Introduction

Ipomoea pes-caprae (L.) Stweet and I. imperati (Vahl) Griseb. (Convolvulaceae) are two creeping herbaceous species that colonize sandy and saline soils in the “Restinga”, a coastal ecosystem. In Brazil, their occurrence has been reported to the phytophysiognomies nearest to the sea, in the creeping halophilous–psammophilous plant formation (Thomaz and Monteiro 1992). In this formation, plants are exposed to a broad range of stress factors, such as excess light, high temperatures, increased salinity, and water shortage (Crawford 2008). These factors may have driven the development of a set of morphological, anatomical, and physiological adaptations on local plants (Dickison 2000). Under stressful conditions such as high irradiation and salinity, secretory structures like glands and laticifers may play an important role in plant survival (Fahn 1979).

In Ipomoea species, some secretory structures have been described, such as glandular trichomes (Silva and Azevedo 2007; Arruda et al. 2009; Martins et al. 2012), laticiferous canals, secretory idioblasts (Metcalfe and Chalk 1957; Martins et al. 2012), floral nectaries on sepals (Keeler and Kaul 1984), and extrafloral nectaries on the petiole (Beckmann and Stucky 1981; Keeler 1977, 1980; Keeler and Kaul 1979, 1984; Martins et al. 2012). All these structures are present in both I. pes-caprae and I. imperati (Keeler and Kaul 1984; Arruda et al. 2009), except for nectaries in the latter. Only a few studies have described the chemical nature of the secretion of these structures and have sought to understand their role in plant survival in the “Restinga” environment. Pongprayoon et al. (1991, 1992) identified an acyclic diterpene (E-phytol) and a sesquiterpene (damascenone) in leaves of I. pes-caprae, but the authors did not correlate their occurrence with environmental factors. These compounds were reported to have spasmolytic activity and to be effective against the dermatitis caused by jellyfish venom.

Salt glands are commonly reported in “Restinga” plants, as well as to others halophytes. These structures secrete solutions of excess mineral salts or organic compounds, and have been described in species from several families, like Plumbaginaceae, Frankeniaceae, Tamaricaceae, and Convolvulaceae (Fahn 1979). They have also been reported in mangrove plants, such as Avicennia (Acanthaceae) (Evert 2006) and Laguncularia (Combretaceae) (Fahn 1979), and in plants from sand-dune environments, like Spartina (Poaceae) (Levering and Thomson 1971). Silva and Azevedo (2007) described glandular trichomes in Ipomoea species that occur on the seashore. Their results have led us to question whether such trichomes can secrete saline solution, and thus be classified as salt glands.

Laticifers are frequently reported in Convolvulaceae (Metcalfe and Chalk 1957), but they are named “laticiferous canals” in some genera, like Calystegia, Convulvulus, Ipomoea, and Dichondra (Metcalfe and Chalk 1957), due to the presence of an epithelium (Arruda et al. 2009). Laticifers are mainly related to the defense against herbivores and microorganisms (Fahn 1979). According to Pickard (2008), laticifers produce secondary metabolites that form up the latex, which is stored inside the living cell(s) that produce(s) them. The latex is a fluid with variable chemical composition and may contain precipitates or suspended colloids, and a variety of solutes (Mahlberg 1993).

Thus, we aimed to characterize the structure and the secretion of glandular trichomes and laticifers in I. pes-caprae and I. imperati, in order to understand their role in the species survival in a stressful environment. For this purpose, we addressed the following questions: (1) are the glandular trichomes salt glands? and (2) which laticifer types are present in the species?

Materials and methods

Study area

Both I. pes-caprae (L.) Stweet and I. imperati (Vahl) Griseb. (Convolvulaceae) occur in the creeping halophilous–psammophilous formation of the Paulo César Vinha State Park. The park is located in Guarapari city, southern coast of Espírito Santo state, Brazil (23°33′–20°38′S and 40°26′–40°26′W), and comprises a coastal plain of approximately 1500 ha composed mainly of Restinga vegetation.

