IAWA Journal, Vol. 28 (3), 2007: 315-324

COMPARATIVE ANATOMY OF THE BARK OF STEMS, ROOTS AND XYLOPODIA OF BROSIMUM GAUDICHAUDII ()

Dario Palhares, Jose Elias de Paula, Luiz Alfredo Rodrigues Pereira and Concei~ao Eneida dos Santos Silveira Department of Botany, University of Brasilia, Darcy Ribeiro Campus, 70919-970, Brasilia-DF, Brazil Correspondence: C. E. S. Silveira [E-mail: [email protected]]

SUMMARY

Brosimum gaudichaudii Trec. occurs in the Atlantic and Amazon for­ ests, and is the only species of Brosimum commonly found in Cerrado vegetation. It is of pharmaceutical interest due to the large accumulation of furocoumarins such as psoralens in the bark of roots and xylopodia. This work describes the bark anatomy of sterns, roots, and xylopodia. Although the external bark morphology of stern and subterranean system are different, anatomically they are similar, with both having wavy and fused rays at the outer region of the phloem and a gradual transition be­ tween pervious (non-collapsed) and collapsed phloem. Tbe stern and bark periderms have three to seven layers of cells. The bark of younger stern regions is different from the bark of older parts of the stern. Younger stern parts have higher abundance of laticifers in the phloem, and gelatinous fibers arranged in bundles. Compared with the younger regions, older sterns have fewer laticifers and the gelatinous fibers are scattered in the phloem. The root and the xylopodium bark are structurally similar to each other, with a higher abundance of laticifers than sterns. Starch was found in the roots, but not in sterns. Key words: Moraceae, Brosimum gaudichaudii, bark anatomy, stern, root, xylopodium.

INTRODUCTION

Even though Brosimum gaudichaudii is found in the Amazon and Atlantic forests (Santos & Kinoshita 2003), it is the only occurring species of the Brosimum in Brazilian Cerrado (Berg 1972), which is a savanna type formation (Mistry 2000). This species has a xylopodium, a woody tuberosity capable of resprouting (Rizzini & Heringer 1961; Appezzato-da-GI6ria & Estelita 2000). The aerial portion of the is not fire resistant, probably because of the thin bark, which is unable to confer a proper protection. As a consequence, the growth habit of mature varies from sub-shrub to tree. Brosimum gaudichaudii trees only occur in places where fire is not frequent (Ribeiro et al. 1985). Latex is abundant in the bark of the subterranean system. In sterns, latex is only abundant in the bark and pith of young sterns and branches. Latex is scarce in both bark and pith of older stern parts and not present in B. gaudichaudii wood (Palhares et al. 2006).

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Brosimum gaudichaudii has been largely exploited by the pharmaceutical industry because of the large accumulation of psoralens in the bark of its subterranean system (Monteiro et al. 2002). Psoralens are furocoumarins with photo-mutagenic and photo­ chemotherapeutic properties used in the treatment of auto-immune skin disorders. While sterns, leaves, flowers, and fruits have only trace amounts of these compounds, the concentration of psoralens in the roots may represent up to 3% of the dry weight (Pires 2004). As the psoralens active principle is primarily found in the subterranean system of B. gaudichaudii, we have carried out an anatomical characterization of the bark of stern, root and xylopodium. The purpose of this study is to prepare a list of characteristics that may help to separate stern and subterranean system barks in the plant material indis­ criminately collected in the field. Also, this study makes a contribution to the knowledge of the genus Brosimum; most Brosimum species are found in wet forests and B. gaudi­ chaudii is the only species commonly found in the Cerrado.

MATERIAL AND METHODS

Five mature plants of shrubby and arboreal size were collected in Cerrado areas of Brasilia. Voucher specimens were deposited in the herbarium of the Vniversity of Brasilia (VB) underregistration numbers VB 12050, VB 12051, VB 12052, VB 13190 and VB 13191. Stern sampIes were obtained at a height of 20 cm, from sterns with a circumference ranging from 5 to 45 cm. The tap roots were 1.5 to 2 m deep with a cir­ cumference of 10 to 15 cm in the middle section where the sampIes were taken. The xylopodia had a circumference varying from 10 to 115 cm, and a length varying from 15 to 25 cm. More details on the extemal morphology of the studied material can be found in Palhares et ai. (2006) and Palhares & Silveira (2007). Transverse, radial and tangential bark sections (20-30!-Lm thick) were dehydrated in alcohol-xylene series un­ der vacuum, and subsequently double-stained with safranin and malachite green (Johan­ sen 1940). Maceration ofthe bark sampIes was accomplished as described by Franklin (1945). Histochemical tests were carried out to detect stareh, phenolic compounds, lignin and alkaloids (Johansen 1940; Bamber 2001). Anatomical terminology follows the nomenclature as described by Trockenbrodt (1990), Lev-Yadun (1991), Junikka (1994) and Richter et al. (1996). A dissociated bark preparation was used to measure the length /width of 20 intact sieve elements as weIl as the sieve plate angle. The sampIe size was determined by the number of intact cells found in the macerations. The values were used to calculate the average and standard error (SE).

