Histochemical observations in Piper malgassicum (Piperaceae) with a special focus on the epidermis

Histochemical observations in Piper malgassicum (Piperaceae) with a special focus on the epidermis


Introduction
The genus Piper L. belongs to the family Piperaceae and includes more than 2000 species with a pantropical distribution (Quijano-Abril et al. 2006), most of them from America (Ulloa Ulloa et al. 2017). Nowadays, the phylogenetic position of Piper as well as of the Piperaceae family, is among the so-called "paleoherbs", a phylogenetically complex basal group of dicots (Loconte and Stevenson 1991;Chase et al. 2016), within the order Piperales (Jaramillo et al. 2008;Palchetti et al. 2018). Piperales are herbaceous or woody plants exhibiting quite primitive morpho-anatomical features (Isnard et al. 2012) Piper species can be described either as shrubs, or more frequently, as creepers and lianas in the equatorial regions (Jaramillo et al. 2001). Many Piper species present two circles of vascular bundles in the stem, sometimes called polystelic organization, considered as a synapomorphy of the Piperaceae family, except for genus Verhuellia (Isnard et al. 2012). The main commercial species are Piper nigrum L. native to India, P. methysticum (L.) G. Forst. typical of West Polynesia, and P. cubeba L. a typical Indonesian species (Maugini et al. 2014). Due to their importance, many species are also cultivated beyond their native geographical region and, in some cases, have become naturalized . However, species belonging to this genus generally display an exclusive pantropical distribution. They present a high number of growth forms and biomechanical organizations (Isnard et al. 2012) with most species having a restricted area of distribution, while others are widespread (Marquis 2004;Quijano-Abril et al. 2006) Many species of genus Piper have a high economic value all around the world and its trade has a long history, dating back to ca. 9,000 years ago. Magnoliids, including Piperaceae, are also characterized by the presence of aromatic compounds like terpenoid essential oils and other odorous volatile substances (Marinho et al. 2011) These substances are found in fruits, seeds, and leaves inside glandular pockets and trichomes, and confer the intense flavour and the characteristic aromatic fragrance in most species of the genus (Ballantini et al. 2018) These chemical components led many species of Piper to become an important resource from a commercial point of view. The genus is poorly represented in continental Africa. P. guineense Schum. & Thonn. and P. capense L. are the only two currently known native and endemic species. In Madagascar, the knowledge of the genus is still incomplete ) According to Weil et al. (2017), the non-cultivated species of Piper present on the island are P. heimii C.DC. and P. pachyphyllum Baker, while the presence of P. borbonense is not confirmed, even though De Candolle (1923, 1869) assigned some samples from Madagascar (and Mauritius) to this species.
In this contribution, we investigated the vegetative structures and the localization of secondary metabolites of a recently described species of the genus Piper L. from Madagascar, P. malgassicum Papini, Palchetti, Gori & Rota Nodari (Palchetti et al. 2018, 2020, which is used for the production of local pepper named "voatsiperifery", probably mixed with P. borbonense (Miq.) C. DC. and P. tsarasotrae Papini, Palchetti, M. Gori & Rota Nodari. For this reason, these species are economically relevant.
Despite extensive knowledge about the secondary metabolites content and a few accounts about the histochemistry of the fruit and seed of Piper, particularly P. nigrum, there is very limited evidence about the presence of secondary metabolites in the leaves and in general in the epidermis of the organs in the same genus and no data, in general, about P. malgassicum. The aim of this study was to correlate anatomical and histochemical features of the epidermis and underlying tissues with secondary metabolite production and defence systems of P. malgassicum. We show the anatomical features of the epidermis in leaves, anthers, and ovary using light microscopy (LM) and histochemical techniques. Specific histochemical staining methods were used to localize different classes of secondary metabolites in the vegetative parts of the plants. Since the presence of secondary metabolites in the anthers has been documented rarely, we also used transmission electron microscopy (TEM) to check the local ultrastructural patterns linked to the route of formation of secondary metabolites in the organelles inside anther cell wall cells.