Collection of plant material

Flowering branches of I. pes-caprae and I. imperati were collected, and voucher specimens were deposited in the VIC herbarium at Universidade Federal de Viçosa (UFV), with numbers 32,586 and 32,585, respectively; and in the VIES herbarium at Universidade Federal do Espírito Santo (UFES), with numbers 18,723 and 18,721, respectively.

Vegetative branches were collected for the anatomical analyses. The characterization of secretory structures was made in ten leaves sampled between the first and fourth nodes from five individuals. Leaf samples were fixed in FAA (formalin, acetic acid, 50 % ethanol, 1:1:18 v/v/v) and stored in 70 % ethanol (Johansen 1940).

Anatomical characterization

After a minimum 24-h storage in ethanol, leaf samples were embedded in histological paraffin with dimethyl sulfoxide (DMSO) (Histosec, Merck, Germany) or in 2-hydroxyethyl methacrylate (Historesin, Leica Instruments, Germany). Longitudinal and transverse sections (5–8 μm) were obtained in a rotary microtome (RM2155 Leica, Deerfield, USA). Paraffin sections were stained with astra blue and safranin (Gerlach 1969), while historesin sections were stained with toluidine blue at pH 4.0 (O`Brien and Mccully 1981).

Sections were photographed in a photomicroscope (AX-70TRF, Olympus Optical, Tokyo, Japan) equipped with an U-photo system (Spot Insightcolour 3.2.0, Diagnostic Instruments Inc., New York, USA). Under epifluorescence, sections were visualized using an HBO50 W mercury vapor lamp and a UV light filter.

Histochemical tests

Histochemical tests for the contents of glandular trichomes and laticifers were performed using fresh, recently collected samples, which were sectioned using a LPC table microtome (Rolemberg & Bhering Trade and Import LTDA, Belo Horizonte, Brazil). The performed tests for lipids, terpenoids, phenolic compounds, alkaloids, polysaccharides, proteins, and sodium are listed in Table 1. For each investigated metabolite group (Table 1), a negative-control test was conducted in parallel, as recommended in the reference of the respective histochemical test. Glass slides were mounted either with the reagent itself or with glycerin jelly.

Table 1 Metabolite groups investigated and respective methodologies adopted in the histochemical study of leaf secretory structures of Ipomoea pes-caprae and Ipomoea imperati
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Results

Glandular trichomes

Ipomoea pes-caprae and I. imperati have multicellular glandular trichomes on both sides of the leaf blade (Figs. 1–3, 10) and in the petiole. The structures are morphologically similar in the two species, having three regions: secretory head, stalk, and basal cell (Figs. 7, 9). The mature secretory head is composed of 12 radially arranged cells (Fig. 8) with thin primary walls (Figs. 7, 15). The stalk is short, being composed of a single rectangular cell with hyaline cytoplasm. The basal cell is thin-walled, isodiametric and vacuolated (Figs. 7, 9), and its conspicuous nucleus is located in the cell periphery (Fig. 9). This cell is located in a small depression on the leaf surface, slightly below the level of the other epidermal cells.

Figs. 1–6
figure 1

Leaf secretory structures of I. pes-caprae (1, 4, 5) and I. imperati (2, 3, 6) in cross section. 1–4 Leaf blade, 5, 6 midrib, 3 detail of secretory trichome, 4 detail of laticifer. Col collenchyma, EAB epidermis of the leaf abaxial surface, EAD epidermis of the leaf adaxial surface, GT glandular trichome, La laticifer, PP palisade parenchyma, S stomata, SP spongy parenchyma, VB vascular bundle, WSP water-storage parenchyma. Bars 100 µm

