Mucilaginous Secretions in the Xylem and Leaf Apoplast of the Swamp Palm Mauritia Flexuosa L.F

Microscopy and Microanalysis (2020), 1–13 doi:10.1017/S1431927620001543

Micrographia

Mucilaginous Secretions in the Xylem and Leaf Apoplast of the Swamp Palm Mauritia flexuosa L.f. (Arecaceae)

Alessandra Flávia Silveira1, Maria Olívia Mercadante-Simões1* , Leonardo Monteiro Ribeiro2, Yule Roberta Ferreira Nunes3, Lucienir Pains Duarte4, Ivana Silva Lula4, Mariana Guerra de Aguilar4 and Grasiely Faria de Sousa4 1Plant Anatomy Laboratory, General Biology Department, State University of Montes Claros, Montes Claros 39401-089, Brazil; 2Plant Micropropagation Laboratory, General Biology Department, State University of Montes Claros, Montes Claros 39401-089, Brazil; 3Plant Ecology Laboratory, General Biology Department, State University of Montes Claros, Montes Claros 39401-089, Brazil and 4Medicinal Plants Study Center, Chemistry Department, Federal University of Minas Gerais, Belo Horizonte 31270-901, Brazil

Abstract Mauritia flexuosa palms inhabit wetland environments in the dry, seasonal Brazilian savanna (Cerrado) and produce mucilaginous secretions in the stem and petiole that have a medicinal value. The present study sought to characterize the chemical natures of those secretions and to describe the anatomical structures involved in their synthesis. Chemical analyzes of the secretions, anatomical, histochemical analyses, and electron microscopy studies were performed on the roots, stipes, petioles, and leaf blades. Stipe and petiole secretions are similar, and rich in cell wall polysaccharides and pectic compounds such as rhamnose, arabinose, xylose, mannose, galactose, and glucose, which are hydrophilic largely due to their hydroxyl and carboxylate groups. Mucilaginous secretions accumulate in the lumens of vessel elements and sclerenchyma fibers of the root, stipe, petiole, and foliar veins; their synthesis involves cell wall loosening and the activities of dictyosomes. The outer faces of the cell walls of the parenchyma tissue in the mesophyll expand to form pockets that rupture and release pectocellulose substances into the intercellular spaces. The presence of mucilage in the xylem, extending from the roots to the leaf veins and continuous with the leaf apoplast, and sub-stomatal chambers suggest a strategy for plant water economy. Key words: arabinose, pectin, plant mucilage, rhamnose, sclerenchymatic fiber, vessel element (Received 18 June 2019; revised 8 April 2020; accepted 27 April 2020)

Introduction An abundant gelatinous secretion exudes from the stipe of M. flexuosa when it is perforated and is harvested by traditional pop- Mauritia flexuosa L.f. (locally “buriti” or “aguaje”) is the most ulations to produce a fermented beverage—buriti wine (Dias & abundant palm tree in Brazil, although it occurs only in hyperhu- Laureano, 2000). The wines produced by the fermented sap of mid habitats and wetlands (Holm et al., 2008; Lorenzi et al., the petioles and floral axes of African and Indian palm trees are 2010). The species originated in the Amazonian rainforest and, used as a beverage and have potential for use as biofuels together with Mauritiella armata (Calamoideae, Mauritiinae), rep- (Tamunaidu et al., 2013; Bi et al., 2016). Despite the importance resent the only swamp palms to adapt to the seasonal Brazilian of those M. flexuosa secretions, there are no studies examining savanna biome with its prolonged dry periods (Lima et al., 2014; their synthesis and distribution, or ecological functions that Reis et al., 2017). M. flexuosa is considered a key species in the could contribute to our botanical knowledge of those palms or Brazilian savanna wetlands (known as “Veredas”), providing food to the development of technologies for their sustainable use. for the local fauna and contributing to the preservation of springs The present work was therefore designed to study the chemi- and other surface waters (Porto et al., 2017). The species provides cal, anatomical, and ultrastructural aspects of the gelatinous secre- important natural resources for traditional populations, as its fruit tions that exude from the stipe and petiole of M. flexuosa, discuss pulp is used in cooking and several plant parts furnish raw mate- their ecological roles, and to respond to the following questions: rials for construction and crafts (Dias & Laureano, 2000; Lorenzi What is the chemical nature of those secretions? Where do they et al., 2010; Virapongse, 2017). Oil extracted from the fruit meso- accumulate in the plant? What are the synthesis and secretion carp is of high quality and valued by pharmaceutical and cosmetic mechanisms? industries—which has stimulated interest in its domestication and agroindustrial production (Koolen et al., 2013). Materials and Methods

