South African Journal of Botany 93 (2014) 27–36

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South African Journal of Botany

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Development, structure and secretion compounds of stipule colleters in lanceolata ()

L.E. Muravnik a,⁎, O.V. Kostina a, A.L. Shavarda b a Laboratory of 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 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 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 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 (1% diphenylboric acid β-aminodiethylester) in 10% (v/v) methanol, 2 min (Heinrich Plants of P. lanceolata were cultivated in the greenhouse of the et al., 2002), or Wilson reagent (5% citric acid (w/v) and 5% boric acid Komarov Botanical Institute RAS, St. Petersburg, Russia. Samples were (w/v) in absolute methanol) for 15 min (Hariri et al., 1991) to detect grown under natural lighting, at temperatures of 20 ± 3/15 ± 3 °C flavonoids, inducting yellow-green fluorescence at 450 nm. For exami- (day/night) in winter and 25 ± 5/15 ± 5 °C (day/night) in summer, nation of auto-fluorescence, the sections were directly viewed under and at a relative humidity of 60/80% (day/night). Stipules were cut in UV light (488 nm). 2010–2012, four times from March to October. Each plant contained 3–5 vegetative apexes; actively functioning colleters were taken from 2.5. Extraction of metabolites the different stipules and used for histochemistry, including eight sam- ples in every reaction. Colleters of P. lanceolata were collected and placed in test-tubes with 30 mL of methanol and extracted this way for one week at room temper- 2.2. Scanning electron microscopy (SEM) ature. Extraction with methanol is the best way for the chromatographic GC–MS analysis when the preliminary derivate is needed (Kanani et al., For morphological study, the stipules were fixed in a solution of 2.5% 2008). The extract was first concentrated by a rotary evaporator to (v/v) glutaraldehyde and 4% (v/v) paraformaldehyde in 0.1 M phosphate about 1.5 mL, then transferred into the special GC-vial with screw cap, buffer with glucose (pH 7.2) for 3 h at 4 °C, washed three times with evaporated completely, and then put to the silylation — standard deriva- phosphate buffer, post fixedinphosphatebuffered2%(w/v)osmiumte- tization procedure (Halket et al., 2005). troxide overnight at 4 °C, and then dehydrated in an ethanol series, critical-point dried in CO2 in Hitachi HCP-2 (Hitachi, Japan), attached to 2.6. Silylation SEM mounts, sputter-coated with carbon and observed with a JEOL JSM-6390 (JEOL, Japan) scanning electron microscope at 6 kV accelerat- Crude methanol extracts were evaporated with vacuum concentrator ing voltage. Digital images were produced using the SEM Control Vacufuge® (Eppendorf US). The remainder after methanol removal was Program associated with microscope. dissolved in the 50 μL of pyridine and converted to the trimethylsilyl de- rivatives by adding 50 μL of N,O-bis-(trimethylsilyl) trifluoroacetamide 2.3. Light microscopy (LM) containing 1% trimethylchlorosilane (Supelco, US). The reaction was per- formed in a closed GC-vial by heating it at 100 °C for 15 min. About 0.5 μL For light study, the stipules were fixed as per the SEM process and, of this reaction mixture was injected into the gas chromatograph. after a standard alcohol and acetone dehydration procedure, infiltrated and embedded in Epon-Araldite epoxy resin. Cross and longitudinal sec- 2.7. Gas chromatography–mass spectrometry (GC–MS) tions were made on a Reichert Ultracut R ultramicrotome (Reichert- Jung GmbH, Heidelberg). Semi-thin sections, 2–3 μm thick, were stained Silylated crude extracts were analyzed using an HP 6850 gas chro- with 1% Toluidine blue O in 0.05% (w/v) borax. Observations were matographinterfacedwithanHP5975Cmassselectivedetector(MSD) carried out with light microscopes SteREO Lumar.V12, equipped with (Agilent Technologies). An HB5-MS capillary column (30 m × 0.25 mm digital imaging AxioCam MRc5 and software AxioVision (Carl Zeiss), I.D., film thickness of 0.25 μm, Agilent, Palo Alto, CA) was used with and AxioImager.А1 (Carl Zeiss), equipped with a filter system (BP helium as the carrier gas at a constant rate of 1.3 mL/min. The injector L.E. Muravnik et al. / South African Journal of Botany 93 (2014) 27–36 29 andMSsourcetemperaturesweremaintainedat320and230°C,respec- mass spectral library of the GC–MS data system (NIST, 2005; McLafferty tively. The column temperature program consisted of injection at 70 °C and Stauffer, 1994). The mass spectra of unknown compounds were with an increase of 4 °C/min to 320 °C followed by an isothermal hold interpreted based on their fragmentation patterns. at 320 °C for 15 min. Naphthalene was used as the internal standard. 10 μg of naphthalene was added to each vial for the quantification of 3. Results analytical results. Quantification was performed by chromatographic software Unichrom™ (http://www.unichrom.com/mainr.shtml). 3.1. Types, distribution, and location of colleters TIC-signal of the MSD was used for the calculation of peak areas. Response Factor was assumed as 1 for all compounds. The obtained amounts were The upper part of a stipule of P. lanceolata is divided into lobes differ- recalculated as μmol considering the added trimethylsilyl groups. The ing by length: the largest lobe is in the center, the length of the other mass fraction of the unidentified compounds was calculated by the meth- lobes decreases towards the stipule edges (Fig. 1A, B, D). The number od of normalization. of lobes per stipule varies from five to eight, and four types of colleters The MS was operated in the electron impact mode with ionization en- may be distinguished on the lobes. The first type, a terminal colleter, is ergy of 70 eV. The scan range was set from 50 to 800 Da at 1.27 scan/s. formed at the tip of each lobe and looks like a cover; three types of The samples were analyzed in the split mode (split ratio: 1/20) Data basal colleters, asymmetrical, conical and standard, are distributed at were acquired and processed with the MSD ChemStation software the base of the stipule lobes (Fig. 1B–E). Colleters of the asymmetrical (Agilent Technologies). Compound identification was performed by type occur between the peripheral lobes of the stipule; whereas, comparison with the chromatographic retention characteristics and colleters of the conical and standard type are found at the base of the mass spectra of authentic standards, reported mass spectra and the central stipule lobes (Fig. 1C–E).