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Figs. 7–13
figure 2

Glandular trichomes and laticifers in leaves of I. pes-caprae and I. imperati. 7 Overview of an immature trichome, 8 front view of a mature trichome, 9 overview of a mature trichome. Notice the large nucleus (arrow) of the basal cell, 10 young leaf in longitudinal section. Notice the mucilaginous secretion (Se) produced by the glandular trichomes (GT) on the leaf adaxial surface, 11 laticifer on the midrib in cross section, 12 young laticifers on the leaf blade in longitudinal section, 13 laticifer on the midrib in longitudinal section. BC basal cell, EAB epidermis of the leaf abaxial surface, EAD epidermis of the leaf adaxial surface, GT glandular trichome, La laticifer, Pa parenchyma, Se secretion, SH secretory head, ST stalk. Bars 30 µm

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Figs. 14–25
figure 3

Histochemical characterization of glandular trichomes (14–20) and laticifers (21–25) in leaves of I. pes-caprae and I. imperati. 14 Nonstained leaf section; 15, 17 autofluorescence. Notice both secretory head (SH) and stalk (ST) cells, 16 NADI test showing the cuticle covering the trichome, in comparison with the thicker cuticle covering the adjacent epidermal cells; 17 thickening of stalk cell anticlinal wall (arrow); 18, 20, 25 positive reactions for pectins; 19 positive reaction for neutral polysaccharides; 20 detail of secretion from glandular trichomes; 21 positive reactions for lipid compounds, both in the wall of laticifer cells (CW) and in laticifer content; 22 positive reaction for terpenoids; 23, 24 positive reactions for rubber in transverse (23) and longitudinal (24) sections. Cu- cuticle, GT glandular trichome, Se secretion, SH secretory head, ST Stalk. Bars 30 µm

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Histochemical tests showed similar results for both species. The negative control showed dense cytoplasm and a yellow-brownish content in the secretory head (Fig. 14). The stalk can be differentiated from the other trichome cells by its anticlinal wall, which has higher thickness than in the other parts of the trichome, as observed through autofluorescence (Figs. 15, 17) and with the NADI test (Fig. 16). The cuticle that covers the secretory head is thinner than in the other epidermal regions (Fig. 16).

Reactions on trichome contents were positive for polysaccharides (Figs. 18, 20), specifically pectins (Fig. 19; Table 2), and negative for all the other compounds, including sodium. This mucilaginous secretion promotes the adherence of the two lobes of the adaxial leaf surface of young leaves (Fig. 10), from the first to fourth nodes, only the abaxial surface remaining exposed to the external environment. This secretion has pectin substances and other polysaccharides, similarly to the observed in glandular trichomes (Fig. 20; Table 2).

Table 2 Histochemical positive results on leaf secretory structures of Ipomoea pes-caprae and Ipomoea imperati
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Laticifers

Laticifers are distributed throughout the mesophyll (Figs. 1, 2, 4) and midrib (Figs. 5, 6). The structures are morphologically similar in both I pes-caprae and I. imperati. On the midrib, laticifers have a large lumen and are clearly delimited by flat cells (Figs. 5, 6 11). On the leaf blade, laticifers occur between cells of the palisade and spongy parenchymas (Figs. 1, 2, 4), but flat cells cannot be visualized adjacent to them, which render them difficult to be identified. These laticifers are classified as articulated and are composed of a single row of cells (Figs. 12, 13), which have large peripheral nuclei (Fig. 12) and lipid-impregnated walls (Fig. 21; Table 2).

Histochemical tests identified the following compounds in the latex of the two Ipomoea species: lipids (Fig. 21), terpenoids (Fig. 22), rubber (Figs. 23, 24), and polysaccharides (pectins) (Fig. 25; Table 2). There was neither production nor accumulation of any investigated chemical compound in the cells that delimit midrib laticifers.

Discussion

Ipomoea pes-caprae and I. imperati occur in coastal environments, where salt tends to be highly concentrated (Suguio and Martin 1993; Scarano 2002; Crawford 2008). For this reason, their multicellular secretory trichomes were initially thought to be salt glands, representing an adaptive strategy that would help in removing the excess salt from the plants. However, no sodium was histochemically detected in these trichomes. Only polysaccharides (neutral ones and pectins) were detected, which demonstrates the mucilaginous nature of the secretion. Martins et al. (2012) observed a mucilaginous composition in the content of glandular trichomes of Ipomoea asarifolia (Desr.) Roem. & Schult. An analysis for sodium detection, however, was not performed in their study.

