Ontogenesis and functions of saxophone stem in Acrocomia aculeata (Arecaceae)

Abstract

Background and Aims The underground saxophone stem systems produced by seedlings of certain palm species show peculiar growth patterns and distinctive morphologies, although little information is available concerning their development and function. We studied the ontogenesis of the saxophone stem in Acrocomia aculeata, an important neotropical oleaginous palm, and sought to experimentally define its function.

Methods Morpho-anatomical evaluations were performed during 240 d on seedlings using traditional methodologies. The tuberous region of the structure was submitted to histochemical tests and evaluated by transmission electron microscopy. The aerial portions of 130 1- to 3-year-old greenhouse plants were removed and their continuous growth capacity was evaluated after 30 d. Severed saxophone stems were also stored at room temperature (average 25 °C) for up to 90 d and then cultured for 60 d to evaluate root and shoot emission.

Key Results The development of the saxophone stem is distinct from other underground systems previously described, and involves three processes: growth and curvature of the cotyledonary petiole, expansion and curvature of the hypocotyl, and expansion of the plumule internodes. The tuberous region stores water and starch, as well as lesser amounts of mucilage and oil. Growth of the aerial portion occurred in 84 % of the separated saxophone stems and in 53 % of the stems held in storage.

Conclusions The saxophone stem represents an important adaptation of A. aculeata to anthropogenically impacted and/or dry environments by promoting the burial of both the shoot meristem and storage reserves, which allows the continuous growth of aerial organs.

INTRODUCTION

In addition to the ecological and economic importance of palms, they are of great botanical interest because of their diverse developmental patterns and adaptive strategies (Tomlinson, 1979, 2006; Dransfield et al., 2008). Unbranched palms undergo an establishment phase (before stipe formation) that is considered critical for plant survival due to the vulnerability of their shoot meristem (Tomlinson, 1990; Tomlinson and Huggett, 2012). The seedlings of genera such as Sabal, Attalea, Syagrus and Acrocomia show a positive geotropic growth obliquely downward into the soil, producing a curved and bulbous underground system known as saxophone stem because of its superficial similarity to the musical instrument (Tomlinson and Esler, 1973; Henderson, 2002; Dransfield et al., 2008). Despite the ecological importance attributed to this structure (McPherson and Williams, 1998; Salm, 2005), there have been no detailed studies focusing on its development and function.

Acrocomia aculeata (the ‘macaúba’ or ‘macaw’ palm) is widely distributed in tropical America (Dransfield et al., 2008), with populations concentrated in the Cerrado (neotropical savanna) biome in Brazil that are usually associated with eutrophic soils and disturbed areas (Motta et al., 2002; Lorenzi et al., 2010). Mature plants grow to approximately 15 m, with high mesocarp and seed oil productivity, and it is considered a species with great potential for biodiesel production in dry tropical regions (Lorenzi et al., 2010; Pires et al., 2013).

Investments in underground organs are common in Cerrado species and are associated with resistance strategies against herbivory, drought and fires (Appezzato-da-Glória et al., 2008; Clarke et al., 2013). There is a considerable diversity of underground systems (reviewed by Appezzato-da-Glória, 2015), although most detailed morpho-anatomical studies have concentrated on dicots and have largely neglected palms. Considering the diversity and peculiarities of Arecaceae seedlings (Henderson, 2006) and their developmental patterns (Henderson, 2002; Tomlinson, 2006), studies of the ontogenesis of saxophone stems can contribute to our understanding of the survival strategies and growth patterns of plants.

We examined the ontogenesis of the saxophone stem in A. aculeata and sought to experimentally determine its functions and to address the following questions. (1) What is the ontogenetic pattern of the saxophone stem and its structure? (2) What are the roles of saxophone stems in the adaptation of this species to its natural environment?

MATERIALS AND METHODS

Collections and preliminary procedures

About 2000 fruits of A. aculeata were collected from 20 individuals in a natural population in the municipality of Montes Claros, Minas Gerais State, Brazil (16 °42′34″S, 43 °52′48″W). The fruits were opened using a manual vice and the seeds removed and sown to germinate in polyethylene containers containing wet vermiculite (Ribeiro et al., 2015). The plantlets were subsequently transferred to 500-mL polyethylene containers with a mixture of clay soil and sand (3 : 1), and then maintained in a greenhouse for 240 d. Seeds/seedlings were removed 0, 1, 5, 10, 15, 20, 30, 45, 60, 120, 180 and 240 d after germination for morphological and anatomical analyses.

