles of the flowers. Expression of both GCHS1 and GCHS4 is high in the epidermal cells of gerbera petals, but only GCHS1 is contributing to flavonoid biosynthesis.
Gerbera contains a family of three CHS encoding genes showing different spatial and temporal regulation. GCHS4 expression in gerbera petals is regulated post-transcriptionally, at the level of either translation elongation or protein stability.
Flavonoids are perhaps the best characterized plant-specific secondary metabolites, and they accumulate in a broad range of plants, from mosses to flowering plants (Koes et al., 1994). Flavonoids are natural products that contain a C6-C3-C6 carbon framework (Marais et al., 2008) and are synthesized by a branched pathway that yields both colored and colorless compounds; some of the latter, however, enhance visible light absorption and therefore act as copigments (Mol et al., 1998; Martens & Mithöfer, 2005). According to the degree of oxidation and saturation of the central pyran ring, flavonoids are classified into many subgroups, such as chalcones, flavones, flavonols, flavandiols, anthocyanins, and proanthocyanidins (Winkel-Shirley, 2001). Flavonoids possess significant and diverse biological functions, including protection against UV radiation, defense against phytopathogens and herbivores, regulation of auxin transport, and signaling between plants and microbes, and, importantly, they are major pigments for flowers, fruits, seeds, and leaves (Stafford, 1991; Mol et al., 1998; Winkel-Shirley, 2002; Bradshaw & Schemske, 2003; Koskela et al., 2011; Ferreyra et al., 2012; Jaakola, 2013).
Decades of extensive studies on flavonoid biosynthesis have been done using maize (Zea mays), snapdragon (Antirrhinum majus), petunia (Petunia hybrida), and arabidopsis (Arabidopsis thaliana) as models. Through studying mutants that affect flavonoid synthesis, a number of structural and regulatory genes have been characterized, and the flavonoid biosynthetic pathway is now well established (Holton et al., 1993; Mol et al., 1998; Koes et al., 2005; Ferreyra et al., 2012). Flavonoids form a branch of phenylpropanoids, where the C6-C3 carbon skeleton is derived from phenylalanine. The first dedicated enzyme in the flavonoid pathway is chalcone synthase (CHS), which catalyzes the stepwise condensation of three molecules of malonyl-CoA to one molecule of 4-coumaroyl-CoA to yield naringenin chalcone. Subsequently, chalcone isomerase (CHI) catalyzes the isomerization of the chalcone to naringenin. The following reaction catalyzed by flavanone 3-hydroxylase (F3H) yields dihydrokaempferol, which is further converted to dihydroquercetin and dihydromyricetin by the actions of flavonoid 3′-hydroxylase (F3′H) and flavonoid 3′,5′-hydroxylase (F3′5′H), respectively. Finally, consecutive reactions catalyzed by dihydroflavonol reductase (DFR) and anthocyanidin synthase (ANS) convert the dihydroflavonols to colored anthocyanidin aglycones, which can be further methylated, glycosylated, and acylated, leading to a large repertoire of species-specific anthocyanins (reviewed by Dooner & Robbins, 1991; Holton & Cornish, 1995). The action of flavone synthase (FNS) and flavonol synthase (FLS) leads to branching of the flavonoid pathway to flavones and flavonols, respectively (Davies et al., 2003; Martens & Mithöfer, 2005).
Many external factors, such as temperature, light, nutrient status, wounding, water stress and pathogen infection, have been reported to affect flavonoid biosynthesis (Christie et al., 1994; Dixon & Paiva, 1995; Chalker-Scott, 1999; Carbone et al., 2009). The structural genes of the pathway are regulated transcriptionally by three major transcription factors (TFs) of MYB, basic helix–loop–helix (bHLH) domain and WD40 protein families (Koes et al., 2005). Other regulatory proteins, such as TFs that contain MADS box, Zn-finger, and WRKY domains, have also been shown to be involved in regulation of flavonoid biosynthesis (Johnson et al., 2002; Nesi et al., 2002; Sagasser et al., 2002; Jaakola et al., 2010).
Chalcone synthase, the key enzyme in flavonoid biosynthesis, belongs to the type III polyketide synthase (PKS) superfamily that also includes stilbene synthases (STS) and in gerbera 2-pyrone synthase (2PS) (Eckermann et al., 1998; Austin & Noel, 2003; Abe & Morita, 2010). Sharing high similarity at the amino acid sequence level, type III PKSs contain a common three-dimensional overall structure and a conserved Cys-His-Asn catalytic triad in the buried active site (Ferrer et al., 1999; Abe & Morita, 2010). Small modifications of only a few amino acids could significantly alter the binding pocket volume and redirect the enzyme function (Ferrer et al., 1999; Jez et al., 2000a). The crystal structure of CHS was first characterized for CHS2 of the legume alfalfa (Medicago sativa) (Ferrer et al., 1999). Loss of CHS enzyme activity results in albino flowers and fruits that lack all flavonoid pigments as well as other flavonoids (van der Krol et al., 1990; Napoli et al., 1990; Schijlen et al., 2007; Ohno et al., 2011; Morita et al., 2012; Dare et al., 2013).
