IAWA Journal, Vol. 29 (1), 2008: 69–77

IAWA Journal, Vol. 29 (1), 2008: 69–77

STEM PROTECTIVE TISSUE IN ERYTHROXYLUM TORTUOSUM (ERYTHROXYLACEAE),

A FIRE TOLERANT SPECIES FROM CERRADO

Alexandre Antonio Alonso and Silvia Rodrigues MachadoDepartamento de Botânica, Universidade Estadual Paulista (UNESP), Botucatu SP, CP 510,

CEP 18618-000, Brazil [E-mail: [email protected]]

SUMMARY

The origin and structure are described of the secondary protective tissue in the stem of Erythorxylum tortuosum Mart., a fire tolerant shrubby species common in Brazilian cerrado. The highly tortuous stems are covered with thick bark which is more developed at the base of the stem. After fire in the cerrado, rhytidome fragments of the burned stem flake off, revealing newly formed cork. The first periderm appears near of the terminal buds and is iniated by periclinal divisions in subepidermal cells giving rise to radial rows of cells. The first phellogen is discernible only after the differentiation of the several radial rows of cork cells. Other phellogens have their origin in successively deeper layers of the cortex. The sucessive periderms are discontinuous around the circumference. The collapsed cells with phenolic substances and the accumulated dead cells cause the formation of discontinuous blackish lines, which delimit the sucessive periderms in the rhytidome. The rhytidome contains large quantities of sclereids developed from cell wall thickening of cortex cells. The occurrence of periderm in the young parts of the stem and of rhytidome in the older parts represents pyrophytic characteristics and may explain, in part, the fire tolerance of this species.

Key words: Bark, cerrado, Erythroxylum tortuosum, fire tolerant species, periderm, rhytidome, stem.

INTRODUCTION

The Brazilian savanna, commonly called cerrado (meaning ʻclosed ̓in Portuguese), is a complex vegetation type, characterized by a mosaic of physiognomies ranging from pure grasslands through open scrubland to dense woodlands (Mistry 1998). For many authors, fire is a key determinant of the cerrado, and it affects vegetation composition and structure, and individual species, many of which have adapted to fire over time (Mistry 1998; Coutinho 2002; Miranda et al. 2002)). The death of woody plants by fire is a rare phenomenon in the cerrado (Mistry 1998). Many species possess pyrophytic characteristics, including strong suberization of trunks and branches (Eiten 1994), dormant apical buds covered by dense, hairy cataphylls (Rachid-Edwards 1956), and capacity to produce sprouts from underground organs following the total carboni-zation of the aerial branches (Rachid-Edwards 1956; Appezzato-da-Glória 2003). Fire

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IAWA Journal, Vol. 29 (1), 200870 71Alonso & Machado — Protective tissue in Erythroxylum tortuosum

protection thus increases the presence of thick-stemmed phorophytes that are highly branched near the base (Eiten 1994; Mistry 1996) as it is the case of Erythroxylum tortuosum Mart., a fire tolerant species (Sambuichi 1991) usually found in cerrado vegetation naturally exposed to recurrent fire during the dry seasons (Mendonça & Amaral Jr. 2002). Erythroxylaceae constitute a family best known for their production of leaf alkaloids, among which Erythroxylum coca L. is the most widely exploited species. The family includes four genera and occurs in relatively warm parts of the world, such as Tropical America and Africa (Heywood 1993). Only Erythroxylum occurs in Brazil, with about 110 species occurring as trees, shrubs and subshrubs distributed among a wide array of habitats, including rainforest, caatinga, rocky grasslands, and cerrado. In the Brazilian cerrado, Erythroxylum species have been exploited regionally for their pigments, the essential oils contained in their bark, and for their medicinal properties (Mendonça & Amaral Jr. 2002). With some exceptions (Beiguelman 1962, 1962a, 1962b, 1962c; Massa & Arens, 1971; Costa et al. 1997; Machado et al. 2005), the bark anatomy of Brazilian cerrado plants has received little attention. Little is know about the cork structure of the Eryth-roxylum cerrado species, since only E. suberosum has been studied (Beiguelman 1962; Massa & Arens 1971). The aim of the present work was to study the stem protective tissue of E. tortuosum and to correlate its structure with cerrado conditions.

MATERIAL AND METHODS

Study site The study was carried out in a cerrado area of Botucatu, state of São Paulo, Brazil (22° 51' S, 48° 26' W). The study site is variable in physiognomy and ranges from open grassland to forest, with a discontinuous layer of deciduous and ever-green trees with a low and shrubby growth form. Most trees and shrubs have a thick bark, tortuous trunks and scleromorphic leaves. Soils are deep, strongly acid dystrophic latosols, with high Al content. The climate is Cwb (mesothermal with dry winters) by Köppenʼs (1931) clas-sification, in which the warmest month presents an average temperature no higher than 22°C, and the month of July is the coldest and driest of the year. The annual rainfall is 1,534 mm, with a distinct dry season from May to September (81–89 mm, respectively). Average (diurnal) relative humidity is around 80% during the rainy season and drops to 55% during the dry season when the daily minimum relative humidity reaches values around 15%. Mean annual temperature is about 20.3 °C. Climatic data were collected from the Meteorological Station of the Faculdade de Ciências Agronômicas (FCA), São Paulo State University (UNESP), Campus Botucatu.