Morphology

Twenty seeds/seedlings were morphologically described and photographed using a digital camera (DSC-49, Sony, Tokyo, Japan) after each time period, evaluating the lengths and diameters of the cotyledonary petiole, haustorium, saxophone stem, the most developed root, the aerial portion and the numbers of leaves. The fresh masses of the seeds and seedling structures were determined using an analytical balance (AUX220, Shimadzu, São Paulo, Brazil); the material was subsequently dried in an oven (Q314 M-242, Quimis, São Paulo, Brazil) at 105 °C for 24 h and the dry masses of the materials were determined and their original water contents calculated (Brasil, Ministério da Agricultura, Pecuária e Abastecimento, 2009).

Anatomy

We evaluated the region involved in the formation of the saxophone stem (cotyledonary petiole, expanded ligule, vegetative axis) for up to 60 d. After 120 d of growth, the saxophone stems (length: approx. 20–40 mm) were divided into three sections: basal, median and apical; each section was then separated into three portions (approx. 2 mm thick) in their central, intermediate and peripheral regions. These portions were fixed in Karnovsky’s solution (Karnovsky, 1965), dehydrated in an ethanol series and subsequently embedded in 2-hydroxyethyl-methacrylate (Historresin, Leica Microsystems, Heidelberg, Germany) following Paiva et al. (2011). Longitudinal sections (5 μm thick) were then prepared using a rotary microtome (Atago, Tokyo, Japan) and were stained with ruthenium red (Johansen, 1940) and 0·05 % toluidine blue, pH 4·7 (O’Brien et al., 1964, modified), and mounted in acrylic resin (Itacril, Itaquaquecetuba, São Paulo, Brazil). The sections were then analysed using a light microscope (Lab. AI/AxionCam TPI 3, Zeiss, Jena, Germany).

Histochemistry

For histochemistry, 120- and 240-d-old fragments of the median region of the tuberous portion of the saxophone stem were processed as described above for the anatomical evaluations. Sections 10 μm thick were stained with lugol solution (Jensen, 1962) to detect starch, with xylidine ponceau (Vidal, 1970) and bromophenol blue (Mazia et al., 1953) to detect proteins, with Sudan IV (Pearse, 1980 modified) to detect lipids, and with ruthenium red (Johansen, 1940) to detect acidic polysaccharides. The images were analysed and photographed as previously described.

Ultrastructure

Cross-sections of fragments similar to those used for histochemical analyses (approx. 0·4 mm thick) were made using a razor blade and subsequently fixed in Karnovsky’s solution, pH 7·2 (Karnovsky, 1965), post-fixed in 1 % osmium tetroxide (0·1 m phosphate buffer, pH 7·2), dehydrated in an acetone series, and embedded in Araldite resin (Roland, 1978). Ultrathin sections (50 nm) were contrasted with uranyl acetate and lead citrate (Robards, 1978; Roland, 1978) and examined using a transmission electron microscope (Philips CM 100, Philips/FEI Corporation, Eindhoven, Netherlands).

The continuous growth capacity of the aerial portion and tolerance to storage

The aerial portions of 130 3-year-old greenhouse-grown plants were cut at ground level, and the number of plants that regenerated their aerial portions after 30 d was tallied.

The leaves and roots were removed from the saxophone stems of 600 young plants grown in a greenhouse that were from 1–3 years old and their masses were determined. The separated saxophone stems were then individually packaged in polyethylene bags (300 µm thickness) and stored in an open shed at room temperature (average 25 °C). The saxophone stems were subsequently planted in polyethylene sacks (500 mL capacity) containing moist vermiculite soil after 0, 5, 15, 30, 60 and 90 d of storage and maintained at 30 °C for 30 d and then for an additional 30 d in a greenhouse. We evaluated the percentage of saxophone stems showing shoot and root emission; the dry masses of the plants were then determined after drying at 105 °C.

The experiments followed a randomized block design with six treatments (initial condition and five storage times) and ten repetitions of ten saxophone stems. We took care to use groups of saxophone stems that had approximately the same masses in the different treatments. Analysis of variance was performed, and the means were compared using the Tukey test at a 5 % level of probability.

RESULTS

Morphology

It was possible to identify three developmental phases of the saxophone stem based on morphological criteria: (1) growth and curvature of the cotyledonary petiole, (2) expansion and curvature of the ligule and (3) formation of the tuberous region (Figs 1 and 2).

Fig. 1.