*Author for correspondence: Mercadante-Simões, E-mail: [email protected]. Secretion Collections Cite this article: Silveira AF, Mercadante-Simões MO, Ribeiro LM, Nunes YRF, Duarte LP, Lula IS, de Aguilar MG, de Sousa GF (2020) Mucilaginous Secretions in Stipe and petiole secretions were collected from three adult the Xylem and Leaf Apoplast of the Swamp Palm Mauritia flexuosa L.f. (Arecaceae). individuals of M. flexuosa growing in the Pandeiros River Microsc Microanal. doi:10.1017/S1431927620001543 Environmental Protection Area in the Brazilian savanna in the

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Fig. 1. Adult individuals of Mauritia flexuosa L. f. (Arecaceae) and mucilaginous secretions. (a) A wetland area of “Vereda” in the Brazilian Savanna Biome. (b,c) Stipe. Secretion (arrow) exuding from perforations. (d,e) Petiole. Secretion (arrow) exuding from a sectioned petiole. st, stipe; pe, petiole.

municipality of Bonito de Minas, northern Minas Gerais State, acid, and the solution was dried at room temperature. The boric Brazil (15°13′48′′S × 44°54′92′′W) (Fig. 1a). The secretions acid was evaporated in the form of trimethyl borate through suc- (Figs. 1b, 1c) were obtained by perforating the stipes (at breast cessive methanol washes, and the alditols formed were acetylated – height) with a hand-held boring tool previously disinfected with in a reaction with 1.0 mL of an Ac2O pyridine mixture (1:1, v/v) 95% alcohol. Petiole secretions (Figs. 1d, 1e) were obtained by for 12 h, with constant stirring at room temperature. Ice was then natural exudation after cutting the leaves. All of the secretions added to the solution and the resulting alditol acetates were were collected in sterile amber glass bottles and immediately extracted with dichloromethane (5 mL). The organic phase was frozen to −20°C. repeatedly washed with 1 M HCl and distilled water. The alditol μ acetates (2.0 L, 2% CHCl3 solution) were analyzed using GC–FID in a HP 7820A (Agilent Technologies, Santa Clara, Neutral Monosaccharide Compositions CA, USA) chromatograph fitted with a BP10 capillary column Stipe and petiole secretions were lyophilized and submitted to (25 m × 0.22 mm i.d.), detector temperature 270°C, injection in total acid hydrolysis. The lyophilized samples (∼5mg) were a split mode (1:20) at 270°C, H2 as the carrier gas (3.0 mL/min), then diluted in 10.0 mL of 2 M TFA with constant stirring and initial temperature 200°C (3 min), then increasing from 200 to refluxed at 100°C for 6 h. After forced-air evaporation, the hydro- 260°C (4°C/min). Glucose, galactose, xylose, mannose, rhamnose, lyzed samples were resuspended in 1.0 mL of distilled water with and arabinose standards were submitted to reduction and 7.5 mg of NaBH4; two drops of basic aqueous solution (1% v/v, acetylation reactions, as previously described. The monosaccha- NH4OH) were then added at room temperature with stirring ride compositions of both secretions were determined using the for 12 h. Excess NaBH4 was eliminated by adding glacial acetic methodology described by Cantu-Jungles et al. (2015), with