RESULTS Stern The general arrangement of Brosimum gaudichaudii bark fits into type 2 in Richter's classification (Richter et ai. 1996), with bark thickness ranging from 0.8 to 3.5 mm. The rays are wavy, funnel-shaped and sometimes fused in the distal part near the cortical zone (Fig. 1-3). The cortical zone is made of a parenchymatous tissue between the phloem and the periderm (Fig. 4).

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There are few differences between the barks of thinner (younger) sterns and thicker (older) sterns. The bark of thinner sterns has remnants of the cortex (Fig. 2 & 4). The secondary phloem has laticifers, as does the cortical zone. However, in all sampies studied laticifers are generally inconspicuous and collapsed (Fig. Ib & 4). Moreover, due to the presence of cortex remnants, it appears that in younger sterns there is a higher abundance of laticifers than older sterns. Bundles of gelatinous fibers are observed in the cortical region of the bark of younger sterns (Fig. la). On the other hand, the bark of thicker sterns does not have cortical remnants (Fig. 3) and the gelatinous fibers are diffusely distributed in the secondary phloem. The outer sieve elements have a higher accumulation of callose than those in more internal regions of the phloem (Fig. 5). In general, sieve elements of B. gaudichaudii stern barks (Fig. 6) are small (138 ± 2 f.tm long, 12.5 ± 0.5 f.tm thick). Sieve plates are oblique, with inclinations ranging from 70° to 80°. The lateral sieve areas are elliptical, and a nacreous wall is frequently observed (Fig. 6 & 7). As mentioned previously, as the phloem parenchyma rays go outward, they become wider (Fig. 3); however, there is no corresponding increase in their height (Fig. 8). Furthermore, besides being perpendicular to the phloem rays (Fig. lb), the laticifers are also found inside the rays, as well as in the axial system of the phloem (Fig. 8). The periderm has three to seven layers of cells. The outer layers easily slough off from the bark (Fig. 9). Phloroglucinol tests (Bamber 2001) revealed the presence of lignified parenchyma cells in the outer region of the barks (Fig. 10 & 11). Phenolic compounds are abundant throughout the bark. Lipids occur in the phellern and crystals of calcium oxalate are occasionally observed in the cortical zone. Starch and alkaloids are absent.

Figures 1-5. Anatomical structure of stern bark. - 1: TS of a young stern bark (0.8 mm thick). Note the laticifers (star) in the inner layers of the phloem and a bundle of gelatinous fibers (square) in the cortical zone. Inset a: higher magnification of a gelatinous fiber bundle. Inset b: detail of a collapsed laticifer in the bark (arrowhead). - 2: TS of a stern bark (1.5 mm thick). - 3: TS of bark in older sterns (3.5 mm thick). Note that there is no cortex remnant. - 4: RLS of the young stern bark (1.0 mm thick). The arrowheads indicate the location of laticifers in the cortical zone and phloem based on the differential staining. - 5: Bark stained with aniline blue and observed under fiuorescent light. High accumulation of callose (arrowheads) in the outer sieve tubes. - Sca1e bars = 100 !-Lm.

Figures 6-9. Sieve elements, laticifers, and periderm of the bark of young sterns. - 6: Macera­ tion showing sieve elements with ellipticallateral sieve areas and compound sieve plate (arrow­ heads). - 7: Naereous walls (arrowhead) observed in TS. - 8: TLS. Note the presenee of lati­ eifers inside and outside of the ray (arrowheads). - 9: TS of the periderm of the stern bark. Note the outer periderm layers sloughing off (star). - Seale bar in 6 = 3 !-Lm; in 7 = 15 !-Lm; in 8&9=100!-Lm.

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Figures 1-5; für the legends, see page 317.

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Figures 6-9; for the 1egends, see page 317.

