Planta DOI 10.1007/s00425-014-2152-9

ORIGINAL ARTICLE

Temporal and spatial regulation of anthocyanin biosynthesis provide diverse flower colour intensities and patterning in Cymbidium orchid Lei Wang • Nick W. Albert • Huaibi Zhang • Steve Arathoon • Murray R. Boase Hanh Ngo • Kathy E. Schwinn • Kevin M. Davies • David H. Lewis

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Received: 22 May 2014 / Accepted: 15 August 2014 Ó Springer-Verlag Berlin Heidelberg 2014

Abstract Main conclusion This study confirmed pigment profiles in different colour groups, isolated key anthocyanin biosynthetic genes and established a basis to examine the regulation of colour patterning in flowers of Cymbidium orchid. Cymbidium orchid (Cymbidium hybrida) has a range of flower colours, often classified into four colour groups; pink, white, yellow and green. In this study, the biochemical and molecular basis for the different colour types was investigated, and genes involved in flavonoid/anthocyanin synthesis were identified and characterised. Pigment analysis across selected cultivars confirmed cyanidin 3-Orutinoside and peonidin 3-O-rutinoside as the major anthocyanins detected; the flavonols quercetin and kaempferol rutinoside and robinoside were also present in petal tissue. b-carotene was the major carotenoid in the

Electronic supplementary material The online version of this article (doi:10.1007/s00425-014-2152-9) contains supplementary material, which is available to authorized users.

yellow cultivars, whilst pheophytins were the major chlorophyll pigments in the green cultivars. Anthocyanin pigments were important across all eight cultivars because anthocyanin accumulated in the flower labellum, even if not in the other petals/sepals. Genes encoding the flavonoid biosynthetic pathway enzymes chalcone synthase, flavonol synthase, flavonoid 30 hydroxylase (F30 H), dihydroflavonol 4-reductase (DFR) and anthocyanidin synthase (ANS) were isolated from petal tissue of a Cymbidium cultivar. Expression of these flavonoid genes was monitored across flower bud development in each cultivar, confirming that DFR and ANS were only expressed in tissues where anthocyanin accumulated. Phylogenetic analysis suggested a cytochrome P450 sequence as that of the Cymbidium F30 H, consistent with the accumulation of di-hydroxylated anthocyanins and flavonols in flower tissue. A separate polyketide synthase, identified as a bibenzyl synthase, was isolated from petal tissue but was not associated with pigment accumulation. Our analyses show the diversity in flower colour of Cymbidium orchid derives not from different individual pigments but from subtle variations in concentration and pattern of pigment accumulation. Keywords Anthocyanin  Carotenoid  Flavonoid  Flower colour  Orchid

Special topic: Anthocyanins. Guest editor: Stefan Martens. L. Wang  N. W. Albert  H. Zhang  S. Arathoon  M. R. Boase  H. Ngo  K. E. Schwinn  K. M. Davies  D. H. Lewis (&) The New Zealand Institute for Plant and Food Research Limited, Private Bag 11 600, Palmerston North 4474, New Zealand e-mail: [email protected] L. Wang Institute of Molecular BioSciences, Massey University, Private Bag 11-222, Palmerston North, New Zealand

Abbreviations ANS Anthocyanidin synthase BBS Bibenzyl synthase CHS Chalcone synthase DFR Dihydroflavonol reductase FLS Flavonol synthase F30 H Flavonoid 30 hydroxylase 0 0 F3 5 H Flavonoid 30 50 hydroxylase PKS Polyketide synthase

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Introduction The genus Cymbidium is part of the Orchidaceae, the largest family within the angiosperms, which includes a large number of genera that exhibit diverse vegetative and floral forms (Bechtel et al. 1992). The diversity of orchids and their aesthetic appeal mean that they have been grown as cultivated plants for hundreds of years (Bechtel et al. 1992; Hew 2001) and some have become significant Fig. 1 Flowers from the selected Cymbidium cultivars illustrating the four main colour groups; pink/red, yellow, green and white. a–h Individual cultivars used in this study illustrating the flower colour range. Pink cultivars: ‘Clarisse Austin South Pacific’ (‘CASP’, a), ‘Lisa Rose Flamingo’ (‘LRF’, b). Yellow cultivars: ‘Gymer Cooksbridge’ (‘GC’, c), ‘Arcadian Sunrise’ (‘AS’, d). Green cultivars: ‘Big Chief Kirawee’ (‘BCK’, e), ‘Vanguard Mas Beauty’ (‘VMB’, f). White cultivars: ‘Virgin’ (‘V’, g), ‘Jungfrau dos Pueblos’ (‘JDP’, h). Floral organs including the outer whorl of sepals, the inner whorl of petals and the modified lip (labellum) are labelled in a

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horticultural crops. Cymbidium orchids are available in a wide range of flower colours and pigment patterns, but the biochemical and molecular basis for these colour morphs is poorly understood compared to other orchid genera. Despite the diversity in flower morphology present in the Orchidaceae, there are characteristic morphological features. The floral parts form in whorls of three (characteristic of monocots), the stigma and style are fused to form the flower column, and the central petal forms an elaborate

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lip (labellum), often with intricate pigmentation patterns (Bechtel et al. 1992). Generally, the flower labellum is larger than the other petals and most frequently lower than the other petals, acting as a landing platform for pollinators (Bechtel et al. 1992). Selection and breeding of Cymbidium has focused on enhancing flower colour and size, particularly via interspecific hybrids (Tomlinson 1985; Bechtel et al. 1992), providing a range of commercial cultivars. Flower colour and pigmentation patterning are important traits for floriculture crops, with novel colours and patterns demanding high prices from consumers. A range of Cymbidium cultivars are available, which are classified into four general flower colour groups: pink, yellow, green and white (Fig. 1). These broad colour groupings are generally determined by the presence or absence of anthocyanins (pink) in the petal tissue along with the presence or absence of chlorophyll (green) and carotenoid (yellow) pigments (Arditti 1992). The basis for differential regulation of anthocyanin pigmentation in Cymbidium is poorly understood, as is the accumulation and retention of chlorophyll and carotenoid pigments. Anthocyanin pigments are derived from the flavonoid biosynthetic pathway and their biosynthesis has been well characterised in model species such as petunia (Petunia hybrida), Antirrhinum (A. majus), Arabidopsis (A. thaliana) and maize (Zea mays) (Grotewold 2006). The first committed step for flavonoid synthesis is catalysed by the polyketide synthase (PKS) family member chalcone synthase (CHS). It is required for the synthesis of all flavonoids, including coloured anthocyanin pigments, as well as colourless flavonoids such as flavonols. CHS is often co-ordinately regulated with the general flavonoid genes chalcone isomerase (CHI) and flavanone 3-hydroxylase (F3H) as well as flavonol synthase (FLS), which is a key branch-point step for flavonol synthesis (Hartmann et al. 2005; Albert et al. 2009). These genes are expressed in both vegetative tissues in response to light (Hartmann et al. 2005; Albert et al. 2009) and in flowers (Weiss 2000). Flavonols perform UV-protective functions, can form nectar guides visible to insects and can act as co-pigments with anthocyanins (Davies et al. 2012). Dihydroflavonol 4-reductase (DFR) catalyses the formation of leucoanthocyanidin and is a key point of regulation for anthocyanin synthesis. Subsequently, the action of anthocyanidin synthase (ANS) generates the chromophore, resulting in coloured anthocyanidins, which then become glycosylated, forming anthocyanins (Tanaka et al. 2008). Flavonoid biosynthesis separates into three distinct lineages depending on the degree of hydroxylation of the flavonoid B-ring, which generates the three basic classes of anthocyanins; pelargonidin (orange/red), cyanidin (pink/magenta) and delphinidin (purple/blue) (Fig. 2).