some modifications (increase of reaction times, evaporation of the 1978) and analyzed in a Tecnai G2-20-SuperTwin transmission reaction mixture after hydrolysis, addition of 1 mL of water after electron microscope (FEI Company, Eindhoven, Netherlands) at NaBH4 reduction, removal of excess NaBH4 by adding glacial ace- 150 kV. tic acid and evaporation of boric acid in the form of trimethyl Root, petiole, and leaf samples for scanning electron micros- borate through successive methanol washes, addition of ice to copy (SEM) studies were obtained by cryofracture in liquid N2, the solution and alditol acetate extraction with dichloromethane, fixed in 2.5% glutaraldehyde (0.1 M phosphate buffer, pH 7.2), – – and use of GC FID instead of GC MS in the quantification of dehydrated in an ethyl series to the critical point with CO2, coated “alditol acetates”). with a gold layer in a Bal-Tec-MD20 metallizer (Leica Microsystems, Heidelberg, Germany) (Robards, 1978), and analyzed using a Quanta 200 scanning electron microscope (FEI Infrared and Nuclear Magnetic Resonance Company, Eindhoven, Netherlands) at 12–20 kV. To detect the Fourier-transformed infrared (FT-IR) spectra (∼1% KBr solution) presence of calcium, the samples were analyzed using X-ray of lyophilized stipe and petiole secretions were obtained using a energy dispersive spectrometry (XEDS) at 200 kW. FTIR 408 spectrometer (Shimadzu Corporation, Kyoto, Japan). The 1D/2D nuclear magnetic resonance (NMR) spectra were recorded on a 400 Avance III HD spectrometer (Bruker Results Corporation, Dresden, Germany) at 9.4 T, observing 1Hat Phytochemical Composition 400.18 MHz. Chemical shifts were expressed as δ, using the reso- nances of HDO at δ 4.69 as the internal reference; all pulse pro- Phytochemical tests of stipe and petiole secretions indicated the grams were supplied by Bruker Corporation (Dresden, Germany). presence of acid and neutral polysaccharides, but not starch, pro- In order to obtain the spectra, 0.7 mL of D2O was added to tein, neutral or acidic lipids, or alkaloids. 100 mg of the lyophilized secretions and filtered into 5 mm NMR tubes. Monosaccharide Composition Plant Material Stipe and petiole secretions are composed of the same neutral monosaccharide units, although in different quantities The plant materials used in the structural, histochemical, and (Table 1). Galactose was the most abundant neutral monosaccha- ultra-structural analyses consisted of fragments of roots, stipes, ride (36.9%) in stipe secretions, followed by rhamnose, arabinose, petioles, and leaf blade regions obtained from M. flexuosa seed- and glucose (21.7, 18.1 and 16.9%, respectively). Xylose (4.3%) lings (~40 cm tall) produced under nursery conditions (Silva and mannose (2.1%) were also present, but in lower proportions. et al., 2014) from seeds collected from adult individuals that Mannose (54.1%) was the predominant neutral monosaccharide had provided the secretions used in the chemical analyses. in petiole secretions, followed by glucose (37.6%) and xylose Testimonial material was deposited in the Montes Claros (4.3%). Rhamnose, arabinose, and galactose were present at con- Herbarium—HMC, Department of General Biology, State centrations below 2%. University of Montes Claros, under number 5777.