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Figures 10-13. Bark strueture ofthe subterranean system. - 10: TS of root bark. Note the presenee oflatieifers (arrowheads) perpendicular to the phloem rays. Also there is a positive reaetion for lignin (phloroglueinol) in the parenehyma eell walls at the outer layer ofthe phloem (asterisk). - 11: Radial seetion of bark showing the presenee oflignified eell walls stained with phloroglu­ einol (asterisks). - 12: Phloem region of the root bark. Observe the latieifers (arrowheads). - 13: TLS of root bark in a region with dilated rays. Observe the laticifers inside and outside the rays (arrowheads). - Seale bar in 10 = 50 ~m; in 11, 12, 13 = 100 ~m.

Root and xylopodium Similar to stern barks, root and xylopodium barks are also of type 2 (Richter et al. 1996), whose thickness varies between 1.7 to 3.5 mm, with wavy and funnel-shaped rays, as weIl as diffuse gelatinous fibers. Sieve elements of sterns and subterranean system are structurally similar. In root and xylopodium barks, the laticifers are abundant in both inner and outer layers ofthe phloem, as weIl as in the cortical zone and phloemrays (Fig. 10,12 & 13). Similar to the sterns, the periderm of the subterranean system has three to seven layers of cells, and the outer layers are weakly bound to the bark. The bark of roots and xylopodia has a high abundance of starch and phenolic compounds. Lipids occur only in the phellern and calcium oxalate crystals occur in the cortex, when present. Alkaloids are not found in either root or xylopodium barks.

DISCUSSION

Richter et al. (1996) studied barks of several Brazilian species and grouped them into three categories. According to them, type 1 barks have indistinct layers of collapsed and pervious (non-collapsed or functional) phloem, with straight linear rays and a periderm

Downloaded from Brill.com09/27/2021 08:10:48AM via free access Palhares et al. - Bark anatomy 0/ Brosimum gaudichaudii 321 with concentric layers. The type 2 barks are also described as having indistinct layers of collapsed and pervious phloem; however, the phloem rays are funnel-shaped and the periderm is reticulate. Finally, type 3 barks have secondary phloem dearly divided into collapsed and pervious layers with rhytidome. Therefore, the structural arrangement of B. gaudichaudii barks, in both stern and subterranean system, dearly falls into type 2 in Richter's description. Additionally, B. gaudichaudii phloem structure has no dear limit between collaps­ ed and pervious phloem, having only a gradual transition between these zones. Sec­ ondary phloem is originated by the cambium, whose activity is infiuenced by several endogenous and exogenous factors such as hormones, deciduousness, temperature, and water. Moreover, it is well established that the seasonality of cambial activity and water availability are directly associated with phloem growth and inactivation, which may result in conspicuous morphological changes in the phloem (Venugopal & Krishna­ murthy 1987). Non-deciduous and perennial Cerrado plants, like B. gaudic,haudii, have a deep root system that allows them to obtain water during the dry season (Sarmiento et al. 1985; Meinzer et al. 1999). This may result in continuous cambial activity through­ out the year, and thereby not resulting in distinguishable morphological modifications between collapsed and pervious phloem. In young sterns of B. gaudichaudii, laticifers abundantly occur in the pith and bark (palhares et al. 2006), while in old sterns these are mostly found in the bark. Laticifers maybe originated from primary initial cells (Mahlberg 1963; Mahlberg & Sabharwal 1966; Rosowski 1968), and from cambial cells (Van Veenendaal & Den Outer 1990). It appears that B. gaudichaudii laticifers have a dual origin, from primary meristems and cambium. Nonetheless, due to the variability of laticifer abundance between barks of young and old sterns, as weIl as in the subterranean system, the origin of these structures remains uncertain. Thus, further investigation is needed to determine the ontogeny of the laticifers in B. gaudichaudii. The latex is composed of secondary metabolites that are commonly toxic, and fre­ quently of medicinal interest. In B. gaudichaudii the psoralens are concentrated in the subterranean system; nevertheless the latex of this species is poor in these metabolites (Pires 2004), indicating that they are not synthesized in the laticifers. Hence, the abundance of latex in the subterranean system bark cannot explain the high abundance of these compounds. In many plant species psoralens are part of a defence mechanism against pathogenic microorganisms (Surico et al. 1987; Downum 1992). It appears that in these species regular parenchyma cells are capable of rapidly synthesizing these compounds, which results in local necrosis of plant tissue (Surico et al. 1987). Moreover, these furocoumarins accumulate in variable amounts depending on the organ, and may become toxic in the presence oflight (Surico et al. 1987; Downum 1992). Therefore it is possible that the parenchyma tissue of the subterranean system of B. gaudichaudii synthesizes these compounds, which would explain the larger amounts of psoralens in these parts of the plant. The bark of B. gaudichaudii has many similarities with those described for the genus, which is characterized by scarce lignified tissues, diffuse distribution of sieve elements, persistent periderm, ray dilatation fused with the cortical zone, absence of