Seeds and seedlings of A. aculeata, indicating (on the left) the development phases of the saxophone stem: (I) growth and curvature of the cotyledonary petiole, (II) expansion and curvature of the ligule and (III) formation of the tuberous region. Longitudinal sections of the seed (A–F) and saxophone stem (K). (A) Embryo inserted within the endosperm of a non-germinated seed; (B) 1 d after germination, showing protrusion of the cotyledonary petiole (arrowhead); (C) 5 d after germination, highlighting the curvature region of the cotyledonary petiole (arrowhead); (D) 10 d after germination; (E) 15 d after germination, showing expansion of the ligule region (arrowhead); (F) 20 d after germination, showing the bending region of the ligule (white arrowhead); (G) 45 d after germination, showing curvature of the base of the saxophone stem (white arrowheads) and the rupture region of the ligule (black arrowhead); (H) 60 d after germination, highlighting the beginning of the formation of the tuberous region (arrow), with upwards growth toward the soil surface – the soil level is indicated by a dashed line; (I) 180 d after germination, showing the base of saxophone stem (white arrow) and the position of the terminal portion of the tuberous region where the senescent primary root is inserted (white arrowhead) – ground level is indicated by a dashed line; (J) 240 d after germination, highlighting the scars of the embryonic leaves (white arrowheads), the remains of the degenerated primary root (white arrow) and the cortical coating (black arrowhead); (K) 240 d after germination, showing the insertion position of the primary root (white arrowhead), and the location of the apical meristem (white arrow). Abbreviations: ar, adventitious root; cp, cotyledonary petiole; ed, endosperm; eo, eophyll; ha, haustorium; le, leaves; li, ligule; me, metaphyll; pr, primary root; s1, first sheath; s2, second sheath; se, seed; sr, secondary root; te, seed coat; tr, tuberous region.

Fig. 2.

Biometric characteristics of the seeds and seedlings of A. aculeata grown in a greenhouse for 240 d. The saxophone stem development phases are separated by dashed lines and indicated by: (I) growth and curvature of the cotyledonary petiole, (II) expansion and curvature of the ligule and (III) formation of the tuberous region. n = 20. Vertical bars indicate standard errors of the means.

Growth and curvature of the cotyledonary petiole (0–10 d)

Acrocomia aculeata seeds are heart-shaped (length: 17·2 ± 1·8 mm; width: 16·0 ± 1·6 mm; thickness: 11·4 ± 1·4 mm; dry mass: 2·0 ± 0·4 g), the seed coat is brown, and the abundant endosperm is whitish with an oily aspect (Fig. 1A). The embryo is linear (length: 4·5 ± 0·4 mm; dry mass: 4·2 ± 0·7 mg) and inserted within the endosperm in the micropilar region. Only the cotyledon of the embryo is visible as a yellowish cylindrical region corresponding to the cotyledonary petiole, and a white, expanded region corresponding to the haustorium located more internally in the seed. Completion of germination is indicated by the elongation and protrusion of the cotyledonary petiole (Figs 1B and 2A). From the first to the tenth day after germination the petiole demonstrates pronounced increases in length, weight and water content (Fig. 2A–C), bending downward sharply in a pattern indicative of positive geotropic growth (Fig. 1B–D). Haustorium expansion is associated with increasing water content and the mobilization and translocation of endospermic reserves (Figs 1B–D and 2D-F). At the end of this phase, the length of the cotyledonary petiole (17·4 ± 3·5 mm) and its water (79·4 ± 2·0 %) content reach close to maximum values, with the concomitant emission of the primary root (Figs 1D and 2A, C).

Ligule expansion and curvature (10–60 d)

This phase shows significant increases in the size and mass of the haustorium and the mass of the cotyledonary petiole (Figs 1E, F and 2B, D, E). At 10 d after germination, the axis of the primary root is perpendicular to the petiole due to the early expansion of the ligule (the region at the end of the petiole involved in the formation of the saxophone stem) (Fig. 1D). Fifteen days after germination, the ligule region initiates a curvature at its base, with the protrusion of the first leaf sheath (Fig. 1E). At twenty days after germination, pronounced root growth is observed; the ligule has expanded more and has become more distinctly curved and adventitious roots grow from it (Figs 1F and 2G, J). By 45 d after germination the distinct saxophone-shaped stem has become identifiable, associated with a marked curvature of the ligule region that shifts the primary root to a higher position (Fig. 1G). The ligule tissues have split in some regions and have taken on a brownish tinge, indicating senescence, and numerous adventitious roots originate from the saxophone stem. At 60 d after germination, the haustorium has grown significantly and the first eophyll is visible (Figs 1H and 2D, E, P). At the end of this phase, the saxophone stem has attained its typical morphology and near maximum diameter (8·7 ± 1·5 mm), although without significant increases in length (8·7 ± 1·4 mm) or mass (43·7 ± 16·5 mg) (Fig. 2G, H). The haustorium has obtained near maximum dimensions (length: 10·7 ± 1·3 mm; diameter: 11·4 ± 1·7 mm) and mass (dry mass: 121·5 ± 0·1 mg) and a significant fraction of seminal reserves has been consumed (Fig. 2D, E, Q).