Fig. 1. Location and types of the stipule colleters in Pentas lanceolata. A. View of the multifid stipule on a shoot. Scale bar: 3 mm. B. Stipule with five lobes after separation from a caulis. The terminal colleters (stars) are on the top of each lobe, the basal colleters (triangles) are on the base of the stipule lobes. Scale bar: 1 mm. C. View of the stipule lobes with colleters (data of scanning electron microscopy). Scale bar: 200 μm. D. Schematics of colleter arrangement on a longitudinal section of a stipule. E. Schematics of colleter arrangement on a cross section of a stipule. Ac — asymmetrical colleters, Cc — conical colleters, Sc — standard colleters, Т — terminal colleters. 30 L.E. Muravnik et al. / South African Journal of Botany 93 (2014) 27–36

3.2. Morphology and anatomy palisade form; usually they lie in one layer. At the same time, the secre- tory cells lying on the adaxial side are isodiametric, they form several The mature colleters have an oblong head. In the terminal colleters layers looking like a bulge on the outside (Fig. 2B, star). Two to four (Fig. 2A), the head's diameter and height average 207 ± 1.2 μmand rows of the secretory cells are formed on the adaxial side of the terminal 375 ± 24.8 μm. Dimensions of a head of the asymmetrical colleters colleters (Fig. 2A), and eight to ten rows on the adaxial side of the asym- averages 320.1 ± 21.2 μm and 331.4 ± 23.5 μm(Fig. 2B). The smaller metrical colleters (Fig. 2B). The conical colleters also have five to seven conical colleters (Fig. 2C) have a head 224 ± 13.8 μm in diameter and rows of isodiametric cells on the adaxial side; however their head ap- 271 ± 17.8 μm in height. Both the asymmetrical and conical colleters pears symmetrical (Fig. 2C). An additional structural feature of the ter- form a short stalk. The standard colleters are rhomboid and smallest minal colleters is the disproportionate arrangement of the majority of (Fig. 2D) (164.9 ± 13.5 μm and 173.6 ± 11.5 μm). secretory cells relative to a horizontal axis of symmetry: on the adaxial The pattern of the structural organization of the terminal and basal side the secretory cells are formed lower than on the abaxial side colleters is identical. It consists of the epidermal (secretory) cells sur- (Fig. 2A, white arrow). The central parenchymal cells are developed to rounding 9–15 layers of the central parenchymal cells. In a longitudinal a greater degree in the asymmetrical colleters. Among the parenchymal section, vertical asymmetry is obvious in the terminal and asymmetrical cells one finds the vascular bundle and many large idioblasts containing types of the colleters: the secretory cells lying on the abaxial side have a crystals (Fig. 2A–C). The crystals have the form of a raphide. In the