Gerbera (Gerbera hybrida), from the family of Asteraceae, is an economically important ornamental species. More than two decades of studies have turned gerbera into an Asteraceae model for studying flower development and secondary metabolism (Elomaa et al., 1993; Helariutta et al., 1996; Yu et al., 1999; Kotilainen et al., 2000; Uimari et al., 2004; Laitinen et al., 2005; Broholm et al., 2008; Tähtiharju et al., 2012). Efforts have been made to elucidate the gerbera flavonoid biosynthetic pathway. A floret cDNA library was constructed as a source for isolation of gerbera CHS and DFR genes for detailed functional studies (Elomaa et al., 1993, 1996; Helariutta et al., 1993, 1995b, 1996). A number of other enzymes in the pathway were also identified, including genes encoding the gerbera FNS II (Martens & Forkmann, 1999), ANS (Wellmann et al., 2006), F3H (Martens et al., 2002) and F3′H (Seitz et al., 2006). In addition, a bHLH-type TF GMYC1 and an MYB domain TF GMYB10 were shown to regulate anthocyanin biosynthesis in gerbera (Elomaa et al., 1998, 2003).
In many plant species, core enzymes in the flavonoid pathway, such as CHS and DFR, are encoded by gene families (Coe et al., 1981; Beld et al., 1989; Koes et al., 1989; Tanaka et al., 1996; Bernhardt et al., 1998; Inagaki et al., 1999; Johzuka-Hisatomi et al., 1999; Himi & Noda, 2004; Shimada et al., 2005; Tuteja & Vodkin, 2008; Martins et al., 2013). Each gene family is considered to be derived as a result of gene duplication events and subsequent positive selection (Helariutta et al., 1996; Yang et al., 2004; Des Marais & Rausher, 2008). The different copies have evolved either to function in different tissues or at different times (Martin & Gerats, 1993; Johzuka-Hisatomi et al., 1999; Durbin et al., 2000; Himi & Noda, 2004; Tuteja et al., 2004) or to specialize in the use of different but related substrates (Des Marais & Rausher, 2008; Martins et al., 2013). The way in which flux is controlled in the branched flavonoid pathway has remained largely unknown. Biochemical evidence suggests that enzymes of the flavonoid pathway interact and form complexes known as metabolons (reviewed by Winkel, 2004). It would be intriguing to discover whether participation in different metabolons was a third tier of the subfunctionalization process in evolution.
We recently isolated a new CHS encoding gene GCHS4 in gerbera (Laitinen et al., 2005). Together with the two previously identified CHS encoding genes, GCHS1 and GCHS3 (Helariutta et al., 1995b), the gerbera CHS gene family is represented by these three members. Previous studies by ectopic expression of GMYB10 enhanced pigment accumulation significantly and induced cyanidin biosynthesis in transgenic lines developed from the gerbera cv Terraregina, which is characterized by flowers containing pelargonidin (Laitinen et al., 2008). Interestingly, GMYB10 ectopic expression did not induce GCHS1 expression, the gene we used to block anthocyanin biosynthesis by antisense transformation (Elomaa et al., 1993). Instead, GCHS4 was strongly induced.
In this study, we characterized the function of the gerbera GCHS4 gene and investigated the spatial and temporal expression patterns of gerbera CHS gene family members. We show that GCHS4 is the only CHS gene expressed in cyanidin-accumulating vegetative tissues. The gene is also expressed strongly in the epidermal cells of gerbera petals, where, unexpectedly, it has no functional role in anthocyanin biosynthesis.
Materials and Methods
Gerbera (Gerbera hybrida (G. jamesonii Bolus ex Adlam × G. viridifolia Schultz-Bip)) cvs Terraregina and President were obtained from Terra Nigra B.V. (De Kwakel, the Netherlands). Gerbera plants were grown as previously described (Elomaa & Teeri, 2001; Deng et al., 2012). The developmental stages of gerbera inflorescence have been described (Helariutta et al., 1993). Gerbera petals are green during the early stages (stages 1–4), start to accumulate pigment at stage 5 and are fully pigmented at stage 6 before the opening of the inflorescence. The basal parts of gerbera leaf petioles and inflorescence stems (scapes) are anthocyanin-pigmented, but the leaf blade is normally green. Occasionally, edges of leaf blades turn red when plants suffer from stresses such as pathogen infection, high light, drought, or flooding. Pigmented leaf blades were sampled without further specifying the causative stress component.
Isolation, sequence, and phylogenetic analysis of GCHS4
GCHS4 was identified as a full-length cDNA clone from the gerbera expressed sequence tag (EST) collection (Laitinen et al., 2005). CHS encoding sequences from other plant species were obtained from GenBank. The sunflower (Helianthus annuus) and lettuce (Lactuca sativa) sequences were extracted from EST libraries (http://compbio.dfci.har-vard.edu/tgi/plant.html) using the keyword ‘chalcone synthase’. Two lettuce sequences were discarded as they had lower similarity scores than others and gave the best BLAST hits to ‘chalcone synthase family’ proteins rather than CHSs. CHS proteins were aligned using the Clustal Omega multiple sequence alignment program (Sievers et al., 2011), and a maximum-likelihood phylogenetic tree based on reverse-translated nucleotides was constructed using RAxML (Stamatakis et al., 2008; http://embnet.vital-it.ch/raxml-bb/). A generalized time-reversible model of molecular evolution was used with a gamma model of rate heterogeneity, but otherwise at default settings. Bootstrapping was automatically performed by RAxML to calculate branch supports. The phylogenetic tree of CHS nucleotide sequences was rooted using pine CHS as outgroup.
