Bilirubin present in diverse angiosperms

Cary Pirone 2 Jodie V. Johnson 1 J. Martin E. Quirke 0 Horacio A. Priestap 2 David Lee 2 Background aims 0 Department of Chemistry and Biochemistry, Florida International University , 11200 SW 8 St., CP-304, Miami, FL 33199, USA 1 Department of Chemistry, University of Florida , PO Box 117200, Gainesville, FL 3261, USA 2 Department of Biological Sciences, Florida International University , 11200 SW 8 St., OE-167, Miami, FL 33199, USA Bilirubin is an orange-yellow tetrapyrrole produced from the breakdown of heme by mammals 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 Paradise 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 functions 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. In this paper, we use HPLC/UV and HPLC/UV/electrospray ionization-tandem mass spectrometry (HPLC/UV/ESI-MS/MS) to search for bilirubin in 10 species across diverse angiosperm lineages. Bilirubin was present in eight species from the orders Zingiberales, Arecales and Myrtales, but only contributed to colour in species within the Strelitziaceae. The wide distribution of bilirubin in angiosperms indicates the need to re-assess some metabolic 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 Strelitziaceae in this study, further sampling may indicate otherwise. Methodology Principal results Conclusions Introduction Tetrapyrroles occur throughout the plant kingdom; this class of molecules includes vital biosynthetic products such as chlorophyll and heme. In plants, the degradation of heme forms first biliverdin-IXa, and subsequently phytochromobilin, the precursor of the phytochrome chromophore, an essential light-sensing molecule (Tanaka and Tanaka 2007). In mammals and some * Corresponding authors e-mail address: vertebrates, biliverdin-IXa is also formed from the degradation of heme, but it is transformed into the yellow-orange pigment bilirubin-IXa. We have identified bilirubin-IXa (henceforth referred to as bilirubin) as the major pigment in the orange arils of Strelitzia nicolai, the White Bird of Paradise Tree (Pirone et al. 2009). Although ubiquitous in animals, this is the first example of bilirubin in a plant. Subsequently, we have discovered this pigment in the sepals and arils of Strelitzia reginae, the Bird of Paradise Flower, indicating that the pigment is not unique to S. nicolai (Pirone et al. 2010). In S. nicolai and S. reginae, bilirubin is a novel biosynthetic source of display colour. As a rule, the colouration of flowers and fruits is achieved with products from three metabolic pathways: the terpenoid (carotenoids), the phenylpropanoid (flavonoids) and the betalain (betalains) (Davies 2004; Grotewold 2006; Lee 2007). Betalain synthesis is restricted to families in the order Caryophyllales, while carotenoids and flavonoids (including anthocyanins) are pervasive in the plant kingdom (Harborne 1967; Goodwin 1988). A rare group of pigments, the phenalenones, has been documented in several species in the Strelitziaceae and related families (Davies 2004). However, to our knowledge, neither the phenalenones nor the other rare pigments play a significant role in colour production. Bilirubin is thus the first product of an additional biosynthetic route, the tetrapyrrole pathway, to produce conspicuous colour in a plant reproductive structure. Chlorophylls, which are also synthesized via the tetrapyrrole pathway, primarily produce colour in foliage, thus forming a green background upon which the contrasting colours of flowers and fruits are displayed. While chlorophylls occasionally produce colour in reproductive structures, these are fairly inconspicuous. Given the presence of bilirubin in Strelitzia, it is interesting to determine whether the pigment is produced by other taxa within the Strelitziaceae, in families closely allied to the Strelitziaceae (as in the Zingiberales), as well as throughout the major groups of the angiosperms. Preliminary high-performance liquid chromatography (HPLC/UV) analyses of aril extracts of an additional species in the Strelitziaceae, Phenakospermum guyanense, showed a pigment with a retention time and UV-Visible spectra that matched those of bilirubin. Here, we use HPLC/UV and HPLC/UV/electrospray ionizationtandem mass spectrometry (HPLC/UV/ESI-MS/MS) to confirm the presence of bilirubin in P. guyanense and investigate the presence of bilirubin in the mature fruits from nine additional species and the flowers of a single additional species. Six species are within the order Zingiberales, and four are from diverse angiosperm orders (Table 1). We discuss our findings within a phylogenetic and biochemical context, and comment on a possible ecological role for bilirubin as a colour signal to attract animal dispersers and pollinators. Materials and methods Plant material was collected from Fairchild Tropical Botanic Garden in Miami, FL, except aril tissue from S. reginae, which was obtained from Ellison Horticulture iiirttttcccveeeeoooaaadnnnnnBBBRRRM..................................... i/-rttccLSSeooPaCnnnHMMllSSeeappa12mm ()n25...................................................................................... 2211,,Y44gggg44nn 21,Ygg44n 2211,,Y44gggg44nn 222111...Ygg100000g10g0010ggmmm 222111...Y0341gggg778537g52gmmm 2211,,Y44gggg44nn 2211,,Y44gggg44nn N N 21,Y44ggn iittcveeoadnBR /LPCVUH ......................... 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