Mucilage may play an important role in wound responses (Fisher et al. 2009), host-pathogen interactions (Pérez-de-Luque et al. 2006), and water transport (Zimmermann et al. 1994; Czarnes et al. 2000; Zimmermann et al. 2007). The mucilaginous secretion in the trichomes of plants from the coastal “Restinga” formation is believed to contribute to the species adaptation to high levels of light irradiation, high temperatures, and low water availability due to low field capacity and/or high soil salinity (Ghanem et al. 2010). The secretion may also provide protection against herbivores (Mafokoane et al. 2007; Rocha et al. 2011; Woodward et al. 2012) and help decreasing transpiration rates (Fahn 1979). Mucilage secretion on the young leaves of both plant species promotes the adherence of the two leaf blade lobes, and therefore reduces the leaf area in direct contact with the external environment. Such reduction may diminish water loss through transpiration, and it could therefore represent a water-saving adaptive strategy.

Laticifers are universally present in the Convolvulaceae (Solereder 1908; Metcalfe and Chalk 1950). The laticifers of Ipomoea are classified as articulated non-anastomosing, as already described by Solereder (1908), Metcalfe (1967), Fahn (1979), and Evert (2006). This same type has also been reported in Convolvulus and Dichondra (Metcalfe and Chalk 1950; Metcalfe 1967; Fahn 1979). In addition, the presence of lipids in laticifers of I. pes-caprae and I. imperati corroborates the report of lipid deposition in the walls of laticifer cells in other Convolvulaceae species (Fineran et al. 1988).

Metcalfe and Chalk (1957) and Arruda et al. (2009) used the term “laticiferous canals” to describe the laticifers in some Ipomoea species. In fact, I. pes-caprae and I. imperati have both been reported to possess an epithelium contouring their laticifers (Arruda et al. 2009). However, our histochemical analyses showed that there is neither production nor accumulation of latex or its components in this group of cells. Furthermore, the occurrence of two types of laticifers (i.e., with an epithelium, on the midrib and petiole; and without an epithelium, on the leaf blade) in the same plant species seems to be rather morphologically improbable. Thus, they are typical laticifers, just with small ordinary parenchymatous cells that delimit the ones occurring on the midrib.

Laticifers can produce chemically complex and diverse types of latex (Van Die 1955; Yoder and Mahlberg 1976; Endress and Bruyns 2000), which may contain sugars, tannins, alkaloids, and/or protein crystals (Heinrich 1967; Fahn 1979). Laticifers of I. pes-caprae and I. imperati produce rubber particles, pectins, lipids, and terpenoids, all of which have already been reported to other Convolvulaceae species, like Calystegia silvatica (Kit.) Griseb. (Condon and Fineran 1989). Terpenoids, which had already been detected on pharmacological studies (Pongprayoon et al. 1992), were found to be restricted to the laticifers in the studied species. Laticifers have been mentioned to perform multiple functions, including regulation of water balance, mediation of oxygen transport, wound healing, and protection against herbivores and pathogens, as well as to compose the plant excretory system (Fahn 1979, 1990; Farrell et al. 1991; Evert 2006).

The secretory structures found in I. pes-caprae and I. imperati contribute to the development of different adaptive strategies that allowed their successful establishment in the coastal sand-dune environment. The secretion of glandular trichomes is purely mucilaginous, and they, therefore, cannot be considered salt glands. The mucilage enables the species to overcome stress by high irradiation, high temperatures, and low water availability. Moreover, the laticifers are typical and can be classified as articulated non-anastomosing, with no production or accumulation of metabolites in the surrounding cells, thus rendering inadequate the use of the term “epithelium” to describe their structure.