CHS enzyme assays
Polymerase chain reaction products (primers listed in Table S1) containing the full open reading frames (ORFs) of GCHS1, GCHS3 and GCHS4 were cloned into NcoI/BamHI digested pHIS8 expression vector, kindly supplied by J. P. Noel (Jez et al., 2000b) and transformed into the Escherichia coli strain BL-21(DE3). For enzyme production, E. coli was grown to OD600 of 0.4–0.6 at 37°C, and induced for 22 h with 0.5 mM isopropyl β-D-1-thiogalactopyranosideat 16°C. The N-terminally 8XHis-tagged proteins were purified (His SpinTrap, GE Healthcare, Uppsala, Sweden) and passed through PD MiniTrap G-25 columns (GE Healthcare, Freiburg, Germany). For enzymatic assays (in 100 μl volumes), 30 μg of purified proteins were added in buffer (HEPES-KOH pH 7.0, 2 mM dithiothreitol (DTT)), with 80 μM 4-coumaryl-CoA (MicroCombiChem e.K., Wiesbaden, Germany) and 160 μM malonyl-CoA (Sigma Aldrich, St Louis, MO, USA), and were incubated for 1 h at 30°C. After incubation, 100 μl of 1M Tris-HCl (pH 8.8) was added, which converted chalcone to naringenin nonenzymatically. Subsequently, reactions were extracted twice with 300 μl ethyl acetate, and evaporated to dryness. Finally the samples were dissolved in 100 μl of methanol and analyzed by high-performance liquid chromatography (HPLC).
Chalcone synthase enzyme activity in gerbera petals was assayed essentially as previously described (Helariutta et al., 1995b) using [14C]malonyl-CoA (Perkin Elmer, Zaventem, Belgium) as labeled substrate. A quantity of petals (0.5 g) was extracted on ice with a mortar and pestle with a few grains of sand and 2 ml of ice-cold, boiled buffer (200 mM HEPES-KOH pH 7.0, 5 mM EDTA, 1 mM DTT, Roche Complete Mini EDTA-free Protease Inhibitor Coctail 1 tablet per 10 ml and DOWEX 1X2-400 ion exchange resin 1 g ml−1), then centrifuged at 13 000 g for 10 min in the cold. Cleared extract with 20 μg petal protein was used in the assay with the same buffer but without protease inhibitor or DOWEX. After a 30 min reaction at 30°C, 100 μl 1 M Tris-HCl, pH 8.8, was added. Reaction products were extracted twice with ethyl acetate, evaporated to dryness, dissolved in 10 μl of ethyl acetate and analyzed by thin-layer chromatography (cellulose plate, Merck KGaA, Darmstadt, Germany; running buffer, 20% acetic acid in water) followed by imaging with a phosphoimager plate.
Virus-induced gene silencing
Tobacco rattle virus (TRV) gateway vectors were kindly supplied by S. Dinesh-Kumar (Liu et al., 2002). Virus-induced gene silencing (VIGS) vectors of TRV2:GCHS1 and TRV2:GCHS4 were constructed as previously described (Deng et al., 2012, 2013) (primers listed in Table S1), and transformed into Agrobacterium tumefaciens strain C58C1(pGV2260) (Deblaere et al., 1985). To avoid off target silencing, gene specific inserts (400 nt in size) were amplified from the 3′ end of GCHS1, and the 5′ end of GCHS4, respectively. Agrobacterium-mediated virus infection was done as described (Deng et al., 2012).
In each experiment, three treatments were done. Ten plants (c. 30 inflorescences) were inoculated with TRV1+TRV2:GCHS1 or TRV1+TRV2:GCHS4, respectively. For control, four plants were inoculated with the vectors TRV1+TRV2 that did not contain a transgene insert. For both cvs Terraregina and President, three independent experiments were carried out.
Total RNA from gerbera leaf and petal tissue was extracted with TRIzol Reagent (Invitrogen) according to the manufacturer's instructions. A DNase (Fermentas, Leon-Rot, Germany) treatment was done to remove any residues of genomic DNA.
Polysome RNA was isolated as described by Barkan (1993) including a few modifications. Gerbera petal tissue (300 mg, stage 6) was ground to powder in liquid nitrogen and collected in 2 ml Eppendorf tubes. The powder was lysed in 1.25 ml ice-cold polysome extraction buffer (200 mM Tris-HCl (pH 9.0), 200 mM KCl, 36 mM MgCl2, 25 mM ethylene glycol tetraacetic acid (EGTA), 50 μg ml−1 cycloheximide, 50 μg ml−1 chloramphenicol, 1% Triton X-100, 1% Brij 35, 1% Tween-40, 1% NP-40, 2% polyoxyethylene tridecyl ether, 1% sodium deoxycholate, 1 mg ml−1 heparin and 40 U ml−1 RNase inhibitor) for 10 min at 4°C. After lysis, the nuclei were removed by centrifugation (12 000 g, 2 min), and the supernatant was layered onto a 11 ml sucrose gradient (20–60% sucrose in 40 mM Tris-HCl, pH 8.0, 20 mM KCl, 10 mM MgCl2) and centrifuged in a SW41Ti rotor (Beckman, Palo Alto, CA, USA) for 4 h at 210 000 g at 4°C. Fractions (12 × 1 ml) were collected into tubes containing 2.2 ml ice-cold 8 M guanidinium-HCl. RNA from each fraction was pelleted by adding one volume of 100% ethanol and centrifugation (12 000 g, 20 min, 4°C). RNA was further purified with phenol : chloroform extraction, and the remaining heparin was removed with LiCl precipitation (Prete et al., 2007).
The first-strand cDNA synthesis and quantitative reverse transcription polymerase chain reaction (RT-PCR) was carried out as previous described (Deng et al., 2012). Relative expression ratios were calculated with the ‘efficiency calibrated mathematical method’ (Pfaffl, 2001), and the gerbera housekeeping gene GGAPDH was used as a reference. Standard error of the mean (SE) was calculated from the biological replicates (n = 3).
