AoB PLANTS http://aobplants.oxfordjournals.org/ Open access – Research article Bilirubin present in diverse angiosperms Cary Pirone1*, Jodie V. Johnson2, J. Martin E. Quirke3, Horacio A. Priestap1 and David Lee1 1 Department of Biological Sciences, Florida International University, 11200 SW 8 St., OE-167, Miami, FL 33199, USA 2 Department of Chemistry, University of Florida, PO Box 117200, Gainesville, FL 3261, USA 3 Department of Chemistry and Biochemistry, Florida International University, 11200 SW 8 St., CP-304, Miami, FL 33199, USA Received: 20 August 2010; Returned for revision: 25 September 2010; Accepted: 24 October 2010; Published: 28 October 2010 Citation details: Pirone C, Johnson JV, Quirke JME, Priestap HA, Lee D. 2010. Bilirubin present in diverse angiosperms. AoB PLANTS 2010: plq020, doi:10.1093/aobpla/plq020 Abstract Background Bilirubin is an orange-yellow tetrapyrrole produced from the breakdown of heme by mammals and aims and some other vertebrates. Plants, algae and cyanobacteria synthesize molecules similar to bilirubin, including the protein-bound bilins and phytochromobilin which harvest or sense light. Recently, we discovered bilirubin in the arils of Strelitzia nicolai, the White Bird of Para- dise Tree, which was the first example of this molecule in a higher plant. Subsequently, we identified bilirubin in both the arils and the flowers of Strelitzia reginae, the Bird of Paradise Flower. In the arils of both species, bilirubin is present as the primary pigment, and thus func- tions to produce colour. Previously, no tetrapyrroles were known to generate display colour in plants. We were therefore interested in determining whether bilirubin is broadly distributed in the plant kingdom and whether it contributes to colour in other species. Methodology In this paper, we use HPLC/UV and HPLC/UV/electrospray ionization-tandem mass spec- trometry (HPLC/UV/ESI-MS/MS) to search for bilirubin in 10 species across diverse angiosperm lineages. Principal results Bilirubin was present in eight species from the orders Zingiberales, Arecales and Myrtales, but only contributed to colour in species within the Strelitziaceae. Conclusions The wide distribution of bilirubin in angiosperms indicates the need to re-assess some meta- bolic details of an important and universal biosynthetic pathway in plants, and further explore its evolutionary history and function. Although colour production was limited to the Strelitzi- aceae in this study, further sampling may indicate otherwise. Introduction vertebrates, biliverdin-IXa is also formed from the Tetrapyrroles occur throughout the plant kingdom; this degradation of heme, but it is transformed into the class of molecules includes vital biosynthetic products yellow-orange pigment bilirubin-IXa. We have identified such as chlorophyll and heme. In plants, the degradation bilirubin-IXa (henceforth referred to as bilirubin) as the of heme forms first biliverdin-IXa, and subsequently major pigment in the orange arils of Strelitzia nicolai, the phytochromobilin, the precursor of the phytochrome White Bird of Paradise Tree (Pirone et al. 2009). Although chromophore, an essential light-sensing molecule ubiquitous in animals, this is the first example of bilirubin (Tanaka and Tanaka 2007). In mammals and some in a plant. Subsequently, we have discovered this pigment * Corresponding author’s e-mail address: cpiro001@fiu.edu AoB PLANTS Vol. 2010, plq020, doi:10.1093/aobpla/plq020, available online at www.aobplants.oxfordjournals.org & The Authors 2010. Published by Oxford University Press. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.5/uk/) which permits unrestricted non- commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. AoB PLANTS Vol. 2010, plq020, doi:10.1093/aobpla/plq020 & The Authors 2010 1 2 AoB PLANTS S. reginae Botanic Garden inPlant Miami, material FL, was exceptMaterials collected aril and methods tissue from from Fairchild Tropical in the sepals and arils of Pirone unique to Paradise Flower, indicating that the pigment is not lains) ( the phenylpropanoid (flavonoids) and the betalainthree (beta- metabolic pathways: theof terpenoid flowers (carotenoids), andthetic source fruits of display is colour. As achieved a rule, with the colouration products from ductive structures, these are fairly inconspicuous. While chlorophylls occasionally produce colourthe contrasting in colours of repro- flowers and fruits arefoliage, displayed. thus formingthe a green tetrapyrrole background pathway,structure. upon primarily Chlorophylls, which produce which colourto are produce in also conspicuous synthesizedadditional colour via biosynthetic in route, a thecolour plant tetrapyrrole