Downloaded from Brill.com09/27/2021 08:10:48AM via free access 322 IAWA Journal, Vol. 28 (3), 2007 rhytidome, and occurrence of a large numbers of laticifers (Roth 1981). As in other Moraceae, the sieve elements of B. gaudichaudii are small (Esau 1969). Moreover, the high accumulation of callose in older sieve elements illustrates the natural developmental sequence in which the increase of callose content together with P-protein precedes the collapse of sieve tube elements (Ehlers et al. 2000). Gelatinous fibers occur in bundles diffusively distributed in the phloem of B. gaudichaudii. Gelatinous fibers are normally found in tension wood, which forms in many Angiosperms as a geotropic response to mechanical stress. Furthermore, these fibers are also a common feature in many Cerrado plants and are found in many plant organs such as leaves and sterns and may be associated or not with vascular tissue (Rodrigues & Machado 2004).1t appears that in Cerrado species the formation of gela­ tinous fibers is not a response to geotropic stimuli. Paviani (1974) considered them to function as a water reservoir. Although the anatomical structure of the stern and the subterranean system barks were similar, the extemal morphology of these organs was found to be quite different (palhares et al. 2006). In the stern, the bark was grey and rough, while in the subterranean system the bark was orange and sloughed off in concentric layers (Palhares et al. 2006). Another interesting point is that the xylopodium and root of B. gaudichaudii are capable of producing shoots and radicles, which is the basis of the vegetative propagation of this plant species (Djalma Silva, Embrapa-Cenargen, personal communication). It has been observed that the B. gaudichaudii wood (palhares et al. 2007) and the sub­ terranean system bark highly accumulate starch, which may likely be used as energy source for resprouting (Bowen & Pate 1993). In many tropical plants that are able to produce root suckers (Hayashi et al. 2001), the cortical zone is the region where the new shoots and radicles are initiated. However, the sprout formation and differentiation in B. gaudichaudii xylopodium and root needs to be further investigated. Due to the high similarity of the bark in the secondary organs of B. gaudichaudii, we have not been able to prepare a list of bark characteristics that would allow a rapid identification of plant material for commercial purposes. Nevertheless, to the best of our knowledge studies on the anatomy of the bark of Moraceae are very scarce. Therefore, the research described here presents valuable scientific data conceming an unknown plant species of pharmacological importance.

REFERENCES

Appezzato-da-Gl6ria, B. & M. Estelita. 2000. The developmental anatomy of the subterranean system in Mandevilla illustris (Vell.) Woodson andM. velutina (Mart. ex Staldem.) Woodson (Apocynaceae). Braz. J. Bot. 23: 2735. Bamber, R.K. 2001. A general theory for the origin of growth stresses in reaction wood: how trees stay upright. IAWA J. 22: 205-212. Berg, e.e. 1972. Flora Neotropica: Monograph 7. Hafner Publishing Company, New York. Bowen, B.J. & J.S. Pate. 1993 The significance of root starch in post-fire shoot recovery of the resprouter Stirlingia latifolia R. Br. (Proteaceae). Ann. Bot. 72: 7-16. Downum, K.R. 1992. Light-activated plant defence. New Phytol. 122: 401-420.

Downloaded from Brill.com09/27/2021 08:10:48AM via free access Palhares et al. - Bark anatomy of Brosimum gaudichaudii 323

