South African Journal of Botany 93 (2014) 27–36 Contents lists available at ScienceDirect South African Journal of Botany journal homepage: www.elsevier.com/locate/sajb Development, structure and secretion compounds of stipule colleters in Pentas lanceolata (Rubiaceae) L.E. Muravnik a,⁎, O.V. Kostina a, A.L. Shavarda b a Laboratory of Plant Anatomy and Morphology, Komarov Botanical Institute of Russian Academy of Sciences, Prof. Popov Street, 2, 197376 St. Petersburg, Russia b Laboratory of Phytochemistry, Komarov Botanical Institute of Russian Academy of Sciences, Prof. Popov Street, 2, 197376, St. Petersburg, Russia article info abstract Article history: Four types of colleters distributed on the stipules in Pentas lanceolata were studied by light and electron micros- Received 25 November 2013 copy, and the metabolites they contain were identified. The terminal colleters of one type are formed at the top of Received in revised form 11 March 2014 the stipule lobes and three types of the basal colleters occur at the base of the lobes. The structure of all colleters Accepted 14 March 2014 matches to the standard type; however, some specific variations also arise. The development of all colleters hap- Available online xxxx pens as a result of anticlinal divisions of initial and daughter protodermal cells followed by periclinal and anticli- Edited by GV Goodman-Cron nal divisions of subprotodermal cells. Maturation of the basal colleters occurs after the terminal ones. The secretory structures produce a complex secretion. Histochemistry and fluorescence microscopy demonstrate Keywords: the presence of proteins, pectins, lipids, terpenoids, phenylpropanoids and tannins in the secretory cells. For Anatomy the first time gas chromatography–mass spectrometry was used to determine the content of metabolites in ex- Histochemistry tracts from isolated colleters. Seventy-four compounds participating in major biochemical processes were iden- Morphology tified. Some iridoids (loganin, tudoside and asperuloside), triterpenes (oleanolic and ursolic acids), sterols fi Metabolite pro ling (campesterol), and phenolics (4-hydroxycinnamic acid) were found in the colleters in larger quantity than in Primary and secondary metabolites the stipules without colleters. Several substances including loganin, campesterol, thymol, caryophyllene, and mollugin have been identified in P. lanceolata colleter extracts for the first time. © 2014 SAAB. Published by Elsevier B.V. All rights reserved. 1. Introduction (Mohan and Inamdar, 1986). Most authors use a special term — colleters (Lersten, 1974a, 1974b; Gonzalez, 1998; Paiva and Machado, 2006a, Among the large diversity of the glandular structures differing by 2006b). location, morphology, function and also secretory products, there is a Colleters are found in sixty-five angiosperm families. Morphology and specific group that includes the glands arising on stipules, petioles, anatomy of the stipular glands were studied in the plants of Apocynaceae sepals, or bracts (Rutishauser, 1984; Robbrecht, 1988; Thomas, 1991). (Thomas and Dave, 1989; Appezzato-da-Gloria and Estelita, 2000), They are responsible for protection of the vegetative and reproductive Aquifoliaceae (Gonzalez and Tarrago, 2009), Caryocaraceae (Paiva and meristems during plant growth. These structures are capable of secret- Machado, 2006b), Fabaceae (Paiva and Machado, 2006a), Orchidaceae ing mucilage, which contains polysaccharides (Lersten, 1974b; Mohan (Leitão and Cortelazzo, 2008), Passifloraceae (Durkee et al., 1984), and Inamdar, 1986), proteins (Thomas and Dave, 1989), specifically, Rubiaceae (Lersten, 1974a; Mangalan et al., 1990; Klein et al., 2004; hydrolytic enzymes (Mangalan et al., 1990; Miguel et al., 2006); resinous Barreiro and Machado, 2007) and Turneraceae (Gonzalez, 1998). substances (Fahn, 1979; Durkee et al., 1984; Leitão and Cortelazzo, 2008), Colleters are finger-like, conical, or pyramidal structures with an oblong and phenolic compounds (Rio et al., 2002; Muravnik and Kostina, 2011). head and a short stalk. The internal cells of a head are located along the During many years of investigation, these glandular structures have been stalk axis, whereas epidermal cells are situated radially. This structural called glandular shaggy hairs (Solereder, 1908), secretory trichomes pattern is regarded as a standard type of organization for all studied (Horner and Lersten, 1968), stipular glands (Van Hove and Kagoyre, colleters. Additionally, variability of the standard type was found in 1974), resin glands (Curtis and Lersten, 1980), or extrafloral nectaries the plants of the Rubiaceae (Lersten, 1974a, 1974b), Turneraceae (Gonzalez, 1998), Apocynaceae (Simões et al., 2006; Martins, 2012), and Aquifoliaceae (Gonzalez and