Plant material
Plants grown from seeds of P. malgassicum obtained from Madagascar were grown in a greenhouse at the Department of Agriculture, Food, Environment and Forestry of the University of Florence (Italy) from March to July 2019 at approximately 25 °C during the day of 14 hours and 15 °C during the night of 10 hours under artificial light in a pot (15 cm diameter) containing universal soil plus garden soil (Vigorplant, Italy), without fertilizers; humidity was kept at 70%. The plants were later transferred to the Botanical Garden of Florence (Giardino dei Semplici) Another plant was grown starting from seed directly in the laboratory at room temperature and leaves and stem of the young plant were used for further analysis (vibratome sectioning) After about one month of growth, when stems, leaves, and flowers had reached a suitable size (i.e., 2 mm diameter for the stem, 5 cm length for the leaf, and 1 cm length for the flower) for the light and TEM analyses, sections of leaf, stem, and anthers were cut with a razor blade and a vibratome.
Herbarium samples were made with material directly collected in the field (Vohiday forest, Ambositra region, Madagascar) and conserved at the Tropical Herbarium of Florence, Italy (FT) They are syntypes of the type. For the images, we used sample PS9, Voidahi (Madagascar).

Light microscopy
Leaf and stem sections for LM were cut with a vibratome (Vibratome 1000 Plus) at a thickness of 40-50 µm. Some sections were stained with selective histochemical dyes and reagents for terpenoids and lipophilic substances using Sudan Black for lipid stain-ing (Lison et al, 1960), NADI for lipids and terpenes (Carde et al. 1964), Sudan Red III-IV for the detection of neutral lipids (Lison et al. 1960), and Fluorol Yellow 088 (FY088) fluorescent staining for lipid detection (Bundrett et al. 1991) Other sections were stained with selective dyes for non-lipophilic molecules, like polysaccharides, polyphenols, and alkaloids. Schiff's reagent and periodic acid-Schiff (PAS) staining were used for detecting polysaccharides (Jensen 1962), staining with FeCl 3 for polyphenols (Strasburger E. 1923) and Wagner's reagent for alkaloids (Wagner and Bladt 1996) Mechanical tissues and other physical support structures were revealed with calcofluor for cellulose staining (Hughes and McCully 1975), phloroglucinol for lignin marking (Johansen 1940), and toluidine blue as generic staining (Beccari and Mazzi 1966) The sections were observed through a Leitz DM-RB "Fluo" light/ fluorescence microscope (Wetzler, Germany) equipped with a digital camera (Nikon DS-L1, Tokyo, Japan)

Transmission electron microscopy
Anther samples (about 2 mm long) were collected and fixed in 1.25% glutaraldehyde, at 4 °C, in 0.1 M phosphate buffer (pH 6.8) for 24 h. The samples were fixed in 1% OsO4 in the same buffer for 1 h. After dehydration in an ethanol series and a propylene oxide step, the samples were embedded in Spurr's epoxy resin (Spurr 1969) Crosssections (about 70-nm thick) were cut with a diamond knife and a Reichert-Jung ULTRACUT ultra-microtome, and then stained with uranyl acetate (Gibbons and Grimstone 1960) and lead citrate (Reynolds 1963) The sections were then examined with a Philips 201 TEM at 80 kV. Semithin sections (1-5 µm), obtained using glass knives, were stained with toluidine blue 0.1%, observed, and photographed.

Macroscopic morphology
The P. malgassicum herbarium sample showed simple elongated ovate leaves, while the inflorescences were cylinder-shaped, about 4 cm long. Infructescences were composed of many drupes carried on peduncles inserted orthogonally to the axis ( Fig. 1 A Autofluorescence of green leaves in blue light (450-490 nm) resulted in red fluorescence of the chloroplasts due to chlorophyll in all chlorenchyma cells (Fig. 3A, B), including the palisade and spongy mesophyll cells. As expected, red fluorescence was absent in the epidermis and hypodermis. Green fluorescence was attributed to the phenolic components in the cuticle of the upper (adaxial) and lower (abaxial) epidermis as well as in glandular trichomes (Fig. 3A, B) Accordingly, the cell walls of xylem elements in the mesophyll appeared green. Lipid droplets appeared as yellow fluorescence within chlorenchyma cells and in the intercellular spaces, both in the palisade and spongy mesophylls (Fig. 3B). Sudan Black staining also revealed the presence of lipids in the cuticle and inside the trichomes of the lower epidermis (   positively stained lipid droplets also in the intercellular spaces of the mesophyll and within cells of both palisade and spongy tissue ( Fig. 3D) As shown in Fig. 3E, NADI staining revealed the presence of a thin NADI-positive layer in correspondence of the leaf cuticle and of aggregates distributed both in the epidermis and in the mesophyll where they were found close to the xylem vessels. NADI-positive trichomes could be observed mainly on the abaxial epidermis (Fig. 3F) Sudan III-IV staining revealed the presence of neutral lipids in the leaf cuticle and in the glandular trichomes which appeared reddish (Fig. 3G) The suberin and cutin components resulted positively stained with Sudan III-IV, appearing brownish. Fluorol Yellow 088 stained the hydrophobic lipid layer (i.e., cuticle) as well as several lipid aggregates in the epidermis and mesophyll (Fig. 4H) Fluorol Yellow 088-positive material was present also in the glandular trichomes (Fig. 3I).
The PAS reaction revealed the presence of polysaccharide material in the abaxial epidermis at the level of the walls and in the glandular trichomes, which showed an intense pink colour (Fig. 4A, B) Leaf cross-sections stained with FeCl3 did not show any positive reaction, suggesting the absence of polyphenolic compounds (Fig. 4C) The Wagner reaction stained positively the epidermal cells (Fig. 4D, E) and some idioblasts in the upper hypodermis (Fig. 4F), suggesting the presence of alkaloids. A limited number of aggregates were also found in trichome basal cells (Fig. 4G)