The hydroxylation state alters the perceived hue of the pigments, which can be further modified or intensified by chemical modifications to the basic anthocyanin molecule (e.g. glycosylation, acylation and methylation) and through co-pigmentation with colourless flavonoids (Schwinn and Davies 2004). The hydroxylation state of the flavonoid B-ring is determined by the activities of flavonoid 30 -hydroxylase (F30 H) and flavonoid 30 50 hydroxylase (F30 50 H), referred to as the ‘red’ and the ‘blue’ genes, respectively (Tanaka et al. 2008), as they are necessary for the production of cyanidin (pink/ magenta) and delphinidin (purple/blue)-based anthocyanins. The absence of orange/red and purple/blue colours in Cymbidium suggests that pelargonidin- and delphinidin-based anthocyanins do not accumulate to significant levels. Targeting the production of these pigment classes could be important for generating Cymbidium cultivars with novel colours. Flavonoid biosynthetic genes have been cloned and characterised, for some monocotyledonous ornamental plants including some orchids, to link patterns of gene expression with pigment accumulation and flower colour. CHS, DFR and ANS genes were isolated and characterised for Anthurium andraeanum (Collette et al. 2004), and CHS and DFR from the Asiatic hybrid lily (Nakatsuka et al. 2003). In terms of the Orchidaceae, both CHS and DFR have been cloned from Bromheadia (Liew et al. 1998a, b), CHS, DFR and a F30 50 H from Dendrobium (Mudalige-Jayawickrama et al. 2005; Whang et al. 2011) and CHS, DFR and F30 50 H from Phalaenopsis (Wang et al. 2006; Ma et al. 2009). In general, flavonoid biosynthetic genes showed patterns of expression in tissues and at stages of bud development consistent with the observed formation of anthocyanin pigments in these species. The Cymbidium flavonoid biosynthetic gene DFR has been isolated (Johnson et al. 1999); however, its pattern of expression during flower development has not been reported. In this study, the pigment composition of anthocyanins, flavonols, carotenoids and chlorophyll was analysed in petal tissue from eight Cymbidium cultivars, across four colour groups. We report the isolation of four flavonoid biosynthetic pathway genes, CHS, ANS, FLS and F30 H and a phenylpropanoid pathway gene, BBS, isolated from Cymbidium flower tissue. The temporal and spatial expression patterns of these flavonoid biosynthetic genes as well as DFR were analysed in sepal/petal and labellum tissues of Cymbidium flowers across six developmental stages and then compared to the observed flower colour. The study has advanced our understanding of pigment profiles and colour in Cymbidium flowers, and will help form possible strategic directions to allow the development of novel flower colours.

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Planta Fig. 2 Schematic representation of the flavonoid biosynthetic pathway. The cross indicates the limitation of pigment biosynthesis in Cymbidium. Dotted arrows represent flavonoids not produced appreciably in Cymbidium

Materials and methods Plant material Eight Cymbidium hybrida cultivars representing four flower colour groups were used in this study (Fig. 1). The cultivars used were the pink cultivars: ‘Clarisse Austin South Pacific’ (‘CASP’) and ‘Lisa Rose Flamingo’ (‘LRF’); yellow cultivars: ‘Gymer Cooksbridge’ (‘GC’) and ‘Arcadian Sunrise’ (‘AS’); green cultivars ‘Big Chief Kirawee’ (‘BCK’) and ‘Vanguard Mas Beauty’ (‘VMB’); white cultivars: ‘Virgin’ (‘V’) and ‘Jungfrau dos Pueblos’ (‘JDP’). Cymbidium flowers and leaf material were obtained from commercial growers. Cut flower stems, each with 6–12 individual flowers, were harvested when the first flower was open on the stem. The cut stems were held in tubes of tap water in a vase-life room maintained at a constant temperature of 20 °C and illuminated with a 12 h photoperiod using cool fluorescent lights (20–25 lmol m-2 s-1). A sequence of six developmental stages was defined to ensure buds and flowers were harvested in a consistent manner (Fig. 3). The harvested

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flowers were then dissected into combined sepal/petal tissues and labellum tissue. Young leaves were taken from the same plants as those used for inflorescence harvests in the same growing season. All samples were frozen in liquid nitrogen and ground to a powder at harvest. Tissue samples for gene expression studies were stored at -80 °C. Tissue samples for pigment analysis were freeze-dried and stored at -20 °C. Anthocyanin and flavonoid analysis Pigment content in petal and labellum tissue from fully open (stage 6) flowers was analysed. Three samples of 50 mg (DW) petal tissue from each cultivar was extracted at 4 °C overnight with 2 ml methanol:acetic acid:water (70:3:27, by vol.). The samples were centrifuged for 4 min at 13,000 g, the supernatant removed and the pellet reextracted in 2 ml methanol:acetic acid:water (90:1:9, by vol.). This process was repeated a third time. The combined supernatants were dried in vacuo on a Savant SC210 Speedvac to approximately 100 ll and made up to a final volume of 2 ml in 80 % methanol [methanol:acetic

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Fig. 3 Flower developmental stages used to assess flavonoid gene expression patterns. The stages shown are from stage 1 (small immature bud) through to stage 6 (open flower) in cultivar ‘Lisa Rose Flamingo’

acid:water (80:2:18, by vol.)]. The extracts were centrifuged and the flavonoids analysed by high-performance liquid chromatography (HPLC), using a Dionex 3000 Ultimate solvent delivery system with a Phenomenex Luna (5 lm, 150 9 4.6 mm) RP-C18 column (column temperature 25 °C) and a Dionex 3000 PDA detector. Elution (0.8 ml min-1) was performed using a solvent system comprising solvent A [HOAc:CH3CN:H3PO4:H2O (20:24:1.5:54.5, by vol.)] and solvent B (1.5 % H3PO4) and a linear gradient starting with 35 % A, increasing to 67 % A at 20 min, 90 % A at 23 min and 100 % A at 29.3 min, remaining at 100 % A for a further 10 min. Flavonoids were detected at 350 nm and anthocyanins at 530 nm. Flavonoid concentrations were determined as quercetin-3O-rhamnoglucoside (Apin Chemicals, Abingdon, Oxon, UK) equivalents, and the anthocyanins as cyanidin 3-Oglucoside (Extrasynthese, Genay, France) equivalents. Carotenoid analysis Two 50 mg dry weight (DW) samples of petal or labellum tissue from each cultivar were moistened with water (100–200 ll) and then extracted in acetone:methanol (7:3, v/v) as described by Albert et al. (2009). After partitioning, the carotenoid extracts were dried under O2-free N2 and then redissolved in 1 ml of 0.8 % butylated hydroxytoluene (BHT)/acetone and analysed by HPLC as previously described (Ampomah-Dwamena et al. 2012). Carotenoids and chlorophyll b were detected at 450 nm, whilst chlorophyll a and other chlorophyll derivatives were monitored at 430 nm. The levels of carotenoids were determined as bcarotene equivalents/g DW of tissue, chlorophyll b was determined using a chlorophyll b standard curve derived from a spinach extract. Chlorophyll a and other chlorophyll derivatives were determined as chlorophyll a equivalents/g DW of tissue, derived from a standard curve using a spinach extract. b-carotene and lutein were identified in the extracts by comparison of retention times and online

spectral data with standard samples. Trans-b-carotene was purchased from Sigma Chemicals (St Louis, MO, USA). Other carotenoids were putatively identified by comparison with reported retention times and spectral data and by comparison with carotenoids present in a spinach extract (Fraser et al. 2000; de Rosso and Mercadante 2007). LCMS analyses Mass spectroscopy was used to identify the main components in both the carotenoid and flavonoid extracts. The LCMS system consisted of a Thermo Electron Corporation (San Jose, CA, USA) Finnigan Surveyor MS pump, Thermo Accela Open Auto sampler (PAL HTC-xt with DLW), Finnigan Surveyor PDA plus detector and a ThermaSphere TS-130 column heater (Phenomenex, Torrance, CA, USA). Flavonoids: A 2 ll aliquot of each prepared extract was separated with a mobile phase consisting of 0.1 % formic acid in water (A) and 0.1 % formic acid in acetonitrile (B) by reverse phase chromatography (Kinetex guard car˚ , 100 9 2.1 mm, tridge and Kinetex C18, 2.6 lm, 100 A Phenomenex) maintained at 30 °C with a flow rate of 250 ll/min. A gradient was applied: min/A%/B% as 0/95/ 5, 3/95/5, 20/65/35, 28/0/100, 30.5/0/100, 31/95/5, and 36/95/5. The eluent was scanned by PDA (200–600 nm) and API-MS (LTQ, 2D linear ion-trap, Thermo-Finnigan, San Jose, CA, USA) with electrospray ionisation (ESI) in the negative and positive mode. Data were acquired for parent masses from m/z 145–2,000 amu with MS3. Data were processed with the aid of XcaliburÒ 2.20 (Thermo Electron Corporation) and Mass FrontierTM 4 or 7 (Thermo Electron Corporation). Carotenoids: A 5 ll aliquot of each prepared extract was separated with a mobile phase consisting of methyl-tertbutyl-ether (MTBE) (A) and methanol (B) by reverse phase chromatography (YMC30, 250 9 2.1 mm) maintained at 30 °C with a flow rate of 200 ll/min (300 ll/min from