Formation of the tuberous region (60–240 d)

Starting at 60 d, the region near the insertion of the primary root begins to grow upwards towards the soil surface, forming a tuberous region, and causing a significant increase in the length and mass of the saxophone stem (Figs 1H and 2G, H). At 180 d, the plant had already emitted the first metaphyll and numerous adventitious roots have been emitted from the saxophone stem (Fig. 1I). The cotyledonary petiole and haustorium have spongy appearances and demonstrate senescence, which is associated with their reduced masses and water contents (Fig. 2B, C, E, F). The saxophone stem maintains a pronounced growth of its tuberous region (Figs 1I and 2G, H), with the axis of the primary root being oblique (almost parallel) to the emerged leaves (Fig. 1I). At 240 d after germination, the remains of the seeds have become detached from the plant and the primary root is atrophied (Figs 1J and 2Q, R). The deposition of reserves in the tuberous region and growth of the aerial portion and roots are associated with reductions in its water content (Fig. 2H–O). Scars of tubular leaves are evident on the tuberous region of the saxophone stem, which is covered by a corky tissue (Fig. 1J). Longitudinal sections of structure clearly demonstrate its similarity to the musical instrument, and that it is composed of the bases of leaves that were issued earlier (the apical meristem is located in the basal region), and the tuberous region, which is slightly fibrous and yellowish (Fig. 1K). The saxophone stem (length: 40·0 ± 7·4 mm, diameter: 15·0 ± 1·4 mm and dry mass: 1·6 ± 0·99 g) continues to grow for several months (beyond the phase evaluated in the present study), associated with the shoot apical meristem remaining underground before the development of the stipe.

Anatomy

Anatomical evaluations confirmed the developmental processes associated with the phases described earlier: (1) the growth and curvature of the cotyledonary petiole, (2) the expansion and curvature of the hypocotyl and (3) the expansion of the plumule internodes.

Growth and curvature of the cotyledonary petiole

The embryonic axis of A. aculeata is nested within the proximal region of the cotyledonary petiole, with an oblique disposition relative to the cotyledon axis (Figs 1A and 3A, B). The embryo axis is composed of the hypocotyl–root axis and the plumule, which lies in a cavity surrounded by a section of the petiole (the ligule). The plumule comprises two leaf sheaths and the apical meristem. On the first day after germination, the cotyledonary petiole (containing the embryonic axis) elongates and protrudes from the seed. This event was related to cell expansion throughout the petiole and to the activity of an intercalary meristem adjacent to the embryonic axis (Figs 1B and 3B). The proximal region of the cotyledonary petiole then initiates its curvature – the result of meristematic activity on one side of that structure (including the ligule) (Fig. 3A, B). Cell division in the root meristem and elongation of the resulting cells, as well as meristematic activity at the extremity of the petiole, result in rupture of the protoderm (Fig. 3B). Vascular bundle differentiation begun in the cotyledonary node leads to the differentiation of vascular bundles, and on the fifth day after germination the notable growth of the cotyledonary petiole is associated with meristematic activity adjacent to the vegetative axis (Figs 1C and 3C, D). The ligule cells show intensive cell division activity that initiates the expansion of that structure. The newly formed root cap demonstrates accumulations of phenolic compounds. On the tenth day after germination, the expansion of ligule is already altering the direction of the vegetative axis, with the plane of the leaf sheath nodes being shifted approx. 150 ° from its original position (compare the position of the dashed orange line in Fig. 3A, E). The leaf sheaths now show significant development. In addition to apical meristem cell division activity, a subapical meristem shows differentiating procambial and ground meristem cells in an atactostelic pattern (Fig. 3F). In the median region of the ligule the cells are vacuolated, with evident proliferation of idioblasts containing raphides. Meristematic activity continues along the periphery of the ligule, contributing to its expansion.

Fig. 3.