Fig. 2. Longitudinal sections of the heads of Pentas lanceolata stipule colleters. A. A terminal type of the colleters. Disproportionate arrangement of the secretory cells relative to a horizontal axis of symmetry (white arrow). B. The asymmetrical type of the colleters. Asymmetric arrangement of secretory cells relative to vertical axis: one layer of the palisade secretorycellsison the abaxial side and several rows of the isodiametric cells are situated on the adaxial side. C. Conical type of the colleters. Symmetrical head contains isodiametric cells on the adaxial and abaxial sides. D. Standard type of the colleters with one layer of the palisade secretory cells. Ab — abaxial side, Ad — adaxial side, R — idioblasts with raphids, S — secretion, St — stalk, Vb — vascular bundle. Scale bar: 100 μm. L.E. Muravnik et al. / South African Journal of Botany 93 (2014) 27–36 31 asymmetrical and conical colleters, the external cells of a stalk are not adaxial side are divided in two directions and form several layers of secretory (Fig. 2B, C, black arrows). The standard colleters are arranged the isodiametric secretory cells (Fig. 3C, D). The subprotodermal cells more simply than the others: one row of secretory cells surrounds four give rise to the central parenchymal cells of the gland. They are orientat- to six rows of parenchymal cells (Fig. 2D). Vascular bundles and idio- ed along the head axis. blasts are absent. After the terminal colleters achieve a quarter of their final length, the asymmetrical and conical colleters are initiated. This takes place on the 3.3. Development stipule of the second node after the apex. Sequence and direction of the first divisions is the same as in the terminal colleters (Fig. 3C, twofold The colleters of all types appear on the stipules at early stages of the arrow). It is impossible to determine what type of basal colleter will de- shoot development. Despite their different morphology and dimen- velop after initiation. Morphological distinctions between the asymmet- sions, development of the colleters happens in an identical way. rical and conical types can be seen when the colleters reach a third of Formation of the terminal colleters starts on the first stipule primor- their final length. The standard colleters arise on the stipules of the dium. One of the protodermal cells located at its top expands and un- third node after the stem apex (Fig. 3E–I). dergoes a number of anticlinal divisions. All arising cells have dense Maturation of the four colleter types occurs sequentially. The cytoplasm (Fig. 3A, arrows). Then, periclinal and anticlinal divisions terminal colleters mature on the third node; secretion appears on the are observed in two to three cells of a subprotodermal layer (Fig. 3B, head surface. The secretory drops are clear, yellowish and viscous arrow), leading to formation of three vertical rows of the internal (Fig. 2A–C). Individual glands secrete for about two to three days. The cells. Subsequently, the protodermal cells at the abaxial side are extend- basal colleters start to secrete on the fourth node, after which they with- ed and acquire a palisade form, whereas the protodermal cells at the er and become dark brown on the fifth and sixth nodes.

Fig. 3. Development of the stipule colleters in Pentas lanceolata. A. The epidermal cells arise as a result of anticlinal divisions of an initial cell and its daughter cells on the top of the stipule primordium (arrows). B. Three columns of cells emerge after the periclinal divisions of three subprotodermal cells (arrow). C. Several layers of the secretory cells occur at the adaxial side of a terminal colleter. The first stage of the asymmetrical or conical type colleter formation is seen in the basal part of the stipule lobe (arrow). D. During maturation the interior cells of a terminal colleter are vacuolated. An advanced stage of the asymmetrical or conical type colleter development is seen on the right (double arrow). E–I. Sequential stages of the standard type colleter formation. Scale bar: 50 μm. 32 L.E. Muravnik et al. / South African Journal of Botany 93 (2014) 27–36