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33.5 to 39 min). A gradient was applied: tmin/A%/B% as t0/ 5/95, t33/62.7/37.3, t33.5–36/90/10, t36.5–39.50/5/95. The eluent was scanned by PDA (300–650 nm) and API-MS (LTQ, 2D linear ion-trap, Thermo-Finnigan) with atmospheric pressure chemical ionisation (APCI) in the positive mode. Data were acquired for parent masses from m/z 400–1,500 amu with fragmentation down to MS3. Data were processed with the aid of XcaliburÒ 2.20 (Thermo Electron Corporation). Isolation of Cymbidium genes All primers used are shown in Supplementary Table ST1. Total RNA was isolated from a mixture of sepal and petal tissues of combined developmental stages of Cymbidium ‘CASP’ and ‘LRF’ using a modified hot borate method (Hunter et al. 2002). First-strand cDNA was prepared using a Superscript II reverse transcriptase (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. Initial gene fragments were isolated by polymerase chain reaction (PCR) using degenerate primers based on conserved domains. The remaining coding sequence of each gene was obtained by rapid amplification of cDNA ends (RACE) PCR, genome walking, or both. The open reading frame (ORF) for each gene was then amplified to verify the contiguous sequence assembled from the RACE and genome walking fragments was for a single gene. The DFR ORF (KM186174) was amplified from ‘CASP’ cDNA using primers based on the published sequence (AF017451, Johnson et al. 1999). The ORF sequence had 22 SNPs and seven amino acid changes compared to the published sequence (Supplementary Fig. S2). 50 RACE used the Invitrogen 50 RACE kit. Genome walking was performed using a GenomeWalker Universal Kit (Clontech Laboratories, Inc., Mountain View, CA, USA) and Triple Master polymerase (Eppendorf, Hamburg, Germany). Genomic DNA for genome walking was extracted from floral tissue using urea buffer (7 M urea, 0.3 M NaCl, 50 mM Tris (pH 8), 20 mM EDTA and 1 % (w/v) N-lauroylsarcosine) and then washed with phenol/chloroform before precipitation. RNA gel blot analysis RNA gel blots were prepared using 20 lg total RNA extracted from stage 1 to 6 of Cymbidium cultivars ‘CASP’, ‘LRF’, ‘BCK’, ‘VMB’, ‘JDP’, ‘V’, ‘GC’ and ‘AS’, as described in Albert et al. (2009). RNA blots were probed with full-length ChCHS, ChFLS, ChDFR and ChANS coding sequence fragments, and a 1.6 kb ChF30 H partial coding sequence fragment. Radiolabelled probes ([a-32P] dATP) were generated by random priming (Feinberg and Vogelstein 1983) cDNA templates of the genes. Unincorporated nucleotides were removed using

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ProbeQuantTM G-50 Micro Columns (GE Healthcare, Little Chalfont, UK). RNA blots were hybridised overnight in Church and Gilbert buffer (Church and Gilbert 1984) at 60 °C and washed to a final stringency of 19 SSC at 60 °C. Images were developed by exposing membranes to a FLA5100 phosphorimager imaging system (Fujifilm, Tokyo, Japan) or X-ray film (Kodak, USA). Blots were stripped after collection of northern analysis images and re-probed for the next gene. Blots were stripped by immersion in boiling 0.1 % SDS and then left to cool to room temperature. Phylogenetic analyses The protein sequences were aligned using ClustalW followed by Maximum-Likelihood analysis using the PhyML programme (Guindon and Gascuel 2003) within the bioinformatics software Geneious (version 6.1.6; Biomatters, Auckland, New Zealand). The default parameters were used: substitution model set to LG; proportion of variable sites set to fixed 0; number of substitution rate categories set to 4; gamma distribution parameter set to estimated; optimise set to topology/length/rate; topology search set to NNI (Default, fast). Bootstrap analysis with 1,000 replicates was used to estimate the confidence of each tree node. Sequences used in the phylogenies with GenBank accession numbers were as follows. (1) Orchidaceae; Bromheadia finlaysoniana CHS (O23729), bibenzyl synthase (CAA10514), Cymbidium floribundum CHS (ABS88695), Dendrobium hybrid CHS (AAU93767), ANS (AGY46124), FLS (AGY46126), F30 50 H (ABI95365), Dendrobium moniliforme F30 50 H (AEB96145), Dendrobium nobile CHS (ABE77392), Doritaenopsis hybrid ANS (AHA36978), Oncidium Gower Ramsey CHS (ABS58499), ANS (AET99288), Paphiopedilum concolor CHS (AFS60082), Phalaenopsis hybrid CHS (AAY83389), bibenzyl synthase (CAA56276), F30 50 H (AAZ79451). (2) Other Asparagales species; Allium cepa CHS (AAO63021), ANS (ABM66367), FLS (AY221247), F30 H (AAS4819), Lycoris chinensis ANS (AGD99672). (3) Non-Asparagales monocotyledonous species; Anthurium andraeanum CHS (ABE01413), ANS (AAP20867), Iris 9 hollandica CHS (BAF99252), ANS (BAF81268), Lilium cernuum ANS (AGC92011), Lilium hybrid CHS (AF169800), F30 H (BAM28972), Musa acuminata CHS (AHB18370), Narcissus tazetta CHS (AEN04070), FLS (AFS63900), Oryza sativa CHS (BAA19186), ANS (CAA69252), Secale cereale CHS (CAA63306), Strelitzia reginae ANS (AGC73738), Triticum aestivum ANS (BAE98276), Tulipa fosteriana ANS (AGL98404), Tulipa gesneriana ANS (BAH98156), Zea mays CHS (CAA42763), ANS (NP_001106074), F30 H (AEF33624). (4) Eudicots; Antirrhinum kelloggii F30 50 H (BAJ16329),

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Antirrhinum majus CHS (P06515), FLS (DQ272591), F30 H (ABB53383, adjusted to remove upstream ATG), Arabidopsis thaliana CHS (NP_196897), ANS (CAD91994), FLS (U84260), F30 H (AAF60189), Arachis hypogaea CHS (AAU43217), stilbene synthase (BAA78617), Cyclamen persicum F30 50 H (ACX37698), Dahlia pinnata F30 H (ACO35754), FNSII (BAM72335), Echinops bannaticus F30 H (ACN65826), Epimedium sagittatum FLS (ABY63659), F30 H (ADE80941), F30 50 H (ADE80942), Eustoma exaltatum ANS (BAJ08926), FLS (BAD34463), F30 50 H (BAK64100), Fragaria 9 ananassa ANS (AFK32781), FLS (AAZ78661), Gerbera hybrida F30 H (ABA64468), FNSII (AF156976), Glycine max ANS (AY382828), FLS (AB246668), FNSII (ACV65037), Malus domestica ANS (ABD04052), FLS (AF119095), Medicago sativa CHS (AAB41559), Perilla frutescens FNSII (BAB59004), Petunia hybrida CHS (CAA32735), ANS (P51092), FLS (Z22543), F30 H (AF155332), F30 50 H (AAC32274), Populus trichocarpa F30 H (EEE95684), F30 50 H (EEE87959), FNSII (XP_002328425), Solanum lycopersicum CHS1 (NP_001234033), Solanum tuberosum ANS (NP_001274859), FLS (NP_001274926), Vitis vinifera CHS (BAB84112), ANS (ABV82967), FLS (XM_002285803), stilbene synthase (ABD64685), F30 H (BAE47006), F30 50 H (ABH06585). (5) Gymnosperms; Ginkgo biloba CHS (AAT68477), ANS (EU600206), FLS (AY496932). Pinus sylvestris CHS (CAA43166), pinosylvin synthase (CAA43165). (6) Bryophyta; Marchantia polymorpha stilbenecarboxylate synthase 1 (AAW30009).

Results Cymbidium orchid flower colour Eight cultivars, representing the main colour groups used for commercial production, were chosen for characterisation of the pigments present in Cymbidium flower tissue (Fig. 1). ‘CASP’ and ‘LRF’ are cultivars with a pink flower colour although that colour is much darker in ‘CASP’, ‘GC’ and ‘AS’ are both yellow cultivars, ‘VMB’ and ‘BCK’ are green cultivars, whilst ‘JDP’ and ‘V’ are whites. Despite being classified as pink, white, yellow or green, all eight cultivars have some red-coloured tissue, particularly in the flower labellum. In some cases, a red blush was observed in the sepal and petal tissue of those cultivars where the predominant colour was not pink. The flower sepals and petals are labelled in Fig. 1a. The flower has a whorl of sepals and then a whorl of petals. The sepals and petals in the Cymbidium flower are pigmented in the same manner; pigment analysis was carried out using a combined sepal/petal sample. The flower labellum is a modified petal and different in colour as well as shape. Flower