Structural and Histochemical Analyses Infrared and NMR Spectroscopy Fragments of the plant material were fixed in Karnovsky’s solu- The FT-IR spectra (Figs. 2a, 2b) of lyophilized stipe and petiole tion (Karnovsky, 1965) for 24 h, dehydrated in an alcoholic series, secretions showed similar profiles, with the presence of alcohol − and embedded in methacrylate resin (Leica Microsystems, groups (O–H) in polymeric interactions (3,000–3,500 cm 1) and Heidelberg, Germany). Transversal and longitudinal sections carbonyls from carboxylate anions (derived from galacturonic − (5 μm) were obtained using a rotary microtome (Atago, Tokyo, acid [broad bands between 1,600 and 1,650 cm 1]). Bands at − − Japan), stained (blue for cellulose, and pink for acid polysaccha- 1,740 cm 1 (stipe, Fig. 2a) and 1,738 cm 1 (petiole, Fig. 2b) rides) with toluidine blue (pH 4.7) (O’Brien et al., 1964, modified, were associated with esterified glucuronic acid residues. These Rodrigues et al., 2018, Ribeiro and Leitão, 2019, Souza et al., 2020) data support the findings in the phytochemistry study, since the and lugol (dark blue for starch) (Johansen, 1940, Royo et al., functional groups detected are present in acidic and neutral 2015), and mounted on glass slides with acrylic resin (Itacril, polysaccharides. Itaquaquecetuba, Brazil). Photographic documentation was per- The heteronuclear multiple bond correlation (HMBC) spectra formed using an AxioCam ICC3 digital camera coupled to an of stipe secretions showed signals at δc 177.8 (Fig. 3a); petiole AI Lab photomicroscope (Zeiss, Oberkochen, Germany). secretions showed signals at δc 172.7 and 173.4 (Fig. 3b) that were associated with the esterified or non-esterified C-6 carboxylic groups of galacturonic acid (Marcon et al., 2005). Electron Microscopy Heteronuclear single quantum coherence spectroscopy Root, stipe, petiole, and leaf samples for transmission electron (HSQC) of the stipe secretion showed that a proton signal at δΗ ’ δ microscopy (TEM) studies were fixed in Karnovsky s solution 1.19 correlated with the C 15.46 carbon signal (Fig. 4a) and δ (Karnovsky, 1965), for 24 h, post-fixed in 1% osmium tetroxide attributed to the H-6/C-6 of rhamnose. The correlations at H/C in 0.1 M phosphate buffer (pH 7.2), dehydrated in an acetone 4.47/102.6, 4.37/103.7, and 5.04/107.9 were characteristic of the series, infiltrated with Araldite resin (Leica Microsystems, H-1/C-1 of galacturonic acid, β-xylopyranosyl, and arabinofura- Heidelberg, Germany), and sectioned using a n UC6 ultramicro- nosyl, respectively (Marcon et al., 2005). The H-1/C-1 correla- δ tome (Leica Microsystems, Heidelberg, Germany); 50 nm sections tions at H/C 4.42/102.4, 4.56/95.9, and 5.33/92.2 of the HSQC were then contrasted with uranyl acetate and lead citrate (Roland, spectrum of the petiole secretions (Fig. 4b) indicated the presence

Table 1. Neutral Monosaccharide Compositions of Stipe and Petiole Secretions of M. flexuosa L.f. (Arecaceae).

Monosaccharide (mol %)

Secretion Rhamnose Arabinose Xylose Mannose Galactose Glucose

Stipe 21.7 18.1 4.3 2.1 36.9 16.9 Petiole 1.9 1.3 4.3 54.1 0.7 37.6

Fig. 2. FT-IR spectra (∼1% KBr solution) of Mauritia flexuosa L.f. (Arecaceae) secretions. (a,b) Both stipe and petiole profiles indicate the presence of alcohol groups − − in polymeric interactions (3,000–3,500 cm 1) and carbonyls from carboxylate anions derived from galacturonic acid (broad band between 1,600 and 1,650 cm 1). − − Bands of the stipe at 1,740 cm 1 (a) and of petiole at 1,738 cm 1 (b) associated with esterified glucuronic acid residues.

Fig. 3. HMBC spectra of Mauritia flexuosa L.f. (Arecaceae) secretions. (a) Stipe—δc 177.8. (b) Petiole—172.7 and 173.4 associated with the esterified or non-esterified C-6 carboxylic groups of galacturonic acid.

Fig. 4. HSQC spectrum of Mauritia flexuosa L.f. (Arecaceae) secretions. (a) Stipe: δΗ 1.19 correlated with the δC 15.46 carbon signal attributed to the H-6/C-6 of rhamnose. δH/C 4.47/102.6, 4.37/103.7, and 5.04/107.9 characteristic of the H-1/C-1 of galacturonic acid, β-xylopyranosyl, and arabinofuranosyl, respectively. (b) Petiole: δH/C 4.42/102.4, 4.56/95.9, and 5.33/92.2 characteristic of the H-1/C-1 of galacturonic acid, glucose/galactose, and sucrose, respectively. Downloaded from https://www.cambridge.org/core. East Carolina University, on 04 Jun 2020 at 11:49:55, subject to the Cambridge Core terms of use, available at https://www.cambridge.org/core/terms . https://doi.org/10.1017/S1431927620001543 6 Alessandra Flávia Silveira et al.