production. pathway, reproductive Bilirubin is thusnor the first the product of other an However, rare to pigments our play knowledge, a neither significant the role phenalenones in 1967 cyanins) are pervasive inlales, while the carotenoids plant and flavonoids kingdomsynthesis (including ( is antho- restricted to families in the order Caryophyl- animal dispersers and pollinators. ecological role for bilirubinand as biochemical a colour context, signal(Table and to comment attract onberales, a and possible fouradditional are species. Six from species diverse arefrom within angiosperm the nine order additional orders Zingi- species andinvestigate the the presence flowers of of bilirubin aconfirm in single the the mature presence fruits tandem of bilirubin mass in we spectrometry use (HPLC/UV/ESI-MS/MS) HPLC/UVUV-Visible and spectra to that HPLC/UV/electrospray matched ionization- thosenense of bilirubin. Here, species in the(HPLC/UV) Strelitziaceae, analyses ofPreliminary aril high-performance extractswell liquid as throughout of the major chromatography groups anallied of the additional to angiosperms. theother Strelitziaceae taxa (as within in theing the Strelitziaceae, to Zingiberales), in determine as families whether closely the pigment is produced by Strelitziaceae andnalenones, has been documented related in several species in the families ( In Given the presence of bilirubin in ; S. nicolai , showed a pigment with a retention time and Goodwin 1988 et al 1 Davies 2004 ). We discuss our findings within a phylogenetic , which was obtained from Ellison Horticulture S. nicolai . — Bilirubin present in diverse angiosperms Vol. 2010, plq020, doi:10.1093/aobpla/plq020 and ; S. reginae ( Pirone Grotewold 2006 ). A rare group of pigments, the phe- et al Strelitzia reginae , bilirubin is a novel biosyn- . 2010 Phenakospermum guya- Strelitzia ; ) Lee 2007 . P. guyanense Davies 2004 , it is interest- , the Bird of ). Betalain Harborne and & ). The Authors 2010 Table 1 Summary of HPLC/UV and MS/MS results of the analysis of BR in 10 angiosperm species. N/A indicates that samples were not treated with diazomethane. Species Family Order Organ BR detection via BR detection via BR detection via BR concentration Mean BR ..................................... diazomethane HPLC/UV HPLC-MS/MS concentration Sample 1 Sample 2 derivative (n 5 2) ........................................................................................................................................................................................................................... M. balbisiana Musaceae Zingiberales Peel N N Y ,44 ng g21 ,44 ng g21 — H. collinsiana Heliconiaceae Zingiberales Fruit N N Y — ,44 ng g21 — C. lucanusianus Costaceae Zingiberales Flower N N Y ,44 ng g21 ,44 ng g21 — R. madagascariensis Strelitziaceae Zingiberales Aril N/A Y Y 0.001 mg g21 0.001 mg g21 0.001 mg g21 P. guyanense Strelitziaceae Zingiberales Aril N/A Y Y 3.041 mg g21 5.787 mg g21 3.725 mg g21 H. coronarum Zingiberaceae Zingiberales Aril N N Y ,44 ng g21 ,44 ng g21 — G. crispa Arecaceae Arecales Fruit N N Y ,44 ng g21 ,44 ng g21 — P. odoratissimus Pandanaceae Pandanales Fruit N N N — — — P. americana Lauraceae Laurales Fruit N N N — — — E. luschnathiana Myrtaceae Myrtales Fruit N N Y ,44 ng g21 —— Pirone et al. — Bilirubin present in diverse angiosperms Pty. Ltd in Allstonville, New South Wales, Australia. Tissue HPLC/UV for each sample and its replicate were composed of HPLC/UV analyses were performed on a Thermo- tissue from one or multiple inflorescences or infructes- Finnigan SpectraSystem HPLC apparatus with a variable cences from a single, sometimes clonal, individual. The wavelength photodiode array detector (SMC1000, replicate aril samples of P. guyanense came from differ- P4000, AS3000, UV6000LP; Thermo Electro Corporation, ent individuals (collected by John Kress; Guyana (South San Jose, CA, USA). The extract was re-dissolved in America), Demerara-Mahaica region). For the names dimethyl sulfoxide (DMSO), partitioned with hexane to and taxonomic affiliations of species sampled, see remove lipids and chromatographed on a reverse Table 1. We sampled species from each banana group phase ODS-A column (150 mm × 4.3 mm, particle size family, except from the Lowiaceae. This monotypic 5 mm; Waters, Milford, MA, USA). Mobile phase A was family consists of 15 rare species within Orchidantha, 0.1% formic acid in methanol, and mobile phase B was and we were not able to obtain enough material for 0.1% formic acid in water. The HPLC gradient (at analysis. We selected Musa balbisiana (Musaceae), one 1.0 mL min21) was started at 40% A and increased line- of the wild progenitors of most cultivated bananas arily