labellum tissue was analysed separately for pigment analysis, as this organ has distinct pigmentation patterns from petals. Anthocyanin accumulation in sepal/petal tissue of pink cultivars began at stage 3 of flower bud development, and accumulated further at stages 4 and 5 (Fig. 3). There was some variation between the two pink cultivars but the general patterns were similar. At the same time, there was a concomitant loss of chlorophyll pigments and green colouration. The loss of chlorophyll also occurred in the white and yellow cultivars but not in the greens. Anthocyanin accumulation in the labellum of green, yellow and red cultivars occur in early stages of bud development. In contrast, pigmentation in the labellum of white cultivars occurs mid to late stages of bud development. Isolation and analysis of chalcone synthase (CHS), flavonol synthase (FLS), flavonoid 30 hydroxylase (F30 H), anthocyanidin synthase (ANS) and the polyketide synthase bibenzyl synthase (BBS) Two CHS-like PKSs, initially designated PKS1 and PKS2, were obtained. PKS1 and PKS2 have coding sequences of 1,173 and 1,182 bp, and encode proteins of 390 and 394 amino acid residues, respectively. Phylogenetic analysis of the deduced amino acid sequences of PKS1 and PKS2 against a range of PKS sequences from other plant species was performed. The protein sequences used included CHS sequences from within the Orchidaceae, the wider Asparagales, and a range of other monocot, eudicot and gymnosperm taxa, as well as sequences for bibenzyl synthase (BBS), stilbene synthase (STS) and pinosylvin synthase (PSS). The CHS sequences included those that were annotated as such in addition to those that have experimental confirmation. The analysis showed that CHS sequences from orchid (with the exception of that for Cymbidium floribundum) formed a separate clade with 99.8 % bootstrap support, and PKS2 grouped within that clade, indicating PKS2 is likely CHS (Fig. 4a). The CHS sequences from other monocots are the next closest neighbours to the orchid sequences. The BBS sequences available from the orchids Phalaenopsis sp. and Bromheadia finlaysoniana formed a separate clade, with 100 % bootstrap support. Cymbidium PKS1 and the partial Cymbidium floribundum sequence (which is annotated as CHS) segregated with BBS sequences from other orchid species, suggesting both PKS1 and the C. floribundum sequence encode a BBS enzyme rather than CHS. As a result of the phylogenetic analysis, PKS1 was renamed as ChBBS (KM186175) and PKS2 as ChCHS (KM186176). The ORF for ChFLS (KM186173) was 999 bp encoding 333 deduced amino acid residues and ChANS (KM186177) was 1,077 bp encoding 359 deduced amino acid residues. The ChANS and ChFLS protein sequences were compared

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a

b

c

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Planta b Fig. 4 Phylogenetic relationships of CHS (a), ANS and FLS (b) and

F30 H (c) sequences from Cymbidium hybrida. The deduced peptide sequences of the candidate C. hybrida clones were compared in phylogenetic alignments with characterised sequences for each enzyme from GenBank. Maximum-likelihood phylogenetic trees were formed from manually optimised ClustalW alignments. Alignments for CHS include examples of stilbene synthase (STS), pinosylvin synthase (from Pinus sylvestris), stilbenecarboxylate synthase 1 (from the liverwort Marchantia polymorpha) and bibenzyl synthase (BBS). The ANS and FLS were aligned in one tree, as they are both in the same protein superfamily (2-oxoglutarate-dependent oxygenases). F30 H is shown in comparison to sequences for F30 H, F30 50 H and FNSII, all of which are CYP450 proteins. Accession details of the sequences used are listed in Materials and methods. Sequences shown in red belong to the Orchidaceae, those in blue to other monocots, those in black to eudicots and those in green to gymnosperms. The scale bar represents substitutions per site and the numbers next to the nodes are bootstrap values from 1,000 replicates. Only bootstrap values over 65 are shown. Species abbreviations used: Allium cepa (Ac), Anthurium andraeanum (Aa), Antirrhinum kelloggii (Ak), Antirrhinum majus (Am), Arabidopsis thaliana (At), Arachis hypogaea (Ah), Bromheadia finlaysoniana (Bf), Cymbidium floribundum (Cf), Cymbidium hybrida (Ch), Cyclamen persicum (Cp), Dahlia pinnata (Dp), Dendrobium moniliforme (Dm), Dendrobium nobile (Dn), Dendrobium hybrid (Dh), 9 Doritaenopsis hybrid (Dx), Echinops bannaticus (Eb), Epimedium sagittatum (Es), Eustoma exaltatum (Ee), Fragaria x ananassa (Fa), Gerbera hybrida (Gh), Ginkgo biloba (Gb), Glycine max (Gm), Iris 9 hollandica (Ih), Lilium cernuum (Lc), Lilium hybrid (Lh), Lycoris chinensis (Lch), Malus domestica (Md), Marchantia polymorpha (Mp), Medicago sativa (Ms), Musa acuminata (Ma), Narcissus tazetta (Nt), Oncidium Gower Ramsey (OGR), Oryza sativa (Os), Paphiopedilum concolor (Pc), Perilla fructescens (Pf), Petunia hybrida (Ph), Phalaenopsis hybrid (Phh), Populus trichocarpa (Pt), Pinus sylvestris (Ps), Solanum lycopersicum (Sl), Solanum tuberosum (St), Secale cereale (Sc), Strelitzia reginae (Sr), Triticum aestivum (Ta), Tulipa fosteriana (Tf), Tulipa gesneriana (Tg), Vitis vinifera (Vv), Zea mays (Zm)

within the same phylogenetic tree, as both enzymes are in the 2-oxoglutarate-dependent oxygenase protein superfamily (Turnbull et al. 2004). Comparisons were made to characterise ANS and FLS sequences from a range of monocot and dicot species. The ANS and FLS sequences formed separate clades as expected, with bootstrap support of 100 % (Fig. 3b). Both ChFLS and ChANS grouped with other orchid sequences with 100 % bootstrap support, within a larger grouping of monocot sequences. The isolated putative ChF30 H (KM186178) has a 1,530 bp coding sequence, encoding 510 deduced amino acid residues. The phylogenetic analysis showed that ChF30 H grouped with the F30 H (CYP75B) subfamily, separated from the F30 50 H (CYP75A) and FNSII (CYP93B) subfamilies with very high bootstrap support (Fig. 4c). However, ChF30 H separates from the other monocot sequences, onion (Allium cepa), Lilium hybrid and maize (Zea mays), and other plant F30 Hs. As no other orchid F30 H sequences are available, it is not possible to tell whether this is typical for orchid sequences. However, the orchid F30 50 H sequences do form a separate sub-clade

within the F30 50 H branch, with 100 % bootstrap support, indicating the orchids may form a separate F30 H sub-clade. Expression analysis of CHS, BBS, FLS, F30 H, DFR and ANS in different cultivars The correlations between the mRNA abundance of flavonoid biosynthetic genes and anthocyanin accumulation in Cymbidium flowers were examined. The expression of CHS, FLS, F30 H, DFR and ANS were examined in floral tissues of eight Cymbidium cultivars representing the four colour groups (Fig. 1) across six developmental stages (Fig. 3). This analysis was also carried out in leaf tissues of cultivars ‘JDP’, ‘LRF’, ‘CASP’ and ‘V’. Gene expression levels were assessed semi-quantitatively using the intensity of signal for rRNA as a reference. In general, CHS, FLS and F30 H showed similar expression patterns, and the expression patterns of DFR and ANS were almost identical (Fig. 5). CHS, FLS and F30 H shared a similar temporal expression pattern, with the strongest expression being detected in developmental stages 1 and 2, with only slight variation between the different colour groups. Strongest expression of all three genes was detected in stage 1 and 2 buds of the white and green cultivars, but strong expression lasted until stage 3 in yellow and pink cultivars. There was a difference in spatial expression between CHS, FLS and F30 H. The expression levels of FLS and F30 H in sepal/ petal and labellum tissues were relatively uniform, whereas CHS showed a stronger expression in labellum tissues. FLS showed two transcripts of *1,580 and *980 nt in all cultivars, but only the smaller transcript was detected in stage 4–6 of ‘V’ (Fig. 5). The smaller FLS transcript was also detected in leaf tissues (Suppl. Fig. S3). In contrast, DFR and ANS transcripts were not abundant in all floral tissues, but rather only detected in floral tissues that accumulated anthocyanins, such as in the labellum tissue of all the cultivars and sepal and petal tissues of the pink cultivars ‘CASP’ and ‘LRF’. DFR and ANS transcripts were detected at early developmental stages, with strongest expression in early stages (s 1–2) of green and yellow cultivars and in later stages (s 4–5) of the white cultivars. Expression was particularly strong in both pink cultivars, and remained strong to stage 4. DFR transcript was also detected in ‘CASP’ stage 6 labellum tissues. Similar to FLS, two ANS transcripts of *2,700 and *1,400 nt were detected, however, in contrast to FLS, both ANS transcripts were expressed uniformly. Expression of the flavonoid biosynthetic genes was monitored in leaf tissue from four of the cultivars included in the study. Low levels of CHS and FLS mRNA were detected in leaf tissues, although only the smaller of the