Diagrams and photomicrographs of anatomical sections of A. aculeata seedlings (1–10 d after germination). Cotyledonary petiole (A–D) and expanded ligule (F). One day after germination (A, B). (A) Diagram of the cotyledonary petiole, showing the constriction caused by the seed coat (arrowhead). (B) Proximal region of the cotyledonary petiole, showing the protoderm ruptured by root growth (white arrow), the peripheral meristematic region of the ligule (white arrowheads) and the cotyledonary node (black arrowhead). Five days after germination (C, D). (C) Diagram of the proximal region of the cotyledonary petiole showing the meristematic region (white arrows) and the expansion region of the ligule (black arrowhead). (D) Longitudinal section of the apical portion of the petiole, highlighting the meristematic regions of the ligule (above right) and petiole (below). Ten days after germination (E, F). (E) Diagram of the expanded ligule region. (F) Longitudinal section of the ligule region, showing vascular bundles (black arrowhead), idioblasts containing raphides (arrow) and the meristematic region of the ligule (white arrowheads). The dashed lines in the diagrams indicate the positions of procambial strands; grey traces indicate the vascular bundles. The dashed orange line indicates the insertion plane of the plumule leaves. Abbreviations: cn, cotyledonary node; cp, cotyledonary petiole; ep, epidermis; hr, hypocotyl–radicle axis; hy, hypocotyl; li, ligule; mz, meristematic zone; pa, parenchyma; pc, procambium; pe, petiole meristem; ph, idioblasts with accumulations of phenolic compounds; pm, pro-meristem; pr, primary root; rc, root cap; rm, root apical meristem; s1, first sheath; s2, second sheath; su, subapical meristem. Scale bars: A, C, E: 1 mm; B, D, F: 200 μm.

Hypocotyl expansion and curvature

At 15 d after germination, elongation of the leaf sheaths, hypocotyl and primary root can be observed (Figs 1E and 4A). The subapical meristem shows the proliferation of procambial strands, while the hypocotyl shows the differentiation of new vascular bundles and the pronounced expansion of parenchymatous cells near the ligule (Fig. 4B). Meristematic activity is observed in the hypocotyledonary region opposite the ligule as well as the development of adventitious root primordia from the vascular bundles (Fig. 4A–C). At 20 d, differentiation of the first eophyll is observed and the hypocotyl has become more elongated and shows significant curvature (Fig. 4D). Cell division activity is still observed in the hypocotyledonary meristematic region, but most hypocotyl cells have already expanded and become vacuolated (Fig. 4E). Adventitious roots emitted from the hypocotyl that originated from the vascular bundles show cells rich in phenolic compounds (Fig. 4D, F).

Fig. 4.

Diagrams and photomicrographs of anatomical sections of A. aculeata seedlings (15–30 d after germination). Fifteen days after germination (A–C). (A) Diagram of the expanded ligule region. (B) Shoot apex and hypocotyl, showing the subapical meristem (orange bracket), dividing cells (white arrowheads), differentiated vascular bundle (black arrowhead) and root primordia (white arrow). (C) Detail of a root primordia, highlighting dividing cells (arrowheads). Twenty days after germination (D–F). (D) Diagram of the vegetative axis. (E) Elongated hypocotyl, showing dividing cells (arrowheads). (F) Detail of adventitious roots being emitted from a vascular bundle (arrow). Thirty days after germination (G, H). (G) Diagram of the vegetative axis. (H) Base of the ligule, showing the peripheral meristem region (arrowheads). The dashed lines in the diagrams indicate the positions of the procambial strands; grey traces indicate the vascular bundles. The dashed orange line indicates the insertion plane of the plumule leaves. Abbreviations: ar, adventitious root; cn, cotyledonary node; cp, cotyledonary petiole; e1, first eophyll; e2, second eophyll; hy, hypocotyl; li, ligule; pa, parenchyma; pc, procambium; ph, idioblasts with accumulations of phenolic compounds; pr, primary root; ri, idioblasts containing raphides; rp, root primordium; s1, first sheath; s2, second sheath; sm, apical meristem; su, subapical meristem; te, tracheal element. Scale bars: A, D, G: 1 mm; B, E, F, H: 200 μm; C: 50 μm.

At 30 d, the curvatures of the cotyledonary petiole and hypocotyl have altered the plane of the embryonic leaf nodes by approx. 180 ° in relation to its initial position (compare the position of the dashed orange line in Fig. 3A with 4G). At this time, the developing saxophone stem is composed of the expanded ligule and the elongated and curved hypocotyl (Fig. 4G). Differentiation of the second eophyll is evident and the parenchymatous cells of hypocotyl and ligule have become even more expanded. A meristematic region can be observed at the periphery of the ligule, with the accumulation of phenolic compounds in the most external cells (Fig. 4H). At 45 d, the curvature of the hypocotyl has elevated the insertion position of the primary root upwards toward the soil surface (Figs 1G and 5A). Procambial strand proliferation is evident in the subapical meristem (Fig. 5B), and the ligule is senescent, with the accumulation of phenolic compounds and the formation of an abscission layer (Figs 1G and 5C).

Fig. 5.

