1 © <2021>. This manuscript version is made available under the CC-BY-NC-ND 4.0 2 license http://creativecommons.org/licenses/by-nc-nd/4.0/ 3 This document is the Accepted Manuscript version of a Published Work that appeared 4 in final form in Plant Science. To access the final edited and published work see 5 https://doi.org/10.1016/j.plantsci.2018.06.013.” 6 7 Digestive glands extraction and precise pigment analysis support the exclusion of 8 the carnivorous plant Dionaea muscipula Ellis from the Caryophyllales order 9 10 Paula Henarejos-Escudero, Berenice Guadarrama-Flores, Francisco García-Carmona, 11 and Fernando Gandía-Herrero* 12 13 Departamento de Bioquímica y Biología Molecular A, Unidad Docente de Biología, 14 Facultad de Veterinaria. Regional Campus of International Excellence “Campus Mare 15 Nostrum″. Universidad de Murcia, 30100 Murcia, Spain 16 17 *Corresponding author (Tel: +34 868 889592; Fax: +34 868 884147; E-mail: 18 [email protected]) 19 1 20 ABSTRACT 21 In the order Caryophyllales, plants synthesize betalains instead of anthocyanins, with only 22 two exceptions, the Caryophyllaceae and Molluginaceae. Dionaea muscipula Ellis was 23 included in the Caryophyllales order but recent research based on genetic studies 24 proposed the consideration of the Droseraceae family into the Nepenthales order. In this 25 work we face the dilemma of the phylogenetic classification of Dionaea from a 26 phytochemical point of view. Dionaea’s pigments were analyzed by using techniques of 27 structural analysis. Extracts from the leaves, mature stem and flowers of different 28 specimens of Dionaea were analyzed, to find possible differences in the types of pigments 29 or in their proportion in different parts of the plant. These extracts were analyzed by 30 spectrophotometry, HPLC co-elution and ESI-MS/MS. In addition, digestive glands were 31 extracted from the snap trap with minor sample manipulation and by reducing the non- 32 pigmented plant tissue. Considering only the digestive glands instead of whole snap traps, 33 the analyses allowed to quantitate and elucidate the structure of the compounds 34 responsible for the red coloration: delphinidin-3-O-glucoside (myrtillin), cyanidin-3-O- 35 glucoside (kuromanin) and a third compound, the aglycone cyanidin, detected in the 36 species for the first time. The unambiguous results of the present work support the 37 exclusion of Dionaea from the Caryophyllales. 38 39 Keywords: Anthocyanins; betalains; bioactive; Dionaea muscipula Ellis; red pigment; 40 digestive gland. 41 2 42 1. Introduction 43 Dionaea muscipula Ellis, commonly known as Venus flytrap, is a carnivorous and 44 autotrophic plant. It commonly occupies relatively closed ecosystems where the soil is 45 poor in nutrient substances, acid and wet, as in its natural habitat in North and South 46 Carolina in the USA. These plants take in and absorb nutrients directly from small animal 47 resources by means of carnivorous leaves [1,2]. The Venus flytrap was already described 48 by Charles Darwin as “one of the most wonderful plants in the world” [3] and evolved 49 from an ancestor that was already carnivorous. Although carnivory may seem an unusual 50 feature for a plant, it has evolved independently at least six times in plants [4,5]. The 51 world of carnivorous plants is diverse, with these plants found in different taxonomic 52 groups [5]. 53 Dionaea muscipula Ellis is a small plant, consisting of a rosette of leaves [1]. It 54 has snap traps consisting of two lobes and with spikes at the end of them. Springing from 55 the upper surface of the two lobes are six slender, mechanosensitive hairs, three on each 56 side in a triangular position. The rest of the surface is covered quite densely with digestive 57 glands [6,7]. The attraction of insects is due to the combination of the shape and color of 58 these traps, in addition to the emission of volatile organic compounds. These compounds 59 might serve as a first signal to attract prey insects from distant locations and entice them 60 towards the plant [8]. Currently, there are very few references about the pigment that this 61 plant has in its lobes, although the red coloration they present is common in Drosera and 62 Dionaea species [2]. 