Tarrago, 2009). In infrequent cases, Abbreviations: Ab, abaxial side; Ac, asymmetrical colleter; Ad, adaxial side; B, basal variations of the standard glandular structures are present on the surface colleter; Cc, conical colleter; Sc, standard colleter; R, idioblasts with raphids; S, secretion; of some organs simultaneously. For example, there are up to four types St, stalk; Т, terminal colleter; Vb, vascular bundle. of colleters in varying places on the petioles or stipules of seven ⁎ Corresponding author. Tel.: +7 8123725466, +7 9213208258. E-mail addresses: [email protected] (L.E. Muravnik), [email protected] (O.V. Kostina), Apocynaceae species, at the leaf base, on the leaf teeth and on the margins [email protected] (A.L. Shavarda). ofthebractsofnineAquifoliaceaespecies.IntheRubiaceaeseveraltypes http://dx.doi.org/10.1016/j.sajb.2014.03.007 0254-6299/© 2014 SAAB. Published by Elsevier B.V. All rights reserved. 28 L.E. Muravnik et al. / South African Journal of Botany 93 (2014) 27–36 of the glandular structures including intermediate, dendroid, brushlike, 450-490, DM 510 and LP 515). The length and width of the colleters and reduced standard were observed, however each plant species stud- with standard error are presented in the text. ied had only one morphological type of colleter (Lersten, 1974b). Until now it is not known whether any Rubiaceae species exist with various 2.4. Histochemistry and fluorescent microscopy (FM) types of colleter occurring on a single organ. If several types of colleter do occur, one of the purposes of this study was to establish the structural Fresh stipules embedded in 5% (w/v) agarose were sectioned by an processes underlying the basis of this variation. automatic precision microtome with a vibrating blade Microm HM- The genus Pentas is an evergreen shrub growing in tropical and 650 V (Microm International GmbH). Longitudinal sections, 25 μm southern Africa, including Madagascar. Plants are erect with simple, thick, were investigated using the following histochemical tests: a con- ovate or lanceolate-oblong opposite leaves and multifid stipules. One centrated nitric acid with addition of saturated ammonium hydrate, of the morphological features of Pentas lanceolata (Forssk.) Deflers com- 1min(Furst, 1979), to detect proteins; a 0.05% (w/v) solution of ruthe- monly known as Egyptian starcluster, is the formation of stipule nium red in water, 5 min (Jensen, 1962), or 2% (w/v) solution of safranin colleters (Schumann, 1891). So far, nothing is known about the struc- O in water, 5 min (Furst, 1979), to detect non cellulosic polysaccharides ture, function and distribution of these glands. with acidic groups; 1% (w/v) solution of pectinase (from Aspergillus)in Due to their high biological activity, the leaf extracts of P. lanceolata water, 24 h, 37 °C, prior to staining of the sections, eliminating pectins, are applied to lymphadenitis, meningitis, abdominal cramps, and arthri- as a control for these reactions; a saturated solution of Sudan III in 70% tis (Giday et al., 2009); the extracts of the flowers are useful for wound (v/v) aqueous ethanol, 20 min (Gahan, 1984), or Sudan black B in 70% healing (Nayak et al., 2005). Formerly, a series of iridoid glucosides was (v/v) aqueous ethanol, 10 min (Gahan, 1984), to detect lipids; a nitrous isolated from the aerial parts of Egyptian starcluster by preparative acid test (10% sodium nitrate in water, 10% acetic acid in water, 10% urea HPLC (Schripsema et al., 2007). It is generally known that the synthesis in water, and 2 N sodium hydroxide), 5 min (Jensen, 1962), or 0.05% To- and accumulation of the secondary metabolites takes place, specifically, luidine Blue 0 in water, 5 min (Gutmann, 1995) to detect phenols; an in the secretory tissues (Hanlidou et al., 1991; Turner et al., 2000; extraction of the sections with methanol–chloroform, 48 h, 20 °C, be- Valkama et al., 2003; Kolb and Muller, 2004; Kobayashi et al., 2008). fore staining and eliminating phenols, as a control for these reactions; To understand whether the stipule colleters of P. lanceolata are involved a 10% solution (w/v) potassium bichromate in water, 5 min (Gahan, in synthesis of the biologically active compounds, the main objectives of 1984), or 1% (w/v) solution of vanillin in hydrochloric acid, 5 min the present work were to study their morphology, anatomy, develop- (Gardner, 1975), to demonstrate tannins; Nadi test (10% α naphtol in ment as well as the chemical nature of their secretion. 40% ethanol and 1% dimethyl para-phenylenediamine chloride in 0.05 M phosphate buffer), 60 min (David and Carde, 1964), to demonstrate 2. Material and methods terpenoids. For fluorescent microscopy, fresh sections were processed using a 2.1. Plants 0.05% (w/v) solution of Natural Product reagent