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Temporal and spatial regulation of anthocyanin biosynthesis provide diverse flower colour intensities and patterning in Cymbidium orchid

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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-O-rutinoside and peonidin 3-O-rutinoside as the major anthocyanins detected; the flavonols quercetin and kaempferol rutinoside and robinoside were also present in petal tissue. β-carotene was the major carotenoid in the 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 3′ hydroxylase (F3′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 F3′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.

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Abbreviations

ANS:

Anthocyanidin synthase

BBS:

Bibenzyl synthase

CHS:

Chalcone synthase

DFR:

Dihydroflavonol reductase

FLS:

Flavonol synthase

F3′H:

Flavonoid 3′ hydroxylase

F3′5′H:

Flavonoid 3′5′ hydroxylase

PKS:

Polyketide synthase

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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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Authors and Affiliations

  1. The New Zealand Institute for Plant and Food Research Limited, Private Bag 11 600, Palmerston North, 4474, New Zealand

    Lei Wang, Nick W. Albert, Huaibi Zhang, Steve Arathoon, Murray R. Boase, Hanh Ngo, Kathy E. Schwinn, Kevin M. Davies & David H. Lewis

  2. Institute of Molecular BioSciences, Massey University, Private Bag 11-222, Palmerston North, New Zealand

    Lei Wang

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  1. Lei Wang
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Special topic: Anthocyanins. Guest editor: Stefan Martens.

Electronic supplementary material

Supplementary Table ST1 Oligonucleotide primers used to amplify Cymbidiumflavonoid biosynthetic genes

Supplementary Fig. S1 Comparison of the deduced amino acid sequence of ChF3′H with other F3′H proteins. Multiple sequence alignments were generated by the Clustal W2.1 program. GenBank accession number is listed after plant species. Numbers indicate position of amino acid from the N-terminus. ChF3′H sequence is listed in pink colour font. The conserved F3′H regions are highlighted in blue boxes, and the conserved domains found in the CYP450 family proteins are highlighted in purple boxes. The conserved amino acids are labelled with asterisks, the highly conservative changes are labelled with double dots, and the conservative changes are labelled with dots

Supplementary Fig. S2 Comparison of the deduced amino acid sequence of ChDFR amplified from Cymbidium ‘Clarisse Austin South Pacific’ (KM186174) with the published DFR protein (AAC17843). Numbers indicate position of amino acid from the N-terminus. The conserved amino acids are labelled with asterisks, the highly conservative changes are labelled with double dots, and the conservative changes are labelled with dots. Different amino acid between the DFR sequence from Cymbidium ‘CASP’ and the published DFR sequence are highlighted in red

Supplementary Fig. S3 Northern blot analysis of five flavonoid biosynthetic genes in leaf tissues of various Cymbidium cultivars. ‘Jungfrau dos Pueblos’ (‘JDP’); ‘Lisa Rose Flamingo’ (‘LRF’); ‘Clarisse Austin South Pacific’ (‘CASP’); ‘Virgin’ (‘V’). Twenty micrograms of total RNA from each leaf sample was loaded on each lane. The lower panel showed the ribosomal RNA stained with ethidium bromide as a loading control

Supplementary Fig. S4 Northern blot showing spatial and temporal expression of BBS in floral and leaf tissues of Cymbidium cultivars ‘Vanguard Mas Beauty’ (‘VMB’), ‘Narella Jennifer Gail’ (‘NJG’), ‘Jungfrau dos Pueblos’ (‘JDP’), ‘Big Chief Kirawee’ (‘BCK’). S, sepal; P, petal; L, labellum. Developmental stages are labelled 1 to 6. The bottom panels indicate the loading of RNA stained using ethidium bromide staining of rRNA bands

Supplementary Fig. S5 Pigment concentration (mg or µg g−1DW) for anthocyanins, flavonoids, carotenoids and chlorophyll in labellum tissue of flowers from 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 Pueblo’s (‘JDP’). Data presented are mean values ± SEM, n = 3 for the anthocyanins and flavonoids, n = 2 for the carotenoids and chlorophyll measurements

Supplementary Fig. S6 Carotenoid profiles presented as a relative proportion of total carotenoids detected in petal tissue of the yellow and green flower colour cultivars. Data presented are the mean values for two independent petal tissue samples

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Wang, L., Albert, N.W., Zhang, H. et al. Temporal and spatial regulation of anthocyanin biosynthesis provide diverse flower colour intensities and patterning in Cymbidium orchid. Planta 240, 983–1002 (2014). https://doi.org/10.1007/s00425-014-2152-9

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