3.4. Histochemistry detected in the colleters. So, neutral lipids are confirmed using Sudan black B that provides a blue coloration in the upper part of the secretory Chemical substances contained in the colleters were identified in ac- cells and a dark-blue color in the basal part (Fig. 4D). In the test with tively secreting glands. It was established that the same compounds are Sudan III, a more intensive orange coloration of lipids is typical for the present in all four types of colleters. Distinctions between them are basal part of the secretory cells too (Fig. 4E). Accumulation of phenols expressed in intensity of coloration or fluorescence of the various cells, in the secretory cells is identified with Toluidine blue by a light-green as well as in the period of function. The largest intensity of coloration color (not shown) or with nitrous acid test by cherry-red color due to various histochemical reagents is found in the terminal secretory (Fig. 4F); the largest concentration of phenols was determined in the cells of the terminal, asymmetrical and conical colleters; in the standard central parenchymal cells. In a control, after extraction of the sec- colleters all secretory cells are colored uniformly. After treatment with tions with methanol–chloroform, eliminating phenols, the nitrous concentrated nitric acid and saturated ammonium hydrate, proteins acid test gives only a weak coloration of the phenols (Fig. 4G). Con- are stained an orange color in the terminal part of a head. They appear densed tannins are found in the lateral secretory and central paren- yellow in other secretory cells and pale-yellow in the parenchymal chymal cells as indicated by a brown color in reaction with cells (Fig. 4А). Acidic polysaccharides are identified in the secretory potassium bichromate (Fig. 4H) or by a red color in reaction with cells, particularly in the cell walls due to Ruthenium red that gives a vanillin in hydrochloric acid (not shown). Terpenoids are detected crimsoncoloration(Fig. 4В). Safranin O also stains polysaccharides, with Nadi reagent as violet coloration in the upper wall of the secre- but an orange color (Fig. 4C). Different lipophilic substances are tory cells (Fig. 4I).

Fig. 4. Detection of the primary and secondary metabolites by light and fluorescent microscopy as well as by histochemistry in fresh sections of the terminal colleters of Pentas lanceolata.A, stained with nitric acid and ammonium hydrate: the secretory cells are stained yellow indicating the presence of proteins. More intensive color is typical of the upper part of the head where proteins give orange color. B, stained with ruthenium red: the secretory cells, particularly the cell walls, are stained pink, indicating the presence of pectins. C, stained with safranin O for pectins: all secretory cells are weakly orange. D, stained with Sudan black B: the apical part of the secretory cells are blue, indicating the presence of neutral lipids; the basal part of the secretory cells are dark blue, demonstrating a greater concentration of neutral lipids. E, stained with Sudan III: the secretory cells are orange which is typical of neutral lipids. F, stained with vanillin in hydrochloric acid: shows the condensed tannins localized mainly in the central parenchymal cells. G, stained with nitrous acid for phenols after an extraction with methanol– chloroform. Phenols are not visible. H, stained with potassium bichromate: condensed tannins are seen in the secretory and central parenchymal cells. I, stained with Nadi reagent for terpenoids: present particularly in outer cell wall. J, stained with Wilson reagent: yellow fluorescence of phenylpropanoids is induced in the secretory cells; K, stained with Natural Product reagent: yellow fluorescence of phenylpropanoids is seen in the secretory cells. L, fresh section of the colleter, without drying: blue autofluorescence in the head cells is a diagnostic feature of flavonoids. Scale bars: 100 μm. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.) L.E. Muravnik et al. / South African Journal of Botany 93 (2014) 27–36 33

Table 1 Metabolites (μM/g dry weight) detected in the methanol extracts of the Pentas lanceolata stipule colleters.

Compounds RT* (min) Stipule without colleters (control) Terminal colleters Basal colleters Identification

Organic acids 1. Lactic acid 4.15 400.41 84.99 65.94 1,2,3 2. Hydroxyacetic acid 4.33 2.87 1.40 1.19 1,2 3. Levulinic acid 5.13 0.99 0.58 0.72 1 4. 3-Hydroxypropionic acid 5.40 0.83 0.38 0.29 1 5. Succinic acid 8.54 1.50 2.92 1.95 1,2,3 6. Glyceric acid 9.08 1.37 1.55 1.82 1,2,3 7. Fumaric acid 9.21 0.17 0.32 0.20 1,2,3 8. Threonolactone 9.73 0.62 0.92 1.10 1 9. Ribonolactone 10.21 0.00 0.13 0.09 2 10. Malic acid 12.24 6.86 28.35 27.67 1,2,3 11. Threonic acid 13.84 3.10 75.71 38.11 1 12. Tartaric acid 14.53 0.28 2.08 1.07 1 13. 2-Ketogluconic acid 17.35 0.00 0.73 0.54 1 14. Ribonic acid 17.62 0.52 2.89 2.92 2 15. Citric acid 18.39 10.32 11.08 19.88 1,2,3 16. Quinic acid 19.21 79.46 295.34 280.18 1,2 17. Gluconic acid 1,5-lactone 19.52 2.85 0.00 0.00 1 18. Gluconic acid 21.44 0.00 2.43 1.80 1 19. Galacturonic acid 26.99 3.79 7.65 5.49 1,2,3