In situ hybridization
Ray flower petals were sampled at stage 6 of inflorescence development (Helariutta et al., 1993) from cv Terraregina, and were fixed, dehydrated, cleared, and paraffin-embedded as previously described (Elomaa et al., 2003). Petal cross-sections of 7–10 μm were cut with a microtome and placed on Superfrost plus slides (N: Menzel-Gläser, Braunschweig, Germany). Nonradioactive hybridizations were carried out as described by Di Laurenzio et al. (1996), except that proteinase K was used at a concentration of 10 μg ml−1. Gene-specific fragments, with the size of 353 bp for GCHS1 and 313 bp for GCHS4, were amplified from the 5′-ends of the genes with primers listed in Table S1, and cloned into pGEM-T easy vector (Promega). RNA probes were synthesized using T7 and SP6 RNA polymerase and labeled with the DIG RNA Labeling Kit (Roche) according to the manufacturer's instructions.
HPLC analysis of flavonoids
Gerbera petal tissues (0.5–1 g) were finely ground in liquid nitrogen and freeze-dried. After determining the DW, samples were extracted with 2.5 ml 80% methanol on an orbital shaker, first at room temperature for 3 h and then at 4°C overnight. The sample was then centrifuged (5500 g, 5 min), and the clear supernatant was collected. Pellets were extracted again with 2 ml 80% methanol. The supernatants were combined and evaporated to dryness in a scanspeed centrifugal evaporator (2000 g, 10°C) (ScanSpeed 40, ScanLAF, Lynge, Denmark). Extracted anthocyanins were hydrolyzed into their corresponding anthocyanidins as described in Zhang et al. (2004). Dried samples were dispersed in water/methanol solution containing 2 N HCl (50 ml methanol + 33 ml water + 17 ml 37% HCl) to the concentration of 25 mg DW ml–1, and incubated at 100°C for 60 min. Hydrolyzed samples were filtered through 0.45 μm GHP syringe filters (Pall, Port Washington, NY, USA) and analyzed by HPLC. HPLC analyses were conducted on Agilent 1100 Series HPLC System as previously described (Laitinen et al., 2008), except that the mobile phases were 0.08% trifluoroacetic acid (TFA) in water (solvent D; Zhang et al., 2004) and 0.08% TFA in acetonitrile (solvent C; Zhang et al., 2004). The elution conditions were from 0 min 18% C to 35 min 40% C using a linear gradient between the time points.
Isolation and analysis of gerbera CHS sequences
We have previously reported two CHS encoding genes, GCHS1 (GenBank: Z38096.1) and GCHS3 (GenBank: Z38098.1) in gerbera (Helariutta et al., 1995b). A third CHS encoding gene, GCHS4 (GenBank: AM906210.1), is present in the ESTcollection of gerbera (Laitinen et al., 2005) and was observed to be highly up-regulated in transgenic plants ectopically expressing the anthocyanin regulatory gene GMYB10 (Laitinen et al., 2008). We recovered GCHS4 in full length from the EST plasmid G0000100008B02. The gene has the shortest ORF (1170 bp) among the CHS-like genes of gerbera and is predicted to encode a 42.9 kDa protein with 389 amino acids and a calculated pI of 6.2 (Table S2). Amino acid sequences of gerbera CHSs, together with arabidopsis CHS and alfalfa CHS2, share high similarity (Table S3). By contrast, the gerbera 2-pyrone synthase G2PS1 (Eckermann et al., 1998), a CHS-like protein evolved from gerbera CHS through gene duplication, has less similarity to CHSs (Table S3).
We made an amino acid sequence alignment for the gerbera CHS-like proteins in comparison to the alfalfa CHS2. All gerbera proteins (including G2PS1) contain the four important catalytic residues of Cys 164, Phe 215, His 303, and Asn 336 (Fig. 1, marked with red background; all numbering of amino acids is as in alfalfa CHS2), which have been shown to be absolutely conserved in all CHS-like sequences (Ferrer et al., 1999). GCHS1, GCHS3, and GCHS4 also contain all of the 13 inert active site residues typical of CHS enzymes (Fig. 1, marked with a yellow background), and the three residues that shape the 4-coumaroly-CoA binding pocket and the polyketide cyclization pocket (Thr 197, Gly 256, and Ser 338; marked with a green background), which are replaced in G2PS1 by Leu, Leu, and Ile, respectively (Fig. 1).
The phylogenetic tree of CHS sequences indicates gene duplications at different evolutionary times (Fig. 2). The most ancient duplication appears to have taken place in a common ancestor of monocots and eudicots. Arabidopsis, for example, has retained only one of these duplications, while alfalfa has retained another (which has further duplicated more recently). Importantly, the CHS gene families of petunia, tomato and morning glory are of independent origin compared with those in Asteraceae, indicating that any functional division within the CHS families is not of shared origin. On the other hand, according to the phylogenetic analysis carried out here, the duplication events leading to the three CHS genes in the taxonomically basal Asteraceae species gerbera may well be shared with the entire sunflower family.
Enzymatic activity of gerbera CHS-like proteins
In order to compare the catalytic properties of GCHS4 with the previously studied GCHS1, GCHS3, and G2PS1, and to verify the sequence-based prediction of GCHS4 enzymatic function, the cDNAs were cloned into the expression vector pHIS8. The recombinant enzymes containing N-terminal histidin-tags were produced in E. coli. To control the E. coli background, the vector with no insert was used.
Consistent with our previous report, GCHS1 and GCHS3 showed a typical CHS enzymatic function, catalyzing the reaction of converting 4-coumaroyl-CoA and malonyl-CoA substrates into naringenin chalcone (Fig. 3a,b). In accordance with its diverged function (Eckermann et al., 1998), G2PS1 was not able to use 4-coumaroyl-CoA as a substrate, but produced triacetic acid lactone (TAL) when supplied with the starter acetyl-CoA and 6-phenyl-4-hydroxy-2pyrone with benzoyl-CoA (data not shown). As predicted, GCHS4 performed a typical CHS function similar to GCHS1 and GCHS3, and produced naringenin chalcone from 4-coumaroyl-CoA and malonyl-CoA (Fig. 3c).