Fig. 5. Root of Mauritia flexuosa L.f. (Arecaceae). (a) Epidermis and outer cortex showing cells with lignified walls and phenolic contents. Median cortex formed by schizolysigenous aerenchyma. Compact and phenolic inner cortex, and endoderm with numerous passage cells. Uniseriate pericycle, polyarch vascular system, and lignified pith. (b) Vessel elements and fibers with mucilage. ae, schizolysigenous aerenchyma; en, endodermis; ep, epidermis; fi, fiber; ic, inner cortex; mu, mucilage; oc, outer cortex; pe, uniseriate pericycle; ph, pith; po, polyarch; pi, pit; ve, vessel.

of galacturonic acid, glucose/galactose, and sucrose, respectively composed of from six to ten tangential layers of cells of varying (Bruyn & Loo, 1991; Marcon et al., 2005). shapes organized radially formed by separation and cellular lysis — giving rise to a schizolysigenous aerenchyma. The inner cortex is composed of from two to three layers of compact isodiametric Anatomy, Histochemistry, and Ultrastructure cells containing phenolic compounds. The endoderm consists of a Roots single layer, with numerous passage cells. The pericycle is uniseri- The epidermal cells of M. flexuosa roots have lignified walls and ate and delimits the polyarch vascular system; the medulla is lig- phenolic contents. The outer region of the cortex is composed nified (Fig. 5a). Vessel elements and sclerenchyma fibers of from four to six layers of compactly arranged isodiametric accumulate mucilage, which is concentrated in pit regions (Figs. cells with lignified walls. The median region of the cortex is 5a, 5b). Vessel element mucilage (Figs. 6a–6d) is composed of

Fig. 6. Root of Mauritia flexuosa L.f. (Arecaceae)—TEM. (a–d) Mucilage accumulated in vessel elements. (a,b) Differentiating loose wall metaxylem and fibrillar material accumulated in the vessel element. (a–d) Fibrillar material from dictyosomes. di, dictyosomes; lw, loose wall; fi, fibrillar material; mx, metaxylem.

Fig. 7. Stipe of Mauritia flexuosa L.f. (Arecaceae). (a) Calibrous vascular bundles, phenolic compounds, and starch grains (box). Large diameter vessel elements. (b) Vessel elements containing mucilage—acidic polysaccharides/pectic compounds and neutral polysaccharides/cellulose. po, acidic polysaccharides/pectic compounds (pink) and neutral polysaccharides/cellulose (blue); ph, phenolic compounds; st, starch grains; vb, vascular bundles; ve, vessel elements.

the loose walls of the differentiating metaxylem (Figs. 6a, 6b) and Figs. 1–3, 7b). The differentiating vessel elements have loose the fibrillar material from the dictyosomes (Figs. 6a–6d). walls—neutral polysaccharides/cellulose and accumulate fibrillar materials—acid polysaccharides—in vacuoles associated with dic- – – Stipe tyosome (Figs. 8a 8d) as in the root (Figs. 5a, 5b, 6a 6d). The stipe accumulates phenolic compounds such as starch, and vascular bundles show large diameters and are scattered through- Petiole out its cross section. The vessel elements have large diameters The epidermal cells of the petiole have thick outer periclinal walls (Fig. 7a) and accumulate mucilage—acidic polysaccharides/pectic and large nuclei. The cortex is composed of from four to five lay- compounds, and neutral polysaccharides—cellulose (Table 1; ers of compactly arranged subepidermal cells with thin walls that

Fig. 8. Stipe of Mauritia flexuosa L.f. (Arecaceae)—TEM. (a–d) Differentiating vessel elements, with loose walls—neutral polysaccharides/cellulose and fibrillar material—acid polysaccharides/pectic compounds associated with dictyossome (b is a magnification of the box in c). di, dictyosome; er, endoplasmic reticulum; fi, fibrillar material; lo, loose wall; mi, mitochondria; nu, nucleus; va, vacuole; ve, vessel element; wa, wall; wt, wall thickening.