Amino acids 20. α-Alanine 4.74 0.52 0.45 0.69 1,2,3 21. Valine 6.73 0.97 0.09 0.17 1,2,3 22. Serine 7.47 1.07 3.06 0.75 1,2,3 23. Threonine 8.20 0.16 0.74 0.12 1,2,3 24. Glycine 8.42 2.30 0.87 0.36 1,2,3 25. Aspartic acid 10.71 0.58 1.12 0.44 1,2,3 26. Pyroglutamic acid 12.59 2.84 6.45 2.18 1,2,3 27. γ-Aminobutyric acid 12.82 2.13 2.52 1.21 1,2,3

Free fatty acids 28. Pelargonic acid 9.37 1.27 0.32 0.41 1 29. Capric acid 11.28 0.90 0.18 0.17 1 30. Lauric acid 14.97 4.02 0.74 0.56 1 31. Tridecylic acid 16.71 1.00 0.95 0.58 1 32. Pentadecilic acid 19.99 7.91 2.84 0.00 1 33. Palmitoleic acid 21.10 9.97 1.52 1.70 1 34. Palmitic acid 21.55 117.10 33.22 26.21 1,3 35. Margaric acid 23.00 1.67 0.41 0.37 1 36. Linolenic acid 23.92 10.20 11.12 8.93 1,3 37. Oleic acid 24.03 54.81 19.06 16.20 1 38. Elaidic acid 24.13 6.04 1.18 0.75 1 39. Stearic acid 24.42 25.33 6.95 5.73 1,3 40. Arachidic acid 27.10 0.88 0.74 0.82 1 41. Lignoceric acid 31.90 1.03 0.55 0.31 1 42. Cerotic acid 34.08 0.00 0.76 0.21 1 43. Methyl palmitate 19.53 8.71 12.60 12.91 1,3 44. Methyl stearate 22.66 0.80 0.26 0.27 1,3

Alcohols 45. Glycerol 7.98 40.35 7.72 9.87 1,2,3 46. Hexadecanol 20.21 2.16 0.52 0.39 1 47. Sorbitol 20.37 2.95 2.22 1.10 1,2,3 48. Dulcitol 20.49 3.01 0.56 0.38 1,2,3 49. Myo-inositol 22.73 6.33 5.87 5.86 1,3 50. Octadecanol 23.19 5.72 0.77 0.59 1

Sugars 51. Anhydroglucose 17.45 0.00 4.27 3.88 1 52. Fructose 27.99 14.71 5.69 1,3 53. Glucose 108.56 105.34 89.22 1,3 54. Methyl glucoside 19.78 8.26 12.26 17.33 1,3 55. Galactose 19.91 0.49 1.55 0.00 1,3 56. Sucrose 30.50 193.60 118.50 92.70 1,3 57. Galactinol 1.60 3.42 2.71 1,3 58. Raffinose 38.71 0.66 0.49 0.57 1,3

Iridoids 59. Loganin 34.24 0.65 2.01 1.59 2 60. Tudoside 35.08 4.91 34.07 53.87 4 61. Asperuloside 36.97 10.30 46.49 22.90 4

Sterols 62. Cholesterol 35.16 8.84 3.69 1.69 1,2 63. Campesterol 36.24 0.00 1.24 1.01 1

(continued on next page) 34 L.E. Muravnik et al. / South African Journal of Botany 93 (2014) 27–36

Table 1 (continued) Compounds RT* (min) Stipule without colleters (control) Terminal colleters Basal colleters Identification

Sterols 64. Stigmasterol 36.55 1.24 1.24 0.86 1 65. b-Sitosterol 37.13 10.85 11.71 5.51 1