Diagrams and photomicrographs of anatomical sections of A. aculeata seedlings (45–120 d after germination). Vegetative axis (A–F) and saxophone stem (G–K). Forty-five days after germination (A–C). (A) Diagram of the vegetative axis with an elongated and curved hypocotyl. (B) Shoot apex and hypocotyl, showing differentiated vascular bundles (arrowheads). (C) Abscission of the ligule region. Sixty days after germination (D–F). (D) Diagram of the vegetative axis, with elongated and curved hypocotyl. (E) Shoot apex and hypocotyl, showing dividing cells (arrowheads). (F) Hypocotyl, highlighting root primordium (arrowhead). One hundred and twenty days after germination (G–K). (G) Diagram of the saxophone stem, with orange rectangles indicating the approximate positions of the anatomical sections (H, K), and dotted orange lines indicating the positions of the embryonic leaf scars and first eophyll. (H) Shoot apex. (I) Second eophyll abscission region, showing idioblasts containing raphides (arrowheads). (J) Periphery of the tuberous region showing the meristem with dividing cells (arrowheads) that give rise to parenchymal cells (right) and cells of the cortical coating rich in phenolic compounds (left). (K) Upper section of the tuberous region, highlighting the vascular cylinder of an adventitious root (dashed line). The dashed lines in the diagrams (A, D) indicate the positions of the procambial strands; grey traces indicate the positions of the vascular bundles. The dashed orange line indicates the insertion plane of the plumule leaves. Abbreviations: az, abscission zone; e1, first eophyll; e2, second eophyll; eo, eophyll; ep, epidermis; hy, hypocotyl; li, ligule; me, metaphyll; pa, parenchyma; pc, procambium; ph, idioblasts with accumulations of phenolic compounds; pr, primary root; ri, idioblasts containing raphides; s1, first sheath; s2, second sheath; sm, apical meristem; su, subapical meristem; tr, tuberous region; vb, vascular bundle. Scale bars: A, D: 1 mm; B, C, E, F, H, I, K: 200 μm; G: 10 mm; J: 50 μm.

Expansion of the plumule internodes

At 60 d after germination, the curvature of the hypocotyl has intensified due to subapical meristem activity, divisions of the parenchymatous cells and cell expansion (Fig. 5D, E). The internode of the first leaf sheath adjacent to the subapical meristem shows marked elongation in the region adjacent to the ligule that is associated with meristematic activity near the node (Fig. 5D). Starting at this time, expansion of the plumule internode begins to contribute (together with the development of hypocotyl) to the formation of the saxophone stem. Remnants of ligule form a corky coating along the periphery of the saxophone stem (Fig. 1H, 5F), and numerous root primordia can be observed differentiating from vascular bundles, especially in the peripheral region of the hypocotyl (Fig. 5F).

At 120 d after germination, the saxophone stem has taken on its distinctive shape (Fig. 5G). The internodes of the plumule (representing the major portion of stem saxophone) and the first eophyll have become significantly elongated and thickened, with evident abscission scars and well-defined rings (these rings are even more evident in more developed plants, see Fig 1J). The formation process of the saxophone stem has rotated the insertion plane of the first leaf sheath node approx. 360 ° from its original position (compare the position of the dashed orange line in Fig. 3A with Fig. 5G) so that the structure resembles a saxophone in profile. The internodes of the second eophyll and the metaphylls have not yet elongated, and only the leaf bases contribute to the underground structure. The metaphylls show vacuolated parenchymatous cells and the proliferation of numerous idioblasts containing raphides (Fig. 5H) that are common throughout the periphery of the basal region of the saxophone stem (Fig. 5I). The second eophyll has become senescent, with a conspicuous abscission zone (Fig. 5I). A peripheral meristem in the tuberous region promotes the thickening of that structure, yielding parenchyma internally, and a coating tissue with a cork-like appearance (containing cells rich in phenolic compounds) externally (Fig. 5J). Numerous adventitious roots can be observed in the distal region of the saxophone stem close to the insertion of the primary root, with well-developed vascular cylinders (Fig. 5K).

Reserve storage in the tuberous region

Histochemical and ultrastructural evaluations showed that the parenchymatous cells in the tuberous region had thin cell walls rich in pectic compounds, with significant starchy reserves stored in the cytoplasm in the form of grains of different shapes and sizes (Fig. 6A, B, D). There were also small amounts of mucilage deposited in the vacuoles (Fig. 6B, D, E) and dispersed drops of lipids in the cytoplasm (Fig. 6C–E). Protein reserves were not detected. Reserve deposition was premature, as there were no significant differences in cell structures in the evaluations performed at 120 and 240 d.

Fig. 6.