Stem anatomy
Piper malgassicum stem showed an epidermis consisting of an outer epidermal layer and an inner hypodermal layer. The outer epidermal cells were tangentially elongated and covered with a thick cuticle (Fig. 5H) Compared to the outer epidermal cells, the hypodermal cells were prismatic and spherical. Beneath the hypodermis, the cortex formed a continuous ring. In the cortex, a 4-5 layered angular collenchyma was present ( Fig. 4G) The vascular system showed isolated vascular bundles arranged in two concentric rings. Each ring of cortical bundles was formed, on average, by 7-9 vascular bundles. A smaller ring of 4-5 (on average) medullary bundles was embedded in the parenchyma of the central cylinder. The medullary bundles were larger than the cortical bundles. In general, bundles of both rings showed phloem and xylem vessels separated by fascicular cambium. Phloem cells were at the external periphery of the bundle. The central part of the central cylinder was formed by parenchyma cells with thin walls and an open space or lacuna (no pith cells) was often observed right in the middle (Fig. 4G) LM observation using a birefringence filter showed an abundance of CaOx crystals in the stem hypodermis (Fig. 5A) as well as in the leaf. In particular, CaOx accumulated abundantly in the hypodermis cells (Fig. 5B) Sudan III-IV positively stained the cuticle covering the external epidermis (Fig. 5C) and lipid droplets occurring in the lumen of the phloem within the vascular bundles ( Fig. 5D) As shown in Fig. 5E, cellulose components were present in the wall of hypodermis cells. Cell walls reacting positively to calcofluor staining were observed in the cortex and the phloem of both cortical and medullar vascular bundles (Fig. 5F) Due to their lignified cell walls, xylem vessels, stained positive to phloroglucinol, but were negative to calcofluor staining (Fig. 5G, H)

Anther anatomy
Piper malgassicum anthers were composed of two locules, joined together by connective tissue. The pollen grains were surrounded by the tapetum and further three cell layers, the epidermis being the most external one (Fig. 6A, B) The anther wall consisted of 2-3 layers of cells, later dehiscing at maturity (Fig. 6A) The tapetum was formed by a monolayered ring of cells, even though, in some points, two cells could be observed (Fig. 6B) Two layers of cells, the middle layer inside and the endothecium outside, just beneath the epidermis, separated the tapetum from the epidermis (Fig. 6B) The microspores in the locule were surrounded by the tapetum, some of whose cells had a higher electron density than the others (Fig. 6C) Small electron-dense particles (orbicles) were lined along the plasmalemma of tapetal cells, while some larger masses of the same electron density as the orbicles could be observed in the locule and on the microspores (Fig. 6C) Along the anther filaments some trichomes were present. Their cytoplasm contained some plastids with large electron-dense bodies (Fig. 6D) Starch grains (black arrows) were also observed inside these plastids (Fig. 6E) In the epidermis electron-dense bodies were observed in the vacuoles, while some plastids with starch grains were present in the cytoplasm (Fig. 6F) Large medium electrondense granular bodies could be found between the vacuole and the external plasma membrane (Fig. 6F) The electron-dense masses in the vacuoles occurred as simple bodies (Fig. 6F), or could be surrounded by a more electron transparent crown (Fig. 6G) In some cells, the electron-dense bodies occupied most of the vacuole (Fig. 6H), while some plastids appeared to host bodies of lower electron density as compared with those of the vacuole (Fig. 6H) The electron-dense bodies surrounded by a less electron-dense crown apparently entered the tonoplast coming from large cisternae in the cytoplasm surrounding the vacuole (Fig. 6G) The medium electron-dense bodies appeared to be surrounded by a membrane inside some plastids that were very often observed close to mitochondria (Fig. 7) The female inflorescence of P. malgassicum is a spike; in our sample, it was 3-8 cm long, with a 1-2 cm long peduncle bearing small sessile flowers on a thin axis. The fleshy ovary was surmounted by a two-branched stigma and contained a single ovule (Fig. 8A) Some resin ducts were observed in the ovary while the stigmas showed papillae in the distal portion ( Figure 8B) Ovary cells located in proximity of the ovule showed CaOx crystal deposits (Fig. 8C) The spike axis showed two concentric circles of vascular bundles, the external ones were smaller in comparison to the most internal bundles and a lacuna was observed at the centre (Fig. 8D) The phloem elements contained material that stained positive with toluidine blue (Fig. 8E)