Expression patterns of gerbera CHS-like genes
The spatial expression patterns of CHS-like genes were analyzed in cv Terraregina using quantitative RT-PCR. Samples were taken from 11 tissue types, including both vegetative and floral parts of the plant. The relative expression ratios were calculated taking into account the PCR efficiencies and cross point (CP) values, against a common control (Pfaffl, 2001). Thus, all relative expression ratios are comparable between the genes analyzed.
All gerbera CHS-like genes had distinct expression patterns. Consistent with previous studies (Helariutta et al., 1995b), G2PS1 was universally expressed in all vegetative and reproductive tissues, with the highest expression in leaf blade, scape, bracts and petals, and the lowest expression in roots, receptacle and stamens (Fig. 4a, left panel). By contrast, GCHS1, GCHS3, and GCHS4 were expressed mainly in the reproductive tissues where flavonoids accumulate (Fig. 4a, Table 1). GCHS3 was specifically expressed in pappus hairs, and showed only a tiny signal in stamen, ovary and young capitula (Fig. 4a, left panel). GCHS1 was also highly expressed in pappus hairs, but showed considerable expression in the petals (Fig. 4a, left panel). Expression of GCHS4 was mainly concentrated in petals and the carpel (Fig. 4a, left panel).
Table 1. Flavonoids in the pigmented tissues of gerbera (Gerbera hybrida) cv Terraregina
Amounts are shown in μg mg−1 DW. Data are means ± SE (n = 3).
ND, not detectable; ─, not measured.
4.27 ± 0.90
0.05 ± 0.02
0.02 ± 0.01
0.05 ± 0.01
0.57 ± 0.15
0.30 ± 0.02
0.62 ± 0.04
4.28 ± 0.42
5.62 ± 0.38
0.20 ± 0.05
0.08 ± 0.08
0.13 ± 0.07
0.28 ± 0.06
1.39 ± 0.34
0.03 ± 0.00
0.02 ± 0.01
0.03 ± 0.01
1.07 ± 0.20
2.67 ± 0.19
More detailed expression patterns were checked during petal development in cv Terraregina. Again, G2PS was highly expressed at all 11 petal stages, with the highest expression at stages 6–9 (Fig. 4a, right panel). Expression of GCHS3 was barely detectable in any of the petal developmental stages (Fig. 4a, right panel). As reported previously (Helariutta et al., 1995b), the expression of GCHS1 correlated with anthocyanin accumulation, with levels increasing steadily at early stages, peaking at stage 6 in fully pigmented petals, and decreasing gradually in later stages (Fig. 4a, right panel). Expression of GCHS4 started later, at stage 5, and expression stayed high during all later stages (Fig. 4a, right panel). The expression patterns of CHS-like genes in cv President were similar to those in cv Terraregina (data not shown).
Previously, in situ expression analysis of gerbera petal cross-sections have indicated that G2PS1 is expressed in all cell types of gerbera petals, and GCHS1 in epidermal cells in particular (Helariutta et al., 1995b). To verify the localization of GCHS4 expression, an in situ hybridization assay was conducted with gene-specific probes for GCHS1 and GCHS4. Petal samples were taken from cv Terraregina at stage 6, when both GCHS1 and GCHS4 are expressed at high levels. The results showed that GCHS4 has a similar expression pattern to GCHS1. Its expression was strongly concentrated in the epidermal cells located on the adaxial side of petals (Fig. 4b). This is correlated with anthocyanin accumulation (Helariutta et al., 1993).
CHS4 is the dominant CHS in red vegetative tissues of gerbera
Except for the pigmentation in flower organs, some vegetative tissues in gerbera, such as the base of the scape and the leaf petiole, and the stressed leaf blade also contain anthocyanins (Fig. 5a, Table 1). HPLC analysis showed that, in cv Terraregina, all colored vegetative tissues contain mainly cyanidin-derived pigments, whereas pelargonidin dominates in petals (Fig. 5b, Table 1).
Quantitative RT-PCR was used to check CHS expression in the red and green vegetative tissues of cv Terraregina. In the green tissues, no CHS mRNA was detected. In all red vegetative tissues, considerable amounts of GCHS4 mRNA was detected, with levels comparable to gerbera petals (Fig. 5c, upper panel). mRNAs of GCHS1 and GCHS3, however, were not detectable (data not shown).
Ectopic expression of the transcription factor GMYB10 in transgenic gerbera leads to accumulation of cyanidin in vegetative tissues and a strong induction of GCHS4 expression (Laitinen et al., 2008). In the stressed and pigmented leaf blade, GMYB10 expression was up-regulated (Fig. 5c, lower panel), indicating a role of this regulatory gene in cyanidin biosynthesis during stress. However, in the unstressed leaf petiole and inflorescence scape, GMYB10 was expressed at similar levels in the red tissues and their neighboring green tissues (Fig. 5c, lower panel). The cyanidin biosynthesis in leaf petiole and scape is still GMYB10-dependent, because in the silenced antisense-GMYB10 lines, both leaf petioles and scapes are nonpigmented (Fig. S1).
Thus, our results indicate that GCHS4 is the dominant CHS in anthocyanin-pigmented vegetative tissues of gerbera and is responsible primarily for cyanidin biosynthesis in those tissues.