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Planta Fig. 5 Spatial and temporal expression of CHS, DFR, FLS, F30 H and ANS in floral tissues of eight cultivars at six defined developmental stages. ‘Clarisse Austin South Pacific’ (‘CASP’); ‘Lisa Rose Flamingo’ (‘LRF’); ‘Gymer Cooksbridge’ (‘GC’); ‘Arcadian Sunrise’ (‘AS’); ‘Big Chief Kirawee’ (‘BCK’); ‘Vanguard Mas Beauty’ (‘VMB’); ‘Virgin’ (‘V’); ‘Jungfrau dos Pueblos’ (‘JDP’); s/p sepal/petal, L labellum. Developmental stages are labelled 1–6. Radiolabelled probes of ChCHS, ChFLS, ChF30 H, ChDFR and ChANS cDNA were hybridized to the blots of total RNA isolated from combined sepal and petal tissues and labellum tissues. Twenty micrograms of total RNA from each sample was loaded on each lane. Panels of ribosomal RNA stained with ethidium bromide are shown as a loading control

two FLS transcripts was detected in leaves. Transcripts for F30 H, DFR or ANS were not detected in leaf tissues (Suppl. Fig. S3). Comparing expression patterns between different colour groups showed that pink cultivars had stronger expression of CHS, DFR and ANS and this was maintained over a longer developmental period than in cultivars of other colours. The expression pattern observed in pink cultivars correlated with the high level of anthocyanin accumulation in flowers (Fig. 5). The expression patterns of the various genes were similar between cultivars in the same colour group, for the green, yellow and pink groups; however, the expression patterns for the two white cultivars, ‘JDP’ and

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‘V’, were slightly different. Whilst CHS, FLS and F30 H were strongly expressed in early stages in both ‘JDP’ and ‘V’, expression of CHS and FLS remained relatively strong to stage 6 in ‘JDP’, but was weaker after stage 2 in ‘V’. Different DFR and ANS expression patterns were also observed between ‘JDP’ and ‘V’. In ‘JDP’, DFR showed higher expression in labellum tissues in later stages of development, beginning in stage 3 and continuing to stage 5. The expression in the labellum was detected throughout development in ‘V’, peaking in stage 4. The difference in expression between the two white cultivars is consistent with different levels of anthocyanin. The labellum tissue of ‘V’ was much more pink than in ‘JDP’ (Fig. 1).

Planta

Pigment profiles in different cultivars

Fig. 6 Pigment concentration (mg or lg g-1 DW) for anthocyanins, flavonoids, carotenoids and chlorophyll in petals of the different Cymbidium orchids characterised in this study. ‘Clarisse Austin South Pacific’ (‘CASP’); ‘Lisa Rose Flamingo’ (‘LRF’); ‘Gymer Cooksbridge’ (‘GC’); ‘Arcadian Sunrise’ (‘AS’); ‘Big Chief Kirawee’ (‘BCK’); ‘Vanguard Mas Beauty’ (‘VMB’); ‘Virgin’ (‘V’); ‘Jungfrau dos Pueblos’ (‘JDP’). Data presented are mean values ± SEM, n = 3 for the anthocyanins and flavonoids, n = 2 for the carotenoids and chlorophyll measurements

Bibenzyl synthase expression was examined in floral tissues at different developmental stages of Cymbidium ‘VMB’, ‘JDP’ and another pink cultivar ‘Narella Jenifer Gail’ (‘NJG’), as well as in the leaf tissues of ‘VMB’ and ‘BCK’. BBS transcripts were most abundant in ‘VMB’ leaf tissue and sepal and petal tissues at stages 4–6 (Suppl. Fig. S4). A very low level of BBS transcript was also detected in the petal tissues of ‘JDP’ at stages 2–4, and in the ‘BCK’ leaf tissues. There was no transcript detected in the ‘NJG’ floral tissue. Overall, BBS expression did not correlate with anthocyanin accumulation.

Pigment profiles in floral tissues of the selected Cymbidium cultivars were analysed as part of the comparison of the different colour groups. The emphasis for this study in terms of gene expression has been on the flavonoid pathway but carotenoids and chlorophyll pigments were also considered. Total anthocyanin, flavonoid, carotenoid and chlorophyll concentrations in both a combined sepal/petal sample and in a separate flower labellum sample are summarised in Fig. 6 and Suppl. Fig. S5. The highest anthocyanin concentration (5.5 mg g-1 DW) was detected in petal/sepal tissue from the cultivar ‘CASP’ and the next highest in the other pink cultivar ‘LRF’. Anthocyanins were detected in all the cultivars although for some cultivars this was only at trace levels. Anthocyanin concentrations were similar in the petals/sepals and flower labellum tissue for the pink cultivars. Anthocyanin concentration was highest in the labellum tissue samples for green, white and yellow cultivars, except for ‘JDP’ where anthocyanin levels were low in both petal and labellum tissue. Six different individual anthocyanins were detected in tissue from the different Cymbidium cultivars (Table 1). The anthocyanins are all derived from cyanidin or peonidin. No mono- or tri-hydroxylated anthocyanins were detected. Not all anthocyanins were detected in all cultivars. A representative chromatogram is shown for an extract from petal tissue of ‘CASP’, the cultivar with the highest anthocyanin concentrations, in Fig. 7b. The relative abundance of the different anthocyanins is summarised in Table 2. The accumulation of particular anthocyanins did vary between cultivars and tissue types. Cyanidin 3-O-rutinoside and peonidin 3-O-rutinoside were the most common anthocyanins detected and present across all cultivars and all samples except for petals of ‘V’. Since this cultivar has white petals and only trace levels of anthocyanin pigment were present in the labellum, the apparent exception may be more due to low concentrations than a clear shift in patterns of anthocyanin biosynthesis. Flavonoid compounds other than anthocyanins were also detected. Flavonoid concentrations detected in petal and labellum tissue are summarised in Fig. 6 and Suppl. Fig. S5. Flavonoid concentration varied between 10 and 35 mg g-1 DW and whilst the difference in total concentration was in some cases two- to threefold that detected in another cultivar, there was no absolute contrast observed for anthocyanin concentration, from quite a high value in the pink cultivars to almost none in other cultivars. No obvious correlation was observed between flavonoid and anthocyanin concentration. Six different flavonoids were detected in petal and flower labellum samples (Table 1). The flavonoids were identified as glycosides of kaempferol, quercetin and the methylated flavonol isorhamnetin. Again,

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Planta Table 1 Major flavonoid and anthocyanin peaks detected at either 350 nm (flavonoids) or 530 nm (anthocyanins) in extracts from Cymbidium orchid petal tissue of the eight cultivars assessed in this study, separated on a C18 liquid chromatography column Peak

Retention time (min)

Spectral maxima

m/z, [M?H]?

ms2

Identity

1

8.5

242, 328

–

–

Unknown

2

12.3

238, 314

–

–

Unknown

3

13.1

256, 355

–

–

Quercetin 3-O-robinoside

4

13.7

256, 355

609

301

Quercetin 3-O-rutinoside

5

15.8

265, 348

593

285

Kaempferol 3-O-robinoside

6

17.7

265, 348

593

285, 257

Kaempferol 3-O-rutinoside

7 8

17.9 18.7

255, 355 255, 355

505 623

463, 301 315

Isorhamnetin 3-O-robinoside Isorhamnetin 3-O-rutinoside

449, 287

Cyanidin 3-O-rutinoside

A1

5.9

280, 516

–

A2

6.5

280, 518

595

Cyanidin 3-O-glucoside

A3

8.9

266, 516

–

A4

9.5

280, 518

609

463, 301

A5

10.4

282, 518

535

287

Cyanidin 3-O-(malonyl) glucoside

A6

14.1

282, 518

549

505, 301

Peonidin 3-O-(malonyl) glucoside

Peonidin 3-O-glucoside Peonidin 3-O-rutinoside

Spectral maxima (nm) derived from the photodiode array detector are reported for peaks at the different retention times. Mass spectroscopy data [molecular mass and mass fragment pattern (ms2)] are reported for major peaks detected using a separate LCMS analysis of these samples. Peak numbers are as labelled on the selected chromatograms shown in Fig. 7. Peak identification is based on comparisons with the spectral data, retention times and mass fragment data as reported previously for flavonoids and anthocyanins (Tatsuzawa et al. 1996; Tian et al. 2005; Mullen et al. 2007). Not all compounds listed in the table were found in all cultivars but summarise the range found across these cultivars

profiles for the different flavonoids did vary somewhat between cultivars but no other flavonoids were detected. A representative flavonoid profile is shown in Fig. 7a. Two unidentified peaks were present in the extracts from the petal and labellum tissue in the chromatogram monitored at 350 nm. These two peaks were present in all extracts. Spectral profiles suggest they may be cinnamic acids but final identity was not determined. Carotenoid concentrations were highest in the yellow cultivars with levels of 198 and 137 lg g-1 DW detected in ‘GC’ and ‘AS’, respectively. Carotenoid pigments were also detected in the two green cultivars and trace levels in the pink ‘CASP’. The major carotenoids detected in the green cultivars were lutein and b-carotene, whilst in the yellows the major carotenoid was b-carotene but lutein, a-carotene and neochrome were also detected (Suppl. Fig. S6). It is possible that a small proportion of the carotenoid content was esterified (approximately \5 %) as there are some carotenoid peaks eluting later than b-carotene but this was not verified following saponification treatment and is not a major part of the carotenoid content. Representative chromatograms showing the carotenoid pigments detected in the yellow and green cultivars ‘GC’ and ‘VMB’ are shown in Fig. 8. Chlorophyll concentration was highest in the two green cultivars, ‘VMB’ and ‘BCK’, at 1,050 and 600 lg g-1 DW, respectively. A small amount of chlorophyll was detected in the yellow cultivar ‘GC’ and chlorophyll was