Photomicrographs of anatomical sections (A–C) and transmission electron microscopy images (D, E) of parenchyma cells from the tuberous region of the saxophone stem of A. aculeata 240 d after germination. (A) Cells stained with lugol solution, showing starch grains (purple). (B) Cell stained with ruthenium red, indicating the accumulation of mucilage (arrowhead) in the vacuole. (C) Cell stained with sudan black, showing lipid droplets (arrowheads) along the periphery of the cytoplasm. (D) Periphery of the cell, with detail of starch grains, and the region shown enlarged in E (orange rectangle). (E) Detail of oil droplets and mucilage with a fibrous appearance accumulated in the vacuole. Abbreviations: cw, cell wall; li, lipid droplet; mi, mitochondria; mu, mucilage; st, starch grain. Scale bars: A: 100 μm; B, C: 20 μm; D: 10 μm; E: 500 nm.

Continuous growth of the aerial portion and storage tolerance

Eighty-four per cent of the plants that had the aerial parts removed showed shoot growth after 30 d. Growth of the aerial portion occurred in 52·5 % of the excised saxophone stems that were immediately planted and then cultivated for 30 d (Fig. 7A, B). Storage did not affect their growth capacity. Only 6 % of saxophone stems that were planted immediately demonstrated the capacity to emit roots (Fig. 7C). Storage for 90 d favoured root emission. The storage of saxophones stems for 15, 60 and 90 d resulted in plants with greater masses after cultivation for 30 d, relative to the zero storage time (Fig. 7D).

Fig. 7.

Percentage of A. aculeata plants showing emission of the aerial portion, shoot length, percentage of plants showing root emission, and the masses of the plants obtained by the cultivation for 30 d of saxophone stems stored for up to 90 d. The same letters indicate no significant differences by the Tukey test, at 5% probability. Vertical bars indicate the standard errors of the means.

DISCUSSION

Little was known about the development or the functions of the saxophone stems of palm trees until recently (Dransfield et al., 2008), and the present work presents the first detailed anatomical evaluations and the first experimental approach identifying the growth patterns and functions of this unique underground system.

Growth patterns

The ontogenesis of the saxophone stem of A. aculeata is complex and takes place in three morphologically defined phases (growth and curvature of the cotyledonary petiole, expansion and curvature of the ligule, and formation of the tuberous region) involving three anatomical processes (growth and curvature of the cotyledonary petiole, expansion and curvature of the hypocotyl, and expansion of internodes of the plumule) (Fig. 8), involving distinct meristematic regions and the differential control of cell expansion.

Fig. 8.

Diagrams of the ontogenesis of A. aculeata saxophone stems, showing the structures and the main meristematic regions involved. Scale bars: 1 mm.

The role of the cotyledonary petiole in the formation of underground systems is a peculiarity of palms. Our results indicate that the growth and initial curvature of the cotyledonary petiole (which determines the positive geotropic growth of the seedling) is associated with the activities of distinct meristematic regions found in the proximal region of the petiole as well as cell expansion. Although the initial pattern of positive geotropic growth has been described for different palm seedlings (Tomlinson, 1979; Henderson, 2002, 2006; Orozco-Segovia et al., 2003; Tomlinson and Huggett, 2012), few anatomical studies have examined this process in detail. While the apical meristems of palm trees have been widely examined (Jouannic et al., 2011), additional meristems that promote early seedling development are still poorly documented. The presence of an intercalary meristem in the petiole was identified in Attalea vitrivir (Neves et al., 2013) and then proposed for the palm Phoenix dactylifera (deMason, 1984), but without anatomical evidence. The peripheral meristematic region, responsible for the expansion of the ligule, was associated with cotyledonary petiole growth in A. aculeata (Ribeiro et al., 2012) and Butia capitata (Oliveira et al., 2013), and the relationship between the expansion of the ligule and the alteration of the root’s position relative to the plumule was also described by Ribeiro et al. (2012). We have shown here that (in addition to the growth of the cotyledonary petiole) the expansion of the ligule is responsible for the burial of the apical meristem and plays an important role in the development of the saxophone stem.

The high cell division rates and expansion of the hypocotyl cells in A. aculeata contribute to the thickening and curvature of the base of the saxophone stem. Another important role of the hypocotyl is related to the emission of large numbers of adventitious roots that give rise to the fasciculated root system and contribute to the fixation of the seedling and its water-absorbing capacity. The participation of the hypocotyl in the formation of underground systems has also been seen in structures classified as rhizophores, xilopodia and hypocotyledonary tubers, although most of these studies were undertaken with dicots (Martins et al., 2011; Appezzato-da-Glória, 2015; Appezzato-da-Glória et al., 2015).