Terpenes 66. Methylthymol 7.02 1.44 0.21 0.19 4 67. Thymol 8.76 1.98 0.65 0.95 1 68. Caryophyllene 10.40 0.29 0.07 0.00 1 69. Oleanolic acid 39.28 3.47 11.01 9.81 1,3 70. Ursolic acid 39.70 18.99 51.75 44.08 1,3

Phenolics 71. 4-Hydroxycinnamic acid 19.86 0.19 0.70 1.03 1 72. Ferulic acid 22.27 1.79 0.80 0.00 1 73. Octyl 4-methoxycinnamate 23.14 0.83 0.03 0.03 1 74. Mollugin 37.34 5.54 5.76 0.00 4

Note. *RT = retention time (min). Identification:

1 NIST 08 2 Agilent Fiehn GC/MS Metabolomics RTL Library (Agilent US) 3RT 4 MS-pattern interpretation

Presence of the phenols is also demonstrated by fluorescence mi- Among organic acids, the content of malic, threonic, tartaric, quinic, croscopy. Wilson reagent gives the yellow-green fluorescence in the ribonic, and gluconic acids in the extracts of the terminal and basal basal part of the secretory cells and in the central parenchymal cells colleters in P. lanceolata is four to ten times larger than in the control ex- (Fig. 4J). Phenylpropanoids are identified with Natural Product reagent tracts of stipules without colleters. For extracts from secretory struc- in the cell walls and in the vacuoles by yellow fluorescence (Fig. 4K). The tures, the increased amount of iridoids (amount of asperuloside is six blue auto fluorescence characteristic of phenolic substances is found to ten and tudoside is four to eleven times greater) (Fig. 5) and terpe- both in the secretory cells and in the parenchymal cells (Fig. 4L). noids (amount of oleanolic and ursolic acids is three times greater) is characteristic in comparison with a control. Extracts of two types of colleters differ in the content of mollugin, belonging to phenolics: 3.5. Metabolite profiling mollugin is found in the terminal colleters but is practically absent in B-type ones (Fig. 5). On the other hand, some substances (for example, The results of the metabolomic analysis of P. lanceolata stipule the free fatty acids and alcohols) present in the extracts of the secretory colleters are shown in Table 1. In the methanol extracts, more than structures occur in far smaller quantities than in the control. 150 compounds were found, of which 74 were identified by their mass-spectra. The stipule extracts contain organic acids, amino acids, 4. Discussion free fatty acids, alcohols, sugars, iridoids, sterols, terpenes, and pheno- lics. The greatest number of the detected metabolites belongs to compo- Existence of the glandular structures on stipules is a typical feature nents of the lipid metabolism. In addition to the usual fatty acids, some of the Rubiaceae (Rutishauser, 1984; Robbrecht, 1988). So far, the stip- sterols, alcohols and compounds of other metabolomic complexes take ule colleters were found in the plants of more than 130 genera. Their part in lipid biogenesis, too. In the stipule extracts, a complex of sugars, morphology has been investigated in Psychotria (Horner and Lersten, resulting from carbohydrate metabolism is well expressed. In addition, 1968), Pavetta (Lersten, 1974a), Neorosa (Lersten, 1974a; Van Hove components of glycolysis, pentose-phosphate cycle, tricarbonic acid and Kagoyre, 1974), Tricalysia (Lersten, 1974a); Gardenia (Mangalan cycle, and glyoxylate cycle are found. et al., 1990), Simira (Klein et al., 2004), Bathysa (Miguel et al., 2006),

OH O Alibertia (Barreiro and Machado, 2007), Galium (Muravnik and ‘ ’ 12 Kostina, 2010), and others. Colleters studied have a so-called standard structure that represents several rows of oblong parenchymal cells Ferulic acid O surrounded by one layer of epidermal, or secretory, cells (Thomas, OH 10 OH O 1991). In some Rubiaceae species, variations of the standard type O

Mollugin were found, but each species studied had only one morphological type O 8 O of colleter. So for example, most Pavetta species contain dendroid or O brushlike colleters, some Tricalysia species have standard or dendroid Octyl-4-methoxycinnamate colleters, among Psychotria species both standard and dendroid OH 6 O O colleters, as well as intermediate forms are met (Lersten, 1974a,

HO O 1974b). An exception is presented by Ramosmania which possessed Loganin OH 4 O O O OH both standard and intermediate types of colleters on the calyx O O OH (Tirvengadum, 1982). For the first time, the morphological diversity of O HO OH OH