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observed in the flower buds from the different cultivars. In the stage 6 fully open flowers that were analysed, however, chlorophyll pigments were only a major component in the two green cultivars. Whilst both chlorophyll a and b were detected, the major components were pheophytin a and pheophytin b, breakdown products of chlorophyll but still green in colour. Chlorophyll and carotenoid pigments were identified by comparison with known retention times and spectral and MS data, as shown in Table 3.

Discussion Pigment profile and flavonoid biosynthetic gene expression were monitored in eight commercial Cymbidium cultivars (Fig. 1) as part of a study to understand the factors influencing flower colour in this crop. Distribution and marketing at a commercial level is based around general groupings of flower colour, concomitant with the predominant pigment that accumulates in petal tissue. At a general level, pink flowers are due to the presence of anthocyanin, white flowers are due to the absence of pigment, yellow flowers accumulate carotenoids, and green flowers accumulate chlorophyll. However, there is a huge range and variety of flower colours amongst the different cultivars. Our results show this diversity derives not so much from different individual pigments but rather from

Planta Fig. 7 HPLC chromatograms at 350 nm for flavonoid (a) and 530 nm anthocyanin (b) extracts from petal tissue of flowers of the Cymbidium ‘CASP’. Peak identities are listed in Table 1

a

b

subtle variations in concentration and when and where the different pigments are produced. Cymbidium flower pigment profiles All three major pigment pathways were detected in the petal tissue of Cymbidium orchid. Anthocyanins were the most widely distributed, and were detected in all the cultivars that were analysed in this study. However, in the yellow, green and white flower colour groups, anthocyanins were mostly detected only in the flower labellum. A blush of anthocyanin was seen in petals of these colour groups in some cases, for example as seen in JDP (Fig. 1h). Anthocyanin concentration varied from approximately 5 mg g-1 DW to trace levels, and the depth and intensity of colour was associated with concentration. The cultivar ‘CASP’ had the highest concentration of anthocyanin and is dark pink in colour. Anthocyanin concentrations were generally higher in labellum tissue than in the other petals

(except for ‘CASP’) and this is linked with a role in pollinator attraction (Bechtel et al. 1992). Six anthocyanins were detected in petal tissue; all were either cyanidin or peonidin glycosides or the acylated forms of these anthocyanins (Table 1). These results are consistent with those reported previously (Sugiyama et al. 1977; Tatsuzawa et al. 1996). Tatsuzawa et al. (1996) describe the cyanidin-based anthocyanins as the major component of the anthocyanin profile in Cymbidium flowers. This was also the case in the tissue sampled for this study, although in ‘GC’ petal tissues, peonidin anthocyanins were the major component. Tatsuzawa et al. (1996) speculated that peonidin-based anthocyanins provide a brighter pink colour. They also examined the anthocyanin profile in the flower labellum as compared to the other petals and showed that, whilst the proportions of the different anthocyanins may vary, no new individual anthocyanins were detected. Anthocyanin profiles have been examined in other orchid genera: Phalaenopsis (Griesbach

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Planta Fig. 8 HPLC chromatograms at 450 nm for carotenoid and chlorophyll extracts from petal tissue of Cymbidium ‘GC’ and ‘VMB’. Peaks are as identified in Table 3

a

b

1990; Tatsuzawa et al. 1997) and Dendrobium (Saito et al. 1994; Kuehnle et al. 1997). The common anthocyanins were cyanidin or peonidin based, although pelargonidin was reported in Dendrobium (Kuehnle et al. 1997) and delphinidin in Oncidium (Liu et al. 2012). The cultivars used in this study are commercial lines, developed as hybrids from a range of Cymbidium species (Tomlinson 1985) Tatsuzawa et al. (1996) included two Cymbidium species in their study (C. insigne and C. tracyanum) and no differences were noted in the individual anthocyanins detected as compared to the commercial cultivars that were also analysed. Other flavonoid compounds were also detected in the petal tissue of the Cymbidium cultivars used in this study. The compounds were identified as flavonols and, specifically, glycosides of kaempferol, quercetin and isorhamnetin. Flavonols can act as co-pigments, influencing the colour associated with the anthocyanin pigments but also have other roles in plant defence and signalling (Shirley

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1996). Flavonol concentration varied between 10 and 40 mg g-1 DW in the different cultivars and concentrations were higher in the labellum tissue as compared to the other petals. Flavonols were present in petal tissue from all the cultivars but did not appear to be linked with anthocyanin production. ‘CASP’ had the highest anthocyanin concentration but flavonol concentration was similar to most of the other cultivars. ‘LRF’ petals had the highest flavonoid concentration and petal colour appears pink rather than maroon. This would be consistent with a ‘‘blueing’’ effect but was not compared against a cultivar with a similar anthocyanin concentration. Other flavonoid compounds do not appear to have been widely studied in other orchid genera. A range of flavonols was detected in Dendrobium (Kuehnle et al. 1997), whilst glycosides of the flavone apigenin were detected in Phalaenopsis (Griesbach 1990) and were linked with a ‘blueing’ of flower colour due to their interaction with the anthocyanin pigments present.

Planta Table 2 Relative proportion of the total anthocyanin content contributed by the different individual anthocyanins detected in petal tissues of Cymbidium orchid Cultivar

A1

A2

A3

A4

A5

A6

‘CASP’ Petal

8.2

40.6

0.6

32.0

11.1

7.5

19.1

32.1

3.4

20.2

16.0

9.2

Petal

11.6

59.7

5.6

23.1

Labellum

23.0

47.7

6.4

22.9

36.4

41.7

12.1

9.8

28.6

37.4

10.0

21.0

3.0

27.7

45.6

11.0

19.8

3.0

Labellum ‘LRF’

‘GC’ Petal Labellum ‘AS’ Petal Labellum

32.6

67.4 7.7

19.0

‘BCK’ Petal Labellum

46.7

53.3

26.3

37.7

2.2

2.0

23.5

34.5

16.0

24.0

13.1

30.1

17.0

29.1

10.7

15.1

17.1

5.5

‘VMB’ Petal Labellum ‘V’ Petal Labellum

100 19.1

43.2 33.8

20.0

46.2

16.0

31.8

23.2

29.0

‘JDP’ Petal Labellum

Anthocyanins are as identified in Table 1 CASP Clarisse Austin South Pacific, LRF Lisa Rose Flamingo, GC Gymer Cooksbridge, AS Arcadian Sunrise, BCK Big Chief Kirawee, VMB Vanguard Mas Beauty, V Virgin, JDP Jungfrau dos Pueblos

Both carotenoid and chlorophyll pigments were detected in petal tissue of some of the cultivars used in this study (Table 3). It has been suggested that for green or yellow cymbidiums this might be expected (Tatsuzawa et al. 1996); however, it has not been directly reported. Carotenoids were detected in both of the yellow and green cultivars and at trace levels in the pink cultivar ‘CASP’. The carotenoid profile was quite different between the yellow and green colour groups. Both yellow cultivars accumulated b-carotene as the major pigment, with some neochrome, lutein, zeaxanthin and a-carotene. The majority of the carotenoid does not appear to be esterified although this was not determined directly but rather from comparison of retention times. Fewer carotenoids were present in the green cultivars and the predominant carotenoid was lutein with some b-carotene. Such a carotenoid profile would be expected in green tissues and associated with chloroplasts rather than chromoplasts (Cazzonelli and Pogson 2010).