GCHS1 is the major functional CHS in gerbera petals
As GCHS1 and GCHS4 are the two CHS genes highly expressed in gerbera petals, we conducted VIGS experiments to study their functions by knocking down GCHS1 and GCHS4 expression separately. To avoid off-target silencing, gene-specific sequences were selected for construction of VIGS vectors of TRV:GCHS1 and TRV:GCHS4. There are no sequence stretches in either of the fragments that would have a perfect match over 15 nucleotides (nt) between GCHS1 and GCHS4, although GCHS1 and GCHS4 are generally highly similar (Fig. S2).
Virus-induced gene silencing was first conducted in cv Terraregina. After inoculation with the VIGS construct TRV:GCHS1, 10 out of 30 treated gerbera inflorescences developed robust silencing phenotypes. The petals became milky white compared with the original bright orange color (Fig. 6a, middle). The white petals typically formed a sector in the inflorescence, on the same side of scape where the inoculation was done. By contrast, gerbera inflorescences inoculated with the VIGS vector TRV:GCHS4 did not show any visible changes and were similar to those infected with the empty TRV vector (Fig. 6a, right) in all three experiments.
Gene-specific silencing of GCHS1 and GCHS4 was detected in most (c. 65%) of the samples consisting of three petals sampled from the same side of scape where inoculation was done (Fig. 6b). Expression levels of both genes decreased to < 10% of the levels in the control samples. Slight off-target knockdown was observed in about one-third of the screened samples, in which untargeted GCHS1 or GCHS4 expression levels decreased to 70–80% of the levels in the control samples.
Since cv Terraregina contains only pelargonidin-derived pigments in petals (Fig. 6c, left panel), it seemed possible that GCHS4 is specifically involved in cyanidin biosynthesis, for example by participating in a different metabolon (Winkel, 2004) than GCHS1. Furthermore, GCHS4, but not GCHS1, was strongly up-regulated by GMYB10 in transgenic gerbera lines accumulating excessive amounts of cyanidin in tissues that are normally green (Laitinen et al., 2008). To investigate this, we used the cv President that accumulates cyanidin instead of pelargonidin in petals (Fig. 6c, right panel) for VIGS. As this cultivar is highly susceptible to TRV infection (Deng et al., 2012), obvious silencing phenotypes following infection with TRV:GCHS1 were achieved more frequently and in larger inflorescence sectors (Fig. 6d, middle) than with cv Terraregina. All inoculated plants of cv President, and 19 out of 30 treated inflorescences, displayed VIGS phenotypes. Petals in the infected inflorescence sector became purely white when GCHS1 was silenced (Fig. 6d, middle). By contrast, by silencing of the GCHS4, petals in the infected sector became only slightly paler than petals opposing the infection site of the inflorescence (Fig. 6d, right) and the color change could only be detected by close observation (Fig. S3). As in cv Terraregina, silencing was gene-specific in cv President (Fig. 6e), indicating that in both cultivars GCHS1 is responsible for anthocyanin biosynthesis, irrespective of whether the pigment produced is pelargonidin or cyanidin.
Samples taken from the silenced petal tissue were further subjected to HPLC analysis. The results showed that, in both cultivars, after the silencing of GCHS1, both colored and colorless flavonoids were markedly decreased to 0–34.7% of the values in the control samples taken from the opposite side of the same inflorescence (Tables 2, 3). By contrast, silencing of GCHS4 did not lead to significant changes in flavonoid concentrations in cv Terraregina (Table 2). Following silencing of GCHS4 in cv President, the concentrations of flavonoids were decreased, as suggested by the slightly paler color. However, these changes were much less dramatic than those in the GCHS1 silenced sectors, and in most cases were not statistically significant (Table 3).
Table 2. Flavonoids in GCHS1- and GCHS4-silenced petals of gerbera (Gerbera hybrida) cv Terraregina
Amounts are shown in μg mg−1 DW. Percentages in brackets are ratios of the levels in gene-silenced samples to the levels in their control samples (taken from the same inflorescence, on the opposite site of the gene-silenced sectors). Data are means ± SE (n = 3).
ND, not detectable. Mean values between gene-silenced and control samples were compared with one-way ANOVA, significant difference at: *, P ≤ 0.05; **, P ≤ 0.01.
Table 3. Flavonoids in GCHS1- and GCHS4-silenced petals of gerbera (Gerbera hybrida) cv President
Amounts are shown in μg mg−1 DW. Percentages in brackets are ratios of the levels in gene-silenced samples to the levels in their control samples (taken from the same inflorescence, on the opposite site of the gene-silenced sectors). Data are means ± SE (n = 3).
ND, not detectable. Mean values between gene-silenced and control samples were compared with one-way ANOVA, significant difference at: *, P ≤0.05; **, P ≤0.01; ***, P ≤0.001.
In conclusion, our data indicate that in both cultivars, biosynthesis of all analyzed flavonoids in petals depends on expression of GCHS1 rather than GCHS4.
GCHS1 is the major CHS enzyme in gerbera petals
For analysis of CHS enzyme activities in petals at different developmental stages, equal amounts of protein extracts from gerbera petals (stages 1–11, cv Terraregina) were subjected to an enzyme activity assay. For tracking the naringenin product, 4-coumaroyl-CoA and [14C]malonyl-CoA were used as substrates. As shown by radioactivity detected in the reaction product, CHS activity was low during the early developmental stages 1–4, increased during the middle stages 5–8 when pigmentation formed, and decreased during the later stages (Fig. 7a). This CHS enzyme activity pattern followed the GCHS1 expression pattern (Fig. 4a) and the anthocyanin pigmentation pattern well (Helariutta et al., 1993). Although GCHS4 is strongly expressed during petal development stages 5–11 (Fig. 4a), it cannot support CHS activity during the late stages. We conclude that GCHS1 is the major CHS enzyme in gerbera petals.