63 Dionaea muscipula Ellis belongs to the Droseraceae family which only recently 64 is considered part of the order Nepenthales [10]. Droseraceae was moved into the 65 Nepenthales from the order Caryophyllales. The order Nepenthales, comprising a 66 carnivorous and non-carnivorous clade, is characterized by the frequent presence of 3 67 acetogenic napthoquinones and by the lack of betalain pigments that characterize their 68 sister group Caryophyllales [9,10]. In the Caryophyllales order, betalains substitute the 69 otherwise ubiquitous anthocyanins [11,12], which are the dominant form of pigmentation 70 across land plants [13,14]. Betalains and anthocyanins are two types of water soluble 71 pigments with analogous coloration and similar functions [15]. Anthocyanins and 72 betalains are mutually exclusive and have never been found together in the same plant 73 [16,17]. The origin of this exclusion is unclear and only recently are investigations 74 beginning to clarify how betalains could have arisen [14]. In the Caryophyllales, only the 75 coloration of the Caryophylaceae and Molluginaceae is due to anthocyanins [18,19], and 76 these are the only two exceptions identified to date [20]. Droseraceae is a family of 77 carnivorous herbs in the Nepenthales considered as “non-core Caryophyllales” (APG IV 78 2016) [9,21]. Dionaea is a monotypic genus, comprising the sole extant especies Dionaea 79 muscipula. Complex evolution has recently been described for carnivorous plants. 80 Contemporary researchers have used molecular systematics to demonstrate that carnivory 81 evolved independently among flowering plants at least 10 times. However, carnivory 82 likely arose once in the Nepenthales [10,22]. Unambiguous determination of pigments 83 present in Dionaea may help in the taxonomic and evolution analysis of the plant. 84 Betalains are water-soluble, nitrogen containing pigments. These are divided into 85 two groups: the violet betacyanins and the yellow betaxanthins. Glycosylation and 86 acylglycosilation of one or two hydroxyl groups are possible in betacyanins, and complex 87 pigment structures can be obtained [23-25]. In contrast, betaxanthins are yellow and no 88 glycosylation has ever been reported. Both groups share betalamic acid as the structural 89 and chromophoric unit. It is condensed with amines and amino acids in betaxanthins and 90 with cyclo-DOPA in betacyanins [26]. Anthocyanins are also water-soluble plant 91 pigments, but have no biosynthetic or structural relationship with betalains. They are 4 92 responsible for the red, purple and blue tones of flowers and fruits of most of the plants. 93 These pigments are flavonoids that are derived from the shikimic acid pathway [27]. 94 Anthocyanins, like betacyanins, are constituted by an aglycone, which is anthocyanidin, 95 to which a sugar is bound by a beta-glycosidic bond. The chromophoric aglycones 96 (anthocyanidins) are red polyhydroxylated salts, which are seldom found in their free 97 form in plant tissues [28]. Despite the structural and biosynthetic differences between 98 betalains and anthocyanins [14], their distribution within the plant and their functions, 99 both vegetative and reproductive, are essentially identical [16]. The two pigments are 100 found in fruits, flowers, leaves, stems and roots [19,29]. In addition, the two pigments 101 have a high antiradical capacity and are potent antioxidants in plants [19,30]. 102 In spite of the abundant scientific literature on the pigments of betalains and 103 anthocyanins, pigments of Dionaea muscipula Ellis have been poorly characterized, with 104 limited references. A first article reports the analysis of its pigment (only one compound 105 was found) through paper chromatography and absorption spectra, identifying a single 106 colored compound with similar properties to cyanidin-3-glucoside [28]. The second, and 107 last, study was carried out using plants grown in vitro in culture medium. It exhibited a 108 second pigment, tentatively identified as delphinidin-3-O-glucoside [2]. This last article 109 does not use plants under natural conditions and does not show structural evidence of the 110 nature of the pigments. Furthermore, in both articles the red pigment was obtained from 111 whole leaves, while it is known that the coloration is restricted to the digestive glands 112 [31]. 113 This paper aims to evaluate the existence of pigments, anthocyanins and betalains 114 in the species Dionaea muscipula Ellis, through the use of modern and sensitive 115 techniques like mass spectrometry and high resolution liquid chromatography (HPLC). 116 Glands were extracted in order to give higher accuracy to the previous partial results. The 5 117 exclusion from its former phylogenetic order is considered in terms of precise pigments 118 analysis and discussed in relation to anthocyanins pigmentation in the Nepenthales order. 119 120 2. Materials and methods 121 2.1. Chemicals 122 Chemicals and reagents were purchased from Sigma (St. Louis, MO, USA). 123 Solvents were from Merck Chemicals Ltd. (Dorset, England). HPLC-grade acetonitrile 124 and methanol were purchased from Labscan Ltd. (Dublin, Ireland). Distilled water was 125 purified using a Milli-Q system (Bedford, MA, USA). 126 2.2. Plant material 127 Dionaea muscipula Ellis plants were obtained from “Viveros Murcia” (Murcia, 128 SE Spain), selecting those that had a deep red color inside the surface of the snap traps. 129 2. 3. Glands extraction 130 A new extraction system was honed to obtain the molecules responsible for the 131 red color, confined in the glands. A scalpel was used and the inner surface of the leaf was 132 scraped to obtain the glands containing the vegetable pigment.