Carotenoids detected in petal tissue of other orchids with yellow flowers include violaxanthin, neoxanthin and bcarotene in Oncidium (Hieber et al. 2006; Chiou et al. 2010), and lutein, violaxanthin, zeaxanthin and b-carotene in Dendrobium (Thammasiri et al. 1986). The carotenoid profile detected in Cymbidium does have some similarities to that seen in other genera, with lutein as a major component in green petals, but the presence of neochrome and the accumulation of b-carotene as the major component in the yellow cultivars is somewhat different. The presence of carotenoid and anthocyanin pigments in petal tissue of some cultivars has been noted for both Phalaenopsis and Dendrobium, and is associated with orange and bronze flower colours (Griesbach 1984; Mudalige et al. 2003). Chlorophyll a and b were detected in petal tissue of the green cultivars but the main chlorophyll pigments detected in the two green cultivars ‘BCK’ and ‘VMB’ were pheophytin a and b. ‘VMB’ petal tissue appeared darker green and this correlated with a higher concentration of chlorophyll pigments. Pheophytin a and b are derivatives that form during chlorophyll degradation (Hendry et al. 1987; Ho¨rtensteiner 2009) although they still retain a green colouration. The presence of these compounds would indicate that chlorophyll degradation had begun and that the photosynthetic capacity of the tissue was declining. Stay-green mutants delayed in leaf senescence have been identified in a number of plants and their phenotypes have been linked to mutations in Stay-green genes that function in the processes of chlorophyll and apoprotein degradation (Ho¨rtensteiner 2009). It is possible that a similar process is involved here. All Cymbidium flower buds are green initially and therefore contain chlorophyll. As flowers mature, the usual pattern observed is a loss of chlorophyll and accumulation of other pigments. It is possible that the retention of chlorophyll in some Cymbidium cultivars is linked with the activity of Stay-green genes. Cymbidium flavonoid biosynthetic genes Anthocyanins, produced from the flavonoid pathway, are important across all Cymbidium flower colour groups, hence the focus on this pathway. Full-length cDNAs for four flavonoid biosynthetic genes, ChCHS, ChFLS, ChF30 H and ChANS, were successfully isolated from Cymbidium orchid. ChDFR cDNA was also cloned for expression studies but this had previously been isolated (Johnson et al. 1999). Chalcone synthase is a member of the type III polyketide synthase (PKS) superfamily. Many secondary metabolite enzymes such as bibenzyl synthase, stilbene synthase (STS) and acridone synthase also belong to the PKS family (Helariutta et al. 1995; Liew et al. 1998b). Using degenerate primers, two possible Cymbidium PKS clones were

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isolated; PKS1 and PKS2. PKS2 has subsequently been identified as ChCHS. ChCHS protein sequence contains the conserved sequence that is specific to CHS but varies in BBS, STS and other polyketide synthases (Austin and Noel 2003). Phylogenetic analysis showed the isolated ChCHS sequence in the CHS clade and clearly grouped with CHS from other orchid genera (Fig. 4). ChCHS gene expression patterns match the accumulation patterns of flavonoids and anthocyanins in both flower buds and leaf tissue. Many plants contain multiple copies of CHS, with each CHS protein potentially associated with different functions and exhibiting specific responses to developmental and environmental signals (Holton and Cornish 1995). It has been shown that at least three CHS members are present in Bromheadia finlaysoniana and Phalaenopsis hybrid orchids (Liew et al. 1998b; Han et al. 2005, 2006b), although only one CHS gene from Phalaenopsis orchid has been functionally confirmed as a flower-specific, anthocyanin accumulation-related CHS (Han et al. 2006a, b). The possibility that Cymbidium has a CHS multigene family was not assessed. Sequence and phylogenetic analysis indicate that PKS1 is a bibenzyl synthase (ChBBS). ChBBS clearly separated from the CHS clade and is closely related to sequences from Phalaenopsis and Cymbidium floribundum. BBS and STS have similar sequences to CHS and use some of the same substrates as CHS but make other compounds such as anti-fungal phytoalexins (Austin and Noel 2003). The expression patterns of the ChBBS appeared to be cultivar specific and did not correlate with anthocyanin accumulation. This study reports the first isolation of a F30 H from a member of the Orchidaceae. This is significant because the F30 H and F30 50 H enzymes are known as the ‘‘red’’ and ‘‘blue’’ genes and directly affects the type of anthocyanins that are produced and have been targeted to modify flower colour. Examples of altering F30 H and/or F30 50 H activity to create novel flower colour in carnation, rose and torenia have been reviewed (Tanaka et al. 2005). The phylogenetic analysis for ChF30 H positions it in the F30 H clade, and the presence of an active ChF30 H gene is consistent with the accumulation of cyanidin-based anthocyanins and quercetin-based flavonols in petal tissue of Cymbidium. Despite the predominance of cyanidin-based anthocyanins in flowers of other orchid genera, F30 H has not been reported, but three sequences for F30 50 H from orchid genera are available on GenBank. The F30 50 H from Phalaenopsis is expressed at high levels at stages when flowers have just opened. The expression correlated with the accumulation of delphinidin in the flower and the expression is stronger in a purple cultivar when compared with a yellow cultivar (Wang et al. 2006). The lack of a functional F30 50 H in Cymbidium orchid is one explanation for the absence of

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delphinidin-based anthocyanin. Only one study has reported the detection of delphinidin-based anthocyanin in emasculated Cymbidium flowers (Woltering and Somhorst 1990), however, whilst mentioned in the results, no spectral data are presented to confirm the observation. F30 H and F30 50 H are both cytochrome P450 enzymes. The amino acid sequences of the two genes in plants are very diverse, but their architecture remains conserved (Hasemann et al. 1995). P450s conserved domains such as the protein-rich ‘‘hinge’’ region PPGPXPXP for orientation of enzyme; the FXXGXRXCXG haem iron-binding site (HBS), the AGTDTS oxygen-binding pocket site (OBS), and the pocket locking motif E–R–R triad are identified in the ChF30 H protein sequence (Suppl. Fig. S1; Hasemann et al. 1995; Chapple 1998). Alignment of ChF30 H with other F30 H indicated that those motifs appear to be more diverse in monocotyledons as compared to dicotyledons (Suppl. Fig. S1). For example, the GGEK motif, which has shown to be conserved only in dicotyledons (except for grapes), has been modified to GGSH in sorghum (Boddu et al. 2004) and maize (Sharma et al. 2011), GGGY in onion, GRMH in rice (Lai et al. 2012) and GSPH in Cymbidium. The VVVAA motif, on the other hand, is conserved in dicotyledons and some monocotyledons, but is modified in lily and Cymbidium. The VDVKG motif also shows some variation in sequence for the clones compared in Suppl. Fig. S1. These differences may explain the lack of F30 H genes characterised for Orchidaceae to date. ANS and FLS enzymes are 2-oxoglutarate-dependent oxygenases. The ChANS and ChFLS sequences clearly aligned with ANS and FLS sequences from a range of monocot and dicot species, with the ANS and FLS sequences on separate branches. The signature residues in a 2-oxoglutarate-dependent oxygenase such as the ironbinding residues (histidine at position 218 for ChFLS and position 233 for ChANS, aspartic acid at positions 220/235, histidine at position 274/290) and 2-oxoglutarate binding residues (arginine at position 284/300 and serine at position 286/302) were observed within the deduced amino acid sequences of ChFLS and ChANS (Martens et al. 2003; Noda et al. 2004). There were two transcripts detected for both FLS and ANS in the northern analyses. This might be due to different polyadenylation sites or a partially processed transcript where an intron has been retained. A number of flavonoid biosynthetic genes are members of multigene families but this was not examined in this study. The ChDFR was cloned as a DFR ORF from the cultivar ‘CASP’ using sequence previously published by Johnson et al. (1999). The ORF was cloned again to allow us to characterise gene expression during flower bud development. DFR is a key enzyme for the formation of anthocyanin pigments. Previous work on the ChDFR indicates that it does not efficiently use dihydrokaempferol as a

Planta Table 3 Major carotenoid peaks detected at 450 nm in extracts from Cymbidium orchid petal tissue of the eight cultivars assessed in this study, separated on a YMC C30 liquid chromatography column. Spectral maxima (nm) derived from the photodiode array detector are reported for peaks at the different retention times Peak

Retention time (min)

Spectral maxima

m/z, [M?H]?

ms2

Identity

1

9.9

421, 448

601

583

Neochrome

2

10.7

421, 448

601

583

3

14.5

443, 471

569

551, 477

Neochrome isomer? Lutein

4

16.3

448, 476

–

5

20.1

449, 476

537

6

23.1

445, 472

–

Zeaxanthin 444

a-carotene Unknown carotenoid

444

b-carotene

7

23.9

450/476

537

8

25.1

446, 472

–

b-carotene isomer?