Fig. 9. Petiole of Mauritia flexuosa L.f. (Arecaceae). (a) Epidermis with cells with thick outer periclinal walls and voluminous nuclei. Cortex with tightly arranged subepidermal layers enveloping bundles of fibers, internal layers with large intercellular spaces, and calibrous vascular bundles flanked by conspicuous sclerenchymatous sheaths. (b–d) Vessel elements with pectic walls and scaliform thickenings. Fibers and vessel elements with acidic polysaccharides. Arrow, acidic polysaccharides; co, cortex; ep, epidermis; fb, fiber bundles; is, intercellular spaces; pw, pectic wall; ss, sclerenchymatous sheath; sc, scaliform thickening; vb, vascular bundle; ve, vessel elements.

surround longitudinally equidistant fiber bundles. Internally, the anchoring the fibrillar material (Table 1; Figs. 1–3, 10a, 10b, cortex has voluminous intercellular spaces and large, isodiametric, 11a, 11b). The fibrillar material is rich in calcium, suggesting thin-walled cells that often contain phenolic substances. The the presence of calcium pectates—acid polysaccharides (Table 1; numerous vascular bundles have large diameters. The phloem Figs. 1–3, 11c, 11d). has large sieve tube elements and is flanked by a conspicuous sclerenchymatous sheath (Fig. 9a). The vessel elements have pec- Leaf Blade tic walls with scalariform thickenings and sclerenchyma fibers The leaf blade is covered by a uniseriate epidermis composed of accumulate acid polysaccharides (Figs. 9b–9d). The vessel ele- cells with lignified walls that accumulate phenolic compounds. ments and differentiating sclerenchymatous fibers accumulate Adjacent to both faces of the epidermis are numerous regularly fibrillar material—acid polysaccharides/pectic compounds— spaced bundles of sclerenchyma fibers. The palisade parenchyma derived from dictyosome vesicles. The cell walls are loosely is composed of from three to four layers of slightly elongated cells. arranged and eroded, and project into the cellular lumen, Phenolic idioblasts occur throughout the bilateral mesophyll. The

Fig. 10. Petiole of Mauritia flexuosa L.f. (Arecaceae)—TEM. (a) Vessel element and differentiating sclerenchymatous fibers with fibrillar material—acidic polysaccharides in the vacuole. (b) Cell wall with a loose arrangement projecting into the cell lumen and anchoring fibrillar material—acidic polysaccharides. fi, sclerenchymatous fiber; fm, fibrillar material; lw, loose wall; pi, pit; va, vacuole; ve, vessel element; wt, wall thickening.

Fig. 11. Petiole of Mauritia flexuosa L.f. (Arecaceae). (a,b) In SEM, fibrillar material—acidic polysaccharides/pectic compounds. Neutral polysaccharides/cellulose. Loose wall erosion (c,d). XEDS. Calcium concentrations in the lumens of vessel elements, suggesting the presence of calcium pectates in the fibrillar material— acidic polysaccharides. Arrow, fibrillar material—acidic polysaccharides/pectic compounds; white arrowheads, loose wall erosion; black arrowheads, neutral polysaccharides/cellulose; asterisk, absence of calcium, loose wall; ve, vessel element.