Ehlers, K., M. Knoblauch & A. J.E van Bel. 2000. Ultrastructural features ofwell-preserved and injured sieve elements: minute clamps keep the phloem conduits free for mass flow. Proto­ plasma 214: 80-92. Esau, K. 1969. The phloem. Handbuch der Pflanzenanatomic. Gebr. Borntraeger, Berlin, Stutt­ gart. Franklin, G. 1945. Preparation of thin sections of synthetic resins and wood-resin composites, and a new macerating method far wood. Nature 155: 1. Hayashi, A., A. Penha, R. Rodrigues & B. Appezzato-da-Gl6ria. 2001. Anatomical studies of shoot bud-forming roots of Brazilian tree species. Aust. J. Bot. 49: 745-751. Johansen, D.A. 1940. Plant microtechnique. McGraw-Hill Book Company, New York. Junikka, L. 1994. Survey of English macroscopic bark terminology. IAWA J. 15: 3-45. Lev-Yadun, S. 1991. Terminology used in bark anatomy: additions and comments. IAWABul1. n.s. 12: 207-209. Mahlberg, P. 1963. Development of non-articulated laticifer in seedling axis of Nerium oleander. Bot. Gaz. 124: 224-231. Mahlberg, P. & P. Sabharwal. 1966. Mitotic waves in laticifers of Euphorbia marginata. Science 152: 518-519. Meinzer, EC., G. Goldstein, A.C. Franco, M. Bustamante, E. Igler, P. Jackson, L. Caldas & P. W. RundeI. 1999. Atmospheric and hydraulic limitations on transpiration in Brazilian cer­ rado woody species. Funct. Ecol. 13: 273-282. Mistry, J. 2000.World savannas. Ecology and human use. Prentice Hall, London. Monteiro, V.EE, L. Mathias, I.J.c. Vieira, J. Schripsema & R. Braz-Filho. 2002. Prenylated coumarins, cha1cone and new cinnamic acid and dihydrocinnamic acid derivatives from Brosimum gaudichaudii. 1. Braz. Chem. Soc. 13: 281-287. Palhares, D., J.E. de Paula, J.A. Rodrigues Pereira & C.E.S. Silveira. 2007. Comparative wood anatomy of stern, root and xylopodium of Brosimum gaudichaudii (Moraceae). IAWA J. 28: 83-94. Palhares, D., J.E. de Paula & C. Silveira. 2006. Morphology of stern and subterranean system of Brosimum gaudichaudii (Moraceae). Acta Bot. Hung. 48: 89-101. Palhares, D. & c.E.S. Silveira. 2007. Morphological aspects ofyoung plants of Brosimum gaudi­ chaudii cultivated in alternative conditions. Braz. J. Med. Plants 9: 93-96. Paviani, T.I. 1974. Ocurrence of gelatinous fibers in Plathymenia reticulata Benth. Cien. & Cult. 26: 783-786. Pires,A.E. 2004. Desenvolvimento e valida<;ao de metodologias para determina<;ao de furanocu­ marinas em medicamentos fitoterapicos. MSc dissertation, Universidade Federal do Mato Grosso de Sul, Brazil. Ribeiro, J., 1. Silva & C. Batmanian. 1985. Fitossociologia de tipos fisionömicos de cerrado em Planaltina. Braz. J. Bot. 8: 131-142. Richter, H. G., S. C. Manzoni-Viveiros, E. S. Alves, A. E. Luchi & C. G. Costa. 1996. Padroniza<;ao de criterios para a descric;ao anatömica da casca: lista de caracteristicas e glossario de termos. IF Serie de Registros 16: 1-25. Rizzini, C. T. & E. P. Heringer. 1961. Underground organs of plants from some southern Brazilian savannas, with special reference to the xylopodium. Phyton 17: 105-124. Rodrigues, T.M. & S.R. Machado. 2004. Comparative anatomy of pulvinus, petiole and rachis of Pterodon pubescens Benth. (Fabaceae-Faboideae). Acta Bot. Bras. 18: 381-390. Rosowski, J. 1968. Laticifer morphology in the mature stern and leaf of Euphorbia supina. Bot. Gaz. 129: 113-120. Roth, I. 1981. Structural patterns of tropical barks. Handbuch der Pflanzenanatomie IX/3. Gebr. Borntraeger, Berlin, Stuttgart.

Downloaded from Brill.com09/27/2021 08:10:48AM via free access 324 IAWA Journal, Vol. 28 (3), 2007

Santos, K. & L.S. Kinoshita. 2003. Flora arbustivo-arb6rea do fragmento de floresta estacional semidecidual do Ribeiriio Cachoeira, municfpio de Campinas, SP. Acta Bot. Bras. 17: 325-341. Sarmiento, G., G. Goldstein & R. Meinzer. 1985. Adaptive strategies of woody species in neo­ tropical savannas. Bio!. Rev. 60: 315-355. Surico, G., L. Varvaro & M. Solfrizzo. 1987. Linear furocoumarin accumu1ation in ce1ery p1ants infected with Erwinia carotovora pv. carotovora. J. Agric. Food Chem. 35: 406-409. Trockenbrodt, M. 1990. Survey and discussion ofthe termino1ogy used in bark anatomy. IAWA BuH. n.s. 11: 141-166. Van Veenendaal, W. & R. den Outer. 1990. Distribution and development ofthe non-articulated branched laticifers of nigra L. (Moraceae). Acta Bot. Neer!. 39: 285-296. Venugopal, N. & K.v. Krishnamurthy. 1987. Seasonal production of secondary phloem in the twigs of certain tropical timber trees. Ann. Bot. 60: 61-67.

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