In order to shed light on the lack of GCHS4-derived CHS enzyme activity in petals, polysomes from gerbera petals at stage 6 (cv Terraregina) were extracted and loaded onto a sucrose gradient for ultracentrifugation. Total RNA was extracted from different fractions and subjected to quantitative RT-PCR. Results showed that mRNAs of both GCHS1 and GCHS4 coincided with fractions 6–12 containing polysomes (Fig. 7b). Thus, mRNAs of GCHS1 and GCHS4 were both loaded onto polysomes for translation.
Flavonoids are important secondary metabolites that color many flowers and fruits, in addition to their several other biological functions. Recent studies have broadened our understanding of the flavonoid biosynthetic pathway and its regulation, and have expanded from the main models to many economically important species, such as potato (Solanum tuberosum; Jung et al., 2009), tomato (Solanum lycopersicum; Schijlen et al., 2007), apple (Malus domestica; Dare et al., 2013; Espley et al., 2013), bilberry (Vaccinium myrtillus; Jaakola et al., 2002), strawberry (Fragaria × ananassa; Lunkenbein et al., 2006), and grape (Vitis species; Davis et al., 2012). Here, we studied CHS enzyme family in gerbera. We found that while GCHS1 is responsible for biosynthesis of all flavonoid groups in gerbera petals, GCHS4 catalyzes anthocyanin biosynthesis in vegetative tissues. Curiously, although GCHS4 is strongly expressed in gerbera petals, it does not seem to have a role in petal pigmentation.
CHS gene family in gerbera comprises of three genes
In this paper, we characterized the third CHS gene in gerbera, GCHS4. Sequence analysis and enzyme activity assay showed that GCHS4 is a true CHS, similar to the previously reported GCHS1 and GCHS3 (Helariutta et al., 1995b), and converts 4-coumaroyl-CoA and malonyl-CoA substrates into naringenin chalcone. No further CHS sequences have been found in large sets of gerbera RNA sequence reads (T. H. Teeri & P. Elomaa, unpublished) or ESTs (Laitinen et al., 2005). Thus, CHS enzymes in gerbera are encoded by a family of three genes: GCHS1, GCHS3, and GCHS4.
In arabidopsis and snapdragon, CHS is encoded by a single gene (Sommer & Saedler, 1986; Burbulis et al., 1996). More commonly, CHS is encoded by a gene family that contains up to 10 members in some species, such as petunia (Petunia hybrida, eight to 10 members) (Koes et al., 1989), maize (Zea mays, two members) (Coe et al., 1981) and morning glory (Ipomoea purpurea, six members) (Johzuka-Hisatomi et al., 1999). Plant CHSs are highly similar in their amino acid sequence. Gerbera CHSs, for example, share 80–88% amino acid sequence identity with each other, and 79–84% with arabidopsis CHS and alfalfa CHS2.
A growing number of other plant-specific PKSs, such as acridone synthase (ACS), phlorisovalerophenone synthase (VPS), STS, and 2PS, have been found to share the same chemical mechanism with CHS but differing in their preference for starter substrate, number of acetate unit extensions and the cyclization of the released procedure (Austin & Noel, 2003; Abe & Morita, 2010). These enzymes also share high sequence similarity with CHS. Thus, many of the CHS sequences in public sequence databases, which are characterized only by their sequence similarity, may actually encode other related enzymes (Schröder, 1997). In gerbera, for example, G2PS1 is c. 70% identical to gerbera CHS for its sequence, but catalyzes biosynthesis of bitter 2-pyrone glucosides that are not phenylpropanoids (Eckermann et al., 1998).
Functions of gerbera CHSs are spatially and temporally regulated
The expression patterns of gerbera CHSs are spatially regulated. Unlike the gerbera G2PS1, which was expressed globally in all tissues, gerbera CHSs were expressed mostly in the reproductive tissues that are rich in flavonoids, such as pappus, petals and carpels (Fig. 4a, Table 1). In contrast to soybean, where CHS genes are most abundantly expressed in roots (Tuteja et al., 2004), gerbera CHS mRNAs are undetectable there. GCHS3 is expressed only in pappus bristles. Together with the concomitantly expressed GCHS1, GCHS3 is involved in the biosynthesis of colorless flavonoids in cv Terraregina (Table 1). Pappus bristles in the Asteraceae plants are proposed to be specialized sepals for seed dispersal (Yu et al., 1999). In some cultivars, such as cv Parade and cv Nero, the pappus is pigmented (Helariutta et al., 1995a). CHSs in the pappus of these two cultivars are obviously involved in anthocyanin biosynthesis. Pappus in cv Terraregina is colorless, as a result of the absence of GDFR expression (Helariutta et al., 1995a).
In addition to tissues from reproductive organs, some gerbera vegetative tissues, such as leaf petioles and the scape bases, are naturally red. Growing under stress conditions, such as water stress, wounding and pathogen infection, gerbera leaf blades turn reddish. The anthocyanins in these red tissues are mainly cyanidin (Fig. 5b, Table 1). GCHS4 is the only CHS that is expressed in the red vegetative tissues. A previously reported R2R3-type GMYB10 TF is induced in the stressed leaves, and in turn regulates the GCHS4 expression. Moreover, ectopic expression of the GMYB10 in gerbera enhanced pigmentation in various tissues such as leaf petioles and scapes (Laitinen et al., 2008).