Tudoside O OH stipule colleters occurring on one organ and functioning sequentially 2 O O OH is described in P. lanceolata in the present research. Four types of the

O OH O glandular structures arise on its stipule lobes: a terminal type is formed Asperuloside O 0 O O O O at the tip of each lobe, and three basal types develop at the lobe bases. OH Control B-colleters T-colleters OH OH Similarly, in some Apocynaceae species four colleter types are distribut- OH ed on different places on one organ, specifically, the petiole (Simões Fig. 5. Relative content of phenolic compounds and iridoids in different types of Pentas et al., 2006) or stipule (Martins, 2012). On the other hand, in some lanceolata colleters. B — basal colleters, Т — terminal colleters. Piriqueta species (Turneraceae) or in Ilex species (Aquifoliaceae) several L.E. Muravnik et al. / South African Journal of Botany 93 (2014) 27–36 35 types of the colleters are present on different organs (leaf primordia, bracts of the different families. They are present in secretory cells prophylls, and bracts; Gonzalez, 1998). of Turnera (Turneraceae — Gonzalez, 1998), Mandevilla illustris and Distinctions between the four colleter types in P. lanceolata are not M. velutina (Apocynaceae — Appezzato-da-Gloria and Estelita, only in location but also in structural organization and time of initiation. 2000), Hymenaea stigonocarpa (Fabaceae — Paiva and Machado, One of the specific anatomical features is an asymmetric structure of the 2006a), as well as in parenchymal cells of Alibertia sessilis colleters colleter head. Asymmetry is typical for the terminal, asymmetrical and (Rubiaceae — Barreiro and Machado, 2007). Localization of phenols conical colleters; it is observed in two directions, namely, relative to in the vacuoles was demonstrated in the glands of Copaifera the vertical and horizontal axes. Usually, formation of the standard langsdorffii (Paiva, 2009)andoftwoGalium species (Muravnik and colleters occurs as a result of a number of anticlinal divisions of the Kostina, 2011). Results of the metabolite profiling demonstrate that protodermal cells as well as of a series of periclinal and anticlinal divi- somefattyacids,phenols,sterols,iridoidsandterpenoidsarepre- sions of the subepidermal cells (Patel and Zaveri, 1975; Dave et al., sented in the methanol extracts of Egyptian starcluster colleters. 1987). In contrast, the adaxial protodermal cells in P. lanceolata undergo Among metabolites, a mollugin was previously identified in the both anticlinal and periclinal divisions forming several layers that result plants of two species (Itokawa et al., 1983). The terpenoid in vertical asymmetry. The same asymmetry of the head was described part of the metabolite profiling includes monoterpenes (thymol before in the glands of Turneraceae species (Gonzalez, 1998). In and methylthymol), sesquiterpenes (caryophyllene), triterpenes colleters of P. lanceolata, there is also an unequal arrangement of most (oleanolic and ursolic acids), and iridoids (loganin, tudoside, secretory cells relative to the horizontal axis when the secretory cells asperuloside). In the colleters of P. lanceolata mono- and sesquiter- on the adaxial side are lower than on the abaxial side, as was also penes are found in smaller quantities than in the control. On the con- shown in Apocynaceae and Aquifoliaceae colleters (Simões et al., trary, triterpenes are localized mainly in the glands. The large 2006; Martins, 2012). From our results, it is obvious that modifications quantity of asperuloside and tudoside and considerable smaller of the standard type of P. lanceolata colleters, in contrast to those previ- quantity of loganin were found in extracts of the glands. Whereas ously described, occur due to the formation of multiple layers of the se- tudoside and asperuloside were identified formerly in P. lanceolata cretory tissue. Apparently, this feature is a peculiar adaptation for (Schripsema et al., 2007), loganin was detected for the first time. It production of the large volume of secretion. Appearance of several is known that flavonoids and especially tannins are insect deter- layers of the secretory cells indicates a more progressive development rents; therefore, a probable biological role for P. lanceolata colleters and later phylogenetic origin of P. lanceolata as compared with other is to defend the young shoots during maturation. Rubiaceae plants studied. Formerly morphological modifications of the It was established here that the four types of the stipule colleters in standard type colleters in Rubiaceae were used as a taxonomic character Egyptian starcluster develop sequentially, namely, at first the terminal at the generic level (Lersten, 1975; Robbrecht, 1988). glands are initiated and then the basal ones; their maturation and senes- It is well known that the chemical content of colleter secretion is