The elongation and thickening of the plumule internodes give rise to the tuberous region and contribute to most of the saxophone stem growth. The elongation of the epicotyl has been shown to be important to the formation of underground systems of rhizomes and rhizophores (Appezzato-da-Glória, 2015), and was seen to be decisive in the formation of saxophone stem in the palm Rhopalostylis sapida (Tomlinson and Esler, 1973). The importance of primary thickening in the formation of saxophone stems was highlighted by Henderson (2002). We observed that the tuberous region was derived from the activity of a subapical meristem at the base of the structure and from a peripheral meristem that promoted thickening and gave rise to reserve parenchyma internally and the cortical coating externally. This peripheral meristem, however, differs from the primary thickening meristem (which is responsible for the radial growth of the stipe and has been well described in palms; see Tomlinson, 1990) as it produces no vascular tissue and is therefore more closely related to phellogen (which is responsible for the development of the periderm in dicots). The formation of a structure similar to the periderm was also seen in a rhizophore coating in the monocot Smilax polyantha (Smilacaceae) (Martins and Appezzato-da-Glória, 2006); the presence of phenolic compounds in this region is related to protection against biotic and abiotic agents (Hutzler et al., 1998; Appezzato-da-Glória and Cury, 2011).

There is no ontogenetic or structural correspondence between the saxophone stem and other underground systems previously described in the literature (Appezzato-da-Glória, 2015), especially in terms of the strict division of its developmental stages, the diversity of meristems and structures involved, its sophisticated morphogenesis and the absence of buds. We therefore propose that the saxophone stems of palm trees should be considered a unique type of underground system.

The functions of the saxophone stem

The saxophone stem favours the survival of natural populations of A. aculeata, especially in anthropogenically impacted areas of the Cerrado, through burial of the apical meristem and water and reserve compound storage. Most palms grow in humid environments, especially rain forests (Dransfield et al., 2008), although approx. 50 species show remarkable adaptation to the seasonal Cerrado climate and its predominantly dystrophic soils (Lorenzi et al., 2010; Ribeiro et al., 2012; Neves et al., 2013; Oliveira et al., 2013). Acrocomia aculeata also demonstrates adaptations to anthropogenic impacts caused by agriculture and pasture formation (Motta et al., 2002; Lorenzi et al., 2010) where the aerial portions of young plants can be suppressed by cultivation, grazing or fire (Rodrigues-Junior et al., 2016).

The saxophone stems of A. aculeata can store up to 60 % of their mass as water, as well as reserve substances such as starch, mucilage and oil. Our experiments showed that continuous growth of the aerial portions (after removal) occurred in over 80 % of the plants, and that the saxophone stem is tolerant of artificial storage (which even favoured root emission and dry matter accumulation in regenerating plants), so that this structure confers resistance to the palms in cases of the removal of the aerial portion and/or exposure to extreme environmental conditions (such as prolonged droughts).

Underground systems with diverse morphological and anatomical features, and often with very complex structures, have been identified in Cerrado taxa of the families Asteraceae (Appezzato-da-Glória et al., 2008), Smilacaceae (Martins et al., 2010, 2011), Apocynaceae (Appezzato-da-Glória and Estelita, 2000) and Cyperaceae (Lima and Menezes, 2009). These organs allow plant survival in environments with limitations related to water stress and the occurrence of fire, by protecting meristems and buds and storing water and nutrient reserves (Rizzini and Heringer, 1961; Appezzato-da-Glória, 2015). Similar functions have been suggested for the saxophone stems of the palms Attalea maripa (Salm, 2005), Rhopalostylis sapida (Tomlinson and Esler, 1973) and Sabal palmetto (McPherson and Williams, 1998), which are not taxonomically related to A. aculeata and occur in different environments. Our results confirm this view and attest to the importance of this structure for the survival of A. aculeata populations and their abundance and wide geographical distribution.

CONCLUSIONS

The development of the saxophone stem of A. aculeata involves three processes: growth and curvature of the cotyledonary petiole, expansion and curvature of the hypocotyl, and expansion of the plumule internodes. This structure promotes the burial of the shoot meristem, is used for storing water, starch, mucilage and oil, and is tolerant of artificial storage when excised. Despite its functional similarity to other underground systems, the stem saxophone is unique in terms of the strict division of its developmental phases, the diversity of meristems and structures involved, its sophisticated morphogenesis and the absence of buds. The saxophone stem represents an important adaptation of A. aculeata to anthropogenically impacted environments in the Cerrado biome as it allows the continuous growth of the aerial portion of the palm that may have been removed by cultivation, fire or grazing during its initial establishment phase.