Discussion
The observations of P. malgassicum herbarium samples showed the typical morphological features of the species and its differences in comparison to the other phylogenetically related species, such as P. tsarasotrae and P guineense. P. malgassicum leaves were Figure 6. P. malgassicum anther A Semithin section stained with toluidine blue showing the shape and structure of the anther. Anther wall (here already dehiscent) formed by 2-3 layers of cells B semithin section showing the microspores surrounded by the tapetum C microspores are in the locule surrounded by the tapetum. Small electron-dense particles (orbicles) are lined along the tapetal cells plasma membrane, while some larger masses can be observed in the locule (black arrow) D anther trichome. Some plastids contain large electron-dense bodies E detail of D The large electron-dense bodies inside the plastid occur together with starch grains F anther epidermis: electron-dense bodies are present in the vacuoles. Some plastids are present with stored starch. Large medium electron dense granular bodies can be found between the vacuole and the external plasma membrane G in a preliminary stage, the electron-dense masses in the vacuole are surrounded by a more electron transparent layer H at a successive stage, the electrondense bodies occupy a large part of the vacuolar volume. Ga: microspores/male gametophyte; L: locule; sp: tapetum/sporophytic tissue; ep: epidermis; end: endothecium, ml: middle layer; st: secretory tapetum; Mcs: microspore; EDB: electron-dense bodies; Mt: mitochondria; Pa: protein aggregate; Ep: elaioplast; yLPT: young lipotubule: Lpt: lipotubule; V: vacuole; Td: terpenic droplets.  hypostomatic as in other Piper species, such as P. aduncum Vell., P. cernuum Vell., P. dilatatum Rich, P. gaudichaudianum Kunth, P. glabratum Kunth, P. lindbergii C. DC., P. solmsianum C. DC., and P. umbellatum Jacq. (Gogosz et al. 2012;Raman et al. 2012;Silva et al. 2017;Santos et al. 2018) However, amphihypostomatic leaves were found in P. sarmentosum Roxb. (Raman et al. 2012) and amphistomatic in P. hispidinervum C. DC (Gogosz et al. 2012) Concerning the anatomical aspects of the leaf, the microscopical analysis revealed how trichomes were not the prevailing secretory structures, but other specialized cells in the hypodermis, called idioblasts, had this function. These cells accumulated lipid-terpenoid aggregates or alkaloids. The general function of these substances and their anatomical localization lead to the hypothesis that their function in P. malgassicum is related to the limitation of infections by fungi and bacteria thanks to terpenoids and to the anti-herbivore activity of alkaloids. Most authors studying the genus Piper from a phytochemical point of view focussed their attention on the fruit, since it is the most relevant part of the plant for economical purposes. Our observations on the leaves are in agreement with other authors who showed these phytochemical features in other species of the Piper genus (Marinho et al. 2011;Pires Jacinto et al. 2018;Silva et al. 2017;Bertocco et al. 2017) From a phytochemical point of view, P. malgassicum is closely related to P. nigrum (Palchetti et al. , 2020 and used traditionally in the same way as a spice. Regarding the stem, P. malgassicum showed the typical features of Piperaceae family anatomy according to Isnard et al. (2012), but some peculiarities of this species were revealed. The stem showed an angular collenchyma under the epidermal layer organized in smaller areas as compared with other closely-related species of this genus. Probably, the low quantity of collenchyma was due to the young age of the observed P. malgassicum stem, which was sectioned after one month of growth from seed. In comparison to P. malgassicum, P. tsarasotrae, and P. guineense showed a higher number of cortical and medullary bundles (Ilodibia et al. 2016;Palchetti et al. 2018) Like P. tsarasotrae and P. guineense, P. malgassicum has larger medullary bundles than cortical vascular ones. Interestingly, P. malgassicum, like P. guineense and P. tsarasotrae, did not exhibit the mucilage canals observed in other Piper species (Ilodibia et al. 2016;Palchetti et al. 2018) A particular anatomical aspect of P. malgassicum was the abundance of CaOx crystals both in the leaves and in the stem. In leaves, these aggregates prevail close to the central rib and in the cells of the spongy mesophyll, while in the stem they were found close to the hypodermis. They were abundant also inside the ovary. Such crystals were also observed in the distantly related P. callosum ) and represent a defence against herbivores due to their