vascular bundles are immersed in the spongy parenchyma and are pectic polysaccharides, including homogalacturonan and rham- enveloped by a conspicuous sclerenchymatous sheath; the vessel nogalacturonan I and II (Carnachan & Harris, 2000; Sáenz elements accumulate mucilage (Figs. 12a, 12b). The spongy et al., 2004; Gorshkova et al., 2012; Cantu-Jungles et al., 2015). parenchyma is composed of from six to eight layers of cells Both secretions also contain galacturonic acid, a component of with pectic walls with bag-shaped expansions (Fig. 12c). The acidic pectins. These chemical analyses corroborate ultrastructural lower face of the leaf epidermis has stomata with wide sub- evidence of dictyosome activity and the occurrence of cell wall stomatal chambers that accumulate mucilage (Figs. 12c, 12d). loosening. Sucrose was detected (by NMR) in petiole secretions, The cells of the spongy parenchyma show regions of their walls possibly derived from the phloem. with the outer layer slightly degraded, releasing a mesh of fibrillar — — material acid polysaccharides into the intercellular spaces. Hydrophilic Nature of the Secretions Vacuoles accumulate dense material suggesting mucilage. The The secretions demonstrated hydrophilic natures and the capacity plastids contain lipophilic bodies, starch grains, and osmiophilic for water retention, apparently due to the presence of hydroxyl bodies, and are associated with numerous mitochondria (Figs. and carboxylate groups (as indicated by their strong FT-IR – 13a 13d). The rupturing of the outer layers of the cell walls of bands and NMR signals). The residues of esterified glucuronic the spongy parenchyma is associated with the presence of bag- acid are responsible for the gelling character of the pectins shaped expansions and the accumulation of mucilage in the inter- (Yapo & Koffi, 2006). cellular spaces (Figs. 14a–14d).

Accumulation and Synthesis of Mucilage Discussion Accumulation in the Xylem Chemical Natures of the Stipe and Petiole Secretions M. flexuosa accumulates mucilaginous secretions within its vessel Cell Wall Components Give Rise to the Secretions elements and the sclerenchyma fibers of the xylem. The structures The secretions exuded from the stipe and the petiole are mucilag- typically responsible for mucilage accumulation in plants are idi- inous compounds with high hygroscopic affinities derived from oblasts, ducts, trichomes, and colleters (Fahn, 1979). Vessel ele- typical cell wall components. Both secretions are composed of ments and xylem fibers, however, can also accumulate mucilage the same neutral monosaccharide units, although in different pro- and act in regulating water transport (Zimmermann et al., 1994; portions. The presence of xylose and mannose indicate their Arend et al., 2008; Ghanem et al., 2010). The presence of mucilage hemicellulose polysaccharide origins (Cordeiro et al., 2015), and in the xylem of mangrove species was found to increase hydraulic their high levels of glucose may be associated with cellulose. resistance, resulting in an increase in the root-to-leaf pressure gra- The occurrence of rhamnose, arabinose, and galactose suggest dient, therefore reducing water flow rates and transpiration losses

Fig. 12. Leaf blade of Mauritia flexuosa L.f. (Arecaceae). (a,b) Uniseriate epidermis, with cells with lignified walls and phenolic compounds. Sclerenchymatous fiber bundles, adjacent to the epidermis, regularly spaced. Palisade parenchyma with slightly elongated cells. Phenolic idioblasts. Vascular bundles enveloped by sclerenchymatous sheaths. Vessel elements with mucilage accumulations. (c) Spongy parenchyma with cells with pectic walls and bag-shaped expansions. (d) Stomata with mucilage in the sub-stomatal chamber. Arrow, bag-shaped expansions; arrow head, mucilage; ep, epidermis; fb, fiber; pi, phenolic idioblast; pp, palisade parenchyma; sb, sclerenchymatous sheath; sc, sub-stomatal chamber; sp, spongy parenchyma; st, stomata; ve, vessel element.

Fig. 13. Leaf blade of Mauritia flexuosa L.f. (Arecaceae)—TEM. (a–d) Cells of the spongy parenchyma with sections of the outer layers of the cell walls slightly degraded. Microfibril network released into intercellular spaces. Vacuoles with the dense material suggesting mucilage. Plastids containing lipophilic bodies, starch grains, and with numerous associated mitochondria. Arrow, broken wall; fm, fibrillar material; lb, lipophilic body; mi, mitochondria; ob, osmiophilic bodies; pl, plastids; st, starch grain; va, vacuole; wa, wall.