9

13.2

466/650

na

Chlorophyll b

10

24.1

435/654

na

Pheophytin b

11

24.8

408/666

na

Pheophytin a

Mass spectroscopy data [molecular mass and mass fragment pattern (ms2)] are reported for major peaks detected using a separate LCMS analysis of these samples. MS analysis was not carried out for the chlorophyll pigments. Peak numbers are as labelled on the selected chromatograms shown in Fig. 8. Peak identification is based on the spectral maxima and retention time relative to other known compounds. Spinach extract was run as a standard to confirm retention times for chlorophyll a and b and for lutein and b-carotene. Carotenoid identification is made on the basis of comparison against previously reported spectral and MS data (de Rosso and Mercadante 2007). Pheophytins were identified using retention time and spectral data as reported by Kamffer et al. (2010). b-carotene and pheophytin b peaks do have the same retention time; these two compounds were only detected in the same tissue for petals of ‘Big Chief Kirawee’ relative content was adjusted to estimate a concentration. Not all compounds were found in all cultivars but summarise the range found across these cultivars na not analysed

substrate (Johnson et al. 1999) and therefore Cymbidium will not accumulate pelargonidin-based anthocyanins. This is consistent with the pigment profiles reported in this study. DFR genes have been isolated from a number of Orchidaceae genera, Bromheadia finlaysoniana (Liew et al. 1998a), Dendrobium (Mudalige-Jayawickrama et al. 2005) and Oncidium (Hieber et al. 2006). Phylogenetic analyses separate DFR sequences from the monocotyledons away from those of the dicotyledons and the Orchidaceae form a separate group within the monocotyledons (Johnson et al. 1999; Mori et al. 2014). Dendrobium and Cymbidium DFR cDNAs share sequence identity of 83 %, and cyanidinbased anthocyanins are the major anthocyanin component in most Dendrobium cultivars too, consistent with an

inefficient use of dihyrokaempferol. The two Dendrobium cultivars in which pelargonidin is the major anthocyanin are considered likely to have a mutation in the F30 H, preventing the formation of dihydroquercetin and consequently cyanidin (Mudalige-Jayawickrama et al. 2005). Gene expression patterns of flavonoid biosynthetic genes The flavonoid biosynthetic genes monitored in petal tissue of different Cymbidium orchids were expressed in a coordinated manner over the period of bud development and in a manner consistent with the patterns of accumulation of anthocyanin pigments. The differences in RNA accumulation were particularly marked between lip and sepal/petal tissues in those cultivars where anthocyanin pigments accumulated to high levels in the labellum (Fig. 5). CHS, FLS and F30 H were expressed across all cultivars and in both petal/sepal and labellum tissue. FLS was expressed across all bud stages, whilst CHS and F30 H varied a little between cultivars, and expression was absent in later bud stages from some cultivars. Expression did decrease for all three genes in later flower stages. CHS is the first enzymatic step in the pathway, and the production of all flavonoids depends on the activity of CHS. FLS and F30 H are also key enzymes in the biosynthetic pathway (Fig. 2); therefore, their expression in most tissues and throughout development is expected. Similar patterns have been seen in other species, initially in work unravelling the flavonoid pathway (Davies et al. 1993; Brugliera et al. 1999) and more recently in flowers of Dendrobium orchid (MudaligeJayawickrama et al. 2005) and Lilium (Lai et al. 2012), both monocotyledonous flower crops. A decrease in expression and activity might be expected as flowers mature and no further flavonoid accumulation is required. CHS and FLS genes were also expressed in leaf tissue (Suppl Fig. S3). Flavonoid compounds also accumulate in vegetative tissue, and the expression of biosynthetic genes in both flower and leaf tissue has been noted in other species (Mudalige-Jayawickrama et al. 2005; Lai et al. 2012). Dihydroflavonol 4-reductase and anthocyanidin synthase showed specific patterns of expression across cultivars and between the petals/sepals and labellum. The differences in expression of these two genes specifically required for the biosynthesis of the anthocyanins (Fig. 2), is a key part of the explanation for the different flower colour groups and the diversity of colours and shades within a group. ‘CASP’ and ‘LRF’ are pink cultivars and expression of these two genes occurred in both tissue types and relatively early in bud development but greatest at stages 3–4. Expression was strongest and prolonged over a longer developmental period in ‘CASP’. This correlates

123

Planta

well with when anthocyanin accumulation begins during bud development (Fig. 3) and with anthocyanin concentration. In all the other cultivars, DFR and ANS were only expressed in the labellum, explaining the absence of anthocyanin pigment in the petals/sepals. Expression patterns for DFR and ANS in labellum tissue across the white, green and yellow cultivars are quite varied. Patterns were generally consistent with the level of anthocyanin observed in labellum tissue. The white cultivar ‘V’ has quite a dark pink labellum and DFR and ANS expression was detected across all bud stages; in contrast ‘JDP’ has limited anthocyanin pigment and expression was limited mainly to stages 4 and 5. The patterns of DFR and ANS expression occurring mid phase of bud development were similar in Dendrobium (Mudalige-Jayawickrama et al. 2005) and Lilium (Lai et al. 2012) although comparisons were not made between and white and red cultivars. In Phalaenopsis, a comparison of two flavonoid biosynthetic genes in a red versus white species showed that DFR was not expressed in the species with white flowers (Ma et al. 2009). The detection of DFR and ANS expression in the labellum of fully open (stage 6) flowers of ‘CASP’ was unexpected, as gene expression had apparently ceased at stage 4. Flower pigmentation in petunia has been shown to involve underlying patterns that are initiated at different stages of flower development. Venation and bud blush patterns develop early, whilst full petal pigmentation occurs closer to flower opening (Albert et al. 2011). Similar patterns may also occur in Cymbidium. The accumulation of anthocyanin pigments in Cymbidium flowers has also been reported after removal of the pollinia and anther cap (Woltering and Somhorst 1990), potentially acting as a signal to pollinators that a flower is no longer a source of pollen or nectar. It is possible that pollinia had been removed or damaged on some of the flowers collected as part of the sample for stage 6 ‘CASP’ flowers and that this initiated further anthocyanin biosynthesis. The differences in expression of flavonoid biosynthetic genes in Cymbidium, both between cultivars and between different floral organs, suggests that regulation of the flavonoid pathway in Cymbidium, as in other species, is complex (Schwinn et al. 2006; Albert et al. 2011, 2014). The presence of anthocyanins in the labellum of all the cultivars examined implies that the lack of anthocyanin in petals and sepals of some cultivars is due to regulatory factors rather than a biosynthetic block. This is strongly supported by the genetic complementation of the white ‘JDP’ by co-expression of R2R3-MYB and bHLH regulatory genes, which restored anthocyanin synthesis to petals (Albert et al. 2010). These findings also raise the possibility that labellum and petal pigmentation are controlled by distinct regulatory gene signals. Vein-associated pigmentation in both Petunia and Antirrhinum, for example, is

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regulated by specific R2R3-MYB factors that are expressed early in flower development, preceding the expression of the R2R3-MYB genes that control full petal pigmentation (Schwinn et al. 2006; Albert et al. 2011). The regulation of anthocyanins is controlled by a MYB-bHLH-WDR (MBW) transcription factor complex in all species studied to date (Baudry et al. 2004; Feller et al. 2011; Davies et al. 2012); it appears likely this will also be the case in orchids. MYB and bHLH transcription factors have been isolated from and shown to be required for anthocyanin production in Phalaenopsis (Ma et al. 2008), and a MYB gene has been linked to flower colour in Oncidium (Chiou and Yeh 2008). Recently a R2R3-MYB transcription factor has been cloned from Cymbidium (Schwinn et al., unpublished) that may be involved in anthocyanin regulation. The pigment profiles and expression patterns for five flavonoid biosynthetic genes have been characterised across eight selected commercial Cymbidium cultivars, relating the observed flower colour to the patterns of pigment accumulation. An apparently general pattern associating a particular colour with a particular pigment pathway overlies more subtle processes linking changes in concentration, patterns of where and when pigment is produced and interactions between pigment groups to provide many variations in colour and the large number of different Cymbidium cultivars. Author contribution DHL, KMD, MRB, HZ and KES conceived of the experiments. LW, NWA, HZ, HN and KES conducted the isolation and sequencing of the flavonoid genes, LW, NWA, HZ, KES and KMD conducted the characterisation and analysis of data sequence data, SA and HZ, and DHL performed the pigment analyses LW, SA and HZ performed the expression studies, LW, KMD, NWA and DHL wrote the manuscript, with assistance from all coauthors. Acknowledgments The authors thank West Coast Orchids, Auckland; Kiwi Orchids, Nelson and Airborne Cymbidiums, Auckland, New Zealand for supplying the flower stems used in these experiments, Stephen Bloor, Ewald Swinny, and Kevin Mitchell (Industrial Research Ltd) and Nigel Joyce (Plant and Food Research) for their assistance with pigment analysis and identification, the members of the Cymbidium Orchid Group for their support and interest in this project. LW thanks Professor Michael McManus (Massey University) for supervision and guidance during her MSc studies.

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