hardness and spikiness. Moreover, they can provide further mechanical support for the plant (Maugini et al. 2014) The anthers of P. malgassicum displayed the typical characteristics of Magnoliids, in particular, those of the order Piperales (Funes and Randall 2001) In accord with previous studies on others species of Piper (Schnarf 1931;Funes and Randall 2001;Valentin-Silva et al. 2015), the anthers of P. malgassicum have a secretory tapetum surrounding the microspores. Some of the tapetal cells showed signs of cytoplasmic and nuclear condensation already at the stage in which microspores are free in the locule. This is evidence of programmed cell death Papini 2018) and in P. malgassicum it appears to occur earlier than in most other angiosperms, where it occurs normally after the first pollen mitotic division (Papini et al. 1999;Varnier et al. 2005;Milocani et al. 2006) LM observations revealed dark globules in the anther epidermis. These organelles with a circular shape could be classified as elaioplasts. The presence of elaioplasts and lipid droplets in anthers is well known (Wu et al. 1999;Suzuki et al. 2013;Quilichini et al. 2014), but mainly within tapetum cells, while in this case they were located in the epidermis. Furthermore, not all the observed dark globules seem to have the typical elaioplast structure. Some of them showed a shape referable to another kind of organelle called lipotubules, according to Kwiatkowska et al. (2012) The histochemical localization of terpenoids and alkaloids in the leaf epidermis was checked by TEM. Results suggest that the terpenoids accumulated in this tissue involved the activity of plastids, whose electron-dense contents appeared to occupy most of the plastid volume. The tentative identification of the lipid droplets as terpenecontaining structures was done on the basis of images from published reports, such as Gersbach (2002, particularly Fig. 7B) and Fahn (1988) The spatial association between plastids and mitochondria may be linked to the functional connection between mitochondrial production of isoprenoids and plastids for the synthesis of terpenoids, namely the 2-C-methyl-D-erythritol-4-phosphate (MEP) pathway observed by Ahn and Pai (2008) in Nicotiana benthamiana plastids.
Wagner positivity indicated the presence of alkaloids that can be correlated with the TEM images showing electron-dense bodies found in the vacuole. We could exclude the possible polyphenolic nature of the bodies thanks to the negativity to FeCl3 staining. The images showed that the alkaloids enter into the vacuoles apparently from membrane cisternae probably produced by the endoplasmic reticulum, first with an electron-dense core and a less electron-dense crown. Other images of the vacuole show almost completely electron-dense bodies that are possibly to be referred to a later stage of alkaloids accumulation . Electron-dense precipitates in the vacuoles were interpreted as alkaloids also in Cataranthus roseus by Neumann et al (1983), even if in this case the electron-dense bodies were more irregular in shape. Also Ferreira et al. (1998) observed electron-dense bodies in the vavuoles of Erythroxylum leaf, interpreting the images (particularly fig. 7 in Ferreira et al. 1998) as accumulation of alkaloids around a more osmiophilic phenolic core. This interpretation may explain the more electron-dense core of the roundish bodies observed in the vacuol of P. malgassicum (our Figure 6G) The observed NADI positivity could be linked to the production of medium electron-dense material in the plastids. We observed first organelles still containing starch grains (hence confirming their plastidial identity) together with electron-dense masses and, in other cells, possibly at a more advanced stage of development, the enlargement of the electron-dense bodies apparently formed from lipids derived from the thylakoids. Overall, the analysis of P. malgassicum has highlighted the presence of peculiar secretory structures in the leaf, thus confirming the aromatic nature of the species and high specialization in the biosynthesis of terpenic substances. However, the abundant accumulation of lipid globules, alkaloids, and CaOx crystals also points to particular bio-functional strategies of defence, support, and attraction of mutualistic organisms.
In conclusion, our results show that P. malgassicum produces secondary metabolites (terpenoids and alkaloids) with defence functions, particularly in the leaf epidermal cells (including trichomes) and in hypodermal idioblasts. This is the first report on the histochemistry of P. malgassicum and the first one on the histochemistry of the epidermis in this genus.