Expression of gerbera CHSs in petals is also developmentally regulated in a way that associates with their anthocyanin pigmentation. GCHS1 and GCHS4 are expressed in the adaxial epidermis where anthocyanins accumulate, and expression of both genes is induced when anthocyanin biosynthesis begins. However, GCHS1 is expressed at some level during all 11 developmental stages while GCHS4 expression is absent in the early stages of petal development. The expression pattern of GCHS1 shows a typical CHS expression pattern (Fig. 4a), similar to those in petunia, snapdragon, and tree peony (Paeonia suffruticosa): the expression levels first increase in the early stages, peak during the middle stages when flower pigmentation starts, and then decrease in the later stages (Koes et al., 1989; Jackson et al., 1992; Zhou et al., 2011). By contrast, GCHS4 expression that starts later at stage 4 is maintained at high levels during all later stages (stages 5–11) (Fig. 4a).
GCHS1 is the dominant CHS in gerbera petals
Although many plants contain several CHS genes, in the main only one or two members of the gene family are transcribed, or account for the majority of CHS mRNAs in a particular tissue (Koes et al., 1986; Fukada-Tanaka et al., 1997; Johzuka-Hisatomi et al., 1999). This is also the case in gerbera. In gerbera pappus bristles, GCHS1 and GCHS3 are expressed, and in the pigmented vegetative tissues, only GCHS4 is expressed. Both GCHS1 and GCHS4 are expressed in gerbera petals. At later stages (stages 5–11) of petal development, GCHS4 is primarily expressed, with its mRNA taking up 64.1% (stage 5) to 96.6% (stage 11) of the total CHS mRNA (Fig. 4a).
Despite the high expression of GCHS4, the VIGS experiments, by the silencing of GCHS1 and GCHS4 expression independently, revealed that GCHS1 is actually the dominant CHS enzyme in gerbera petals. In both cv Terraregina (a pelargonidin cultivar) and cv President (a cyanidin cultivar), silencing of GCHS1 resulted in the development of white petals, in which all the major flavonoid products were decreased. By contrast, silencing of GCHS4 caused almost no change in flower color and the major flavonoid products of either cultivar.
GCHS4 in gerbera petals is post-transcriptionally regulated
Structural genes in the flavonoid biosynthetic pathway are mostly regulated transcriptionally, by the combination and interaction of the R2R3-MYB, bHLH and WD40 TFs (Koes et al., 2005). Regulation of anthocyanin-related genes at the post-transcriptional level has been reported in only a few cases (Franken et al., 1991; Pairoba & Walbot, 2003). Our data have provided evidence that GCHS4 is regulated post-transcriptionally in gerbera petals. Although GCHS4 is predominantly transcribed in gerbera petals at stages 5–11, it does not contribute to either color or extractable CHS activity. The post-transcriptional control of GCHS4 in gerbera is tissue-specific, taking place in gerbera petals but not in vegetative tissues. This is similar to the case in maize, where post-transcriptional control of the maize CHS gene Whp was observed in aleurone cells but not in the tassel (Franken et al., 1991). The control of maize Whp in aleurone, which the authors suggest is at the translational level, is dependent on the expression of the anthocyanin intensifier gene In (Franken et al., 1991). Another maize anthocyanin gene, Bronze2, is also under post-transcriptional control, being specifically down-regulated in tissues lacking anthocyanins, but not in tissues accumulating these pigments (Pairoba & Walbot, 2003). The regulation of Bronze2, however, is partly related to the failure of mRNA splicing (Pairoba & Walbot, 2003).
The mechanism of GCHS4 post-transcriptional control in gerbera petals is still unknown. Mapping of RNA sequence reads isolated from petals of cv Terraregina and from the 35S-MYB10 transgenic line on the GCHS4 cDNA shows that transcript splicing is similar and complete in both cases (T. H. Teeri & P. Elomaa, unpublished). We showed that strong GCHS4 expression does not contribute to CHS activity during the late stages of petal development. In Western blot analysis of gerbera anti-G2PS1 lines (where the interfering G2PS1 protein is not made), the pattern of CHS protein accumulation follows that of enzymatic activity, indicating that GCHS4 is not controlled through post-translational modification of the protein molecule (data not shown). GCHS4 mRNA is still loaded onto polysomes at a similar efficiency to GCHS1 mRNA (Fig. 7b). Thus, the regulation of GCHS4 probably occurs by inhibition of translation elongation or by protein degradation after translation.
For both historical and technical reasons, post-transcriptional regulation has been less intensively studied than transcriptional regulation, in spite of its importance. RNA sequencing becomes increasingly affordable and provides an efficient and unbiased snapshot of steady-state mRNA levels. The finding of post-transcriptional regulation of GCHS4 in gerbera petals provides a clear example of the limitations of mRNA analysis and sheds new light on the regulation of flavonoid biosynthesis.
We would like to thank Dr Dinesh-Kumar for sharing TRV Gateway VIGS vectors and the breeding company Terra Nigra B.V. (the Netherlands) for supplying the gerbera cultivars. We thank our laboratory technicians Eija Takala, Anu Rokkanen, and Marja Huovila for their skilled technical assistance, the glasshouse technicians Sanna Peltola, Johanna Boberg, and Sini Lindström for taking care of the gerbera plants, our colleagues Dr Takeshi Kurokura and Dr Suvi Broholm for their help with the in situ hybridization experiment, and Prof. Elliot Meyerowitz and Dr Katri Eskelin for valuable discussions on polysome extraction. This work was supported by the Finnish Doctoral Program in Plant Science (to X.D. and H.B.), Academy of Finland (grants 1253126 to J.P.T.V. and 139513 to T.H.T.), and Cairo University internal scholarship (for H.B., 2009).