com- cence happen in the same order. Ontogenetic data are confirmed with the plex. Usually it includes proteins (Horner and Lersten, 1968; Mohan and metabolite profiling data: increasing content of the amino acids correlates Inamdar, 1986; Thomas and Dave, 1989; Vieira et al., 2006), acid polysac- with a later stage of development (Newsholme et al., 2011); that is to say, charides (Klein et al., 2004; Paiva and Machado, 2006b), ethers of fatty the terminal colleters are older than the basal colleters. During a life cycle, acids, phenolics (Appezzato-da-Gloria and Estelita, 2000; Muravnik and alternation of the functional activities takes place in the colleters of Kostina, 2011), as well as terpenoids (Esau, 1965). In the present research, P. lanceolata: initially secretion of the polysaccharides occurs, and then it localization of proteins was found in reaction with concentrated nitric is replaced with production of the secondary metabolites. Distinct secre- acid in all secretory cells of P. lanceolata colleters. However, their concen- tory phases were also described in the calycine colleters of Odontadenia tration was largest in the apical part of the colleter head. Presence of pro- lutea (Martins, 2012). Based on the results of the present research, it is teins, including the hydrolytic enzymes, in the cells and secretion of other possible to assert that the secretory structures, which are formed on the colleters has been shown by various methods: using Coomassie brilliant stipules in P. lanceolata, carry out the same functions as the colleters in blue (Mangalan et al., 1990), bromophenol blue (Barreiro and Machado, other species. The proteins and polysaccharides found in them protect 2007), Gomori's cytochemical method (Mangalan et al., 1990), and elec- the growing shoot against desiccation and mechanical damage during trophoresis (Klein et al., 2004; Miguel et al., 2006). Some authors consider growth, and phenols, iridoids, and terpenoids play a role as an antibacte- the hydrolytic enzymes as agents for active transport of the secretory sub- rial, fungicide and insecticidal barrier. stances across the plasmatic membrane and the cell wall (Mangalan et al., 1990); others explain their presence by function to protect a meristematic 5. Conclusions tissue from fungi (Miguel et al., 2006), insects (Curtis and Lersten, 1974) or bacteria (Carvalho et al., 2001). For the first time, four types of colleters occurring simultaneously on In the colleters of P. lanceolata acid polysaccharides were also located. P. lanceolata stipules are found and described. Despite varying morpholo- They occur mainly in the walls of the secretory cells and in the drops of gy of the mature colleters, their initiation and development happens in an secretion. Until now, Ruthenium red has revealed polysaccharides in identical way; and the structure of all colleters is a modification of the most glandular structures (Mohan and Inamdar, 1986; Thomas and standard type. The intensification of secretion is carried out due to signif- Dave, 1989; Mangalan et al., 1990). In the present research, it was realized icant increase in the number of secretory cells. Proteins, pectins, lipids, that a metabolomic analysis of the colleter extracts allowed the identifica- phenols, iridoids, and terpenoids were found in the content of the secre- tion of numerous simple sugars, which are used in polysaccharide synthe- tion. Since the development and activation of the glandular structures of sis. According to Thomas and Dave (1989), polysaccharides provide all types occur in turn, secretion from the colleters provides continuous defense of the growing shoot from mechanical damage or drying. defense of a shoot during a plant's vegetative growth period. In addition to amino acid and carbohydrate metabolism, the se- cretory structures of P. lanceolata are characterized by intensive Acknowledgments lipid metabolism. On the semi-thin sections in reactions with histo- chemical reagents deposition of the lipids was found in the basal We appreciate the Core Centre “Cell and Molecular Technology in part of the secretory cells, perhaps in the vacuoles, as well as in the the Plant Science” at the Komarov Botanical Institute (St. Petersburg) cell wall and cuticle. The largest concentration of the condensed tan- for provision of equipment for light and electron microscopy, and the nins is distinctive in the vacuoles of the central parenchymal cells; Resource Centre at the St. Petersburg State University, Russia, for pro- flavonoids and terpenoids are found in the cell wall of the secretory viding us with GC–MS equipment. We also thank the two anonymous cells. Phenols are ordinary compounds in the glands of stipules and reviewers for their comments on this manuscript. 36 L.E. Muravnik et al. / South African Journal of Botany 93 (2014) 27–36

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