Fig. 14. Leaf blade of Mauritia flexuosa L.f. (Arecaceae)—SEM. (a–d) Broken bag-shaped expansions (arrow) fibrillar material (mucilage) released into the intercellular spaces (asterisk).

(Zimmermann et al., 2001). The rates of water elevation were observed here. We noted large numbers of dictyosomes during found to decrease in tree species of several botanical families vessel element and xylem fiber differentiation in M. flexuosa. due to the presence of mucilage in their xylem (Zimmermann, Dictyosome profusion is a common feature in mucilage- 2001). Mucilage accumulation in the long lived vessel elements producing cells (Lyshede, 1977; Mastroberti & Mariath, 2008; and xylem fibers has been reported even among palm trees growing Lacchia & Carmello-Guerreiro, 2009; Mercadante-Simões & in humid environments, such as Amazonian species of Oenocarpus Paiva, 2013), and those cell structures are also as sites of calcium (Silva & Potiguara, 2009; Gorshkova et al., 2012). Fibers play an pectate synthesis (non-cellulosic plant cell wall components). important role in water movement through pits to the xylem ves- sels, which are normally subjected to high negative pressures Mucilage in the Mesophyll imposed by transpiration. This radial diffusion of the water is facil- We observed a peculiar structure in the mesophyll of M. flexuosa itated by the presence of mucilage adhering to the inner surfaces of that has apparently not yet been reported in palm trees. The cells the fiber walls (Arend et al., 2008), which helps prevent cavitation of the spongy parenchyma exhibit a desquamation of the outer face (the interruption of water flow due to the entry of air into the con- of their cell walls, releasing a mucilaginous fraction into the inter- ducting elements) (Zwieniecki et al., 2001). cellular space that extends into the sub-stomatal chamber. The contribution of the cell wall to mucilage formation inside The Secretion Process the plants during duct formation has been reported in species of The mucilage found in the vessel elements and xylem fibers of M. Anacardiaceae— where the loosening and degradation of the flexuosa originates from the loosening of cellulosic walls and the internal tangential walls of the secretory epithelium lining the activities of dictyosomes during xylem differentiation. Electron lumen has been observed, contributing to the formation of the transmission microscopy images of bean leaves showed the accu- mucilaginous secretion fraction (Fahn & Evert, 1974; Nair et al., mulation of cell wall residues with pectates having a fibrillar 1983; Bhatt & Mohan Ram, 1992; Venkaiah, 1992). Cell walls, aspect in regions not protected by lignification during the autol- despite their apparent rigidity, constitute an extracellular matrix ysis of the protoplasts of tracheal elements (O’ Brien, 1970). The that can be readily modified during tissue development (Barnes gelatinous substances that developed in the integument of mixo- & Anderson, 2017). The formation of mucilage from cell wall spermic species during germination were able to absorb signifi- components is known in seed coats (Mosti et al., 2012) and medi- cant amounts of water. That material was found to be ates seed contact with soil moisture. Considering that the meso- composed of loosely arranged cellulosic walls surrounding and phyll is in close contact with the atmosphere during stomatal anchoring masses of pectic compounds in Lunaria annua opening, mucilage may mediate interactions between the plant (Brassicaceae) (Mosti et al., 2012), very similar to the material and its environment (Zimmermann et al., 2007) such as the

stomatal plug observed in several tropical and subtropical species Author contributions statement. MO Mercadante-Simões conceived and that capture water from the atmosphere (Westhoff et al., 2009). designed research, and performed the ultra-structural analyses. LM Ribeiro The vacuoles of the mesophyll cells of M. flexuosa were also conducted the nursery work and analyzed data. YRF Nunes conducted the observed to be rich in mucilage, a typical characteristic of plant field work and analyzed data. AF Silveira carried out the phytochemical, ana- leaves adapted to xeric environments such as cacti (Mauseth, tomical, and histochemical analyses. LP Duarte, IS Lula, MG Aguilar, and GF Sousa performed the chemical analyses. All of the authors collaborated to the 1980; Monrroy et al., 2017) that rely on water reserves to survive bibliographical revision and writing the text. long periods of drought. 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