Plant material Samples were taken from the most developed stem of six plants of Erythroxylum tortuosum during the dry season and the subsequent rainy season (October–November 2003–2005). Vouchers specimens were deposited at the BOTU Herbarium of the Botany Department at UNESP.



Anatomical studies The materials were fixed with FAA 50 for 48 hours, and then stored in 70% ethanol (Johansen 1940). The materials were cut on a Ranvier microtome. The sections (10–13 µm thickness), were bleached with a 20% sodium hypochlorite solution, washed in distilled water, stained with aqueous solutions of 1% astra blue and 1% safranin, and mounted in a solution of glycerine : distilled water (1 :1). For histochemistry, sections of fresh material were treated with Sudan IV to detect lipids; 10% ferric chloride to detect phenolic compounds; lugol reagent for starch grains (Johansen 1940), aqueous 0.02% ruthenium red for pectic substances (Jensen 1962), and acid phloroglucin for the lignin (Johansen 1940). Schematic representations were made of the histological material observed in the Zeiss® microscope coupled to a light camera. Photomicrographs were taken with a Zeiss® photomicroscope.

Figure 1. Shoot system of Erythroxylum tortuosum. – A: Adult plant. – B: Outer aspect of the rhyti-dome. – C: Terminal internodes with periderms. – D: Schematic representations of a cross section of terminal internodes (dashed line of Fig. 1C) showing collenchyma (CO), parenchyma (PA) in the protuberances and depressions, vascular bundle (VB) and cortical bundles (CB). — Scale bar = 350 μm.



RESULTS

General aspects Individual shrubs of Erythroxylum tortuosum have two to three tortuous stems, with one of them usually more developed than the others, and are 2–2.3 m tall. All plants examined displayed stems with thick reddish bark, fissured in the outer surface; the bark was highly developed at the base of the stem near the ground (Fig. 1A, B). The developed bark remains firmly attached to the wood all along the stem for a long period. After fire in the cerrado, rhytidome fragments of the burned stem flake off, revealing newly formed cork.

Anatomy The internodes subjacent to the terminal bud (Fig. 1C), at cross section, have an ir-regular surface, with protuberances and depressions of variable depths (Fig. 1D). The epidermis is glabrous, uniseriate, with a thick cuticle, while the cortex consists of about 10–20 layers of collenchyma and 5–15 layers of parenchyma. The vascular system is composed of contiguous collateral bundles, which form an irregular cylinder surround-ing a parenchymatous pith. Four to five cortical bundles are also present (Fig. 1D). The first periderm appears near the terminal buds (Fig. 1C), and is iniated by peri-clinal divisions in subepidermal cells (Fig. 2A, B), giving rise to radial rows of cells (Fig. 2C), some of which expand radially more than others (Fig. 2D). Some expanded collenchymatic cells also divide in the anticlinal plane (Fig. 2D, arrow). Several radial rows of cells with thinner walls are produced on each stem protuberance. In sequence, the same pattern of the periderm installation is observed all along each depression (Fig. 2E). This condition was found around the circumference of the stem (Fig. 2F) and, as a result, protuberances and depressions are not discernible on the stem circumference. In the actively dividing rows of cells (Fig. 2C–F) a phellogen is not discernible. By the time the first phellem has been formed and meristematic activity has ended, the phel-logen is clearly discernible (Fig. 3B). Approximately 9–13 radially arranged layers of rectangular to radially elongate suberized cells (Fig. 3A, B) are aligned with their respec-tive rows in the phellogen. The phelloderm is poorly developed in all analyzed samples. Other phellogens have their origin in successively deeper layers of the cortex (Fig. 3C–G). The sucessive periderms are discontinuous around the circumference (Fig. 4A, B).The collapsed cells with phenolic substances and the accumulated dead cells (Fig. 4B) cause the formation of discontinuous blackish lines, which delimit the sucessive periderms in the rhytidome (Fig. 4A–C). The rhytidome contains large quanti-ties of sclereids developed from cell wall thickening of cortex cells (Fig. 4A, B).

←Figure 2. Cross sections showing development initial of secondary covering tissues in the stem branches of Erythroxylum tortuosum. – A: Young stem with thick cortex and pith, and primary vascular tissues. – B: Periclinal cell divisions (arrowheads) beneath the epidermis. – C: Tiers of cells arranged in horizontal series on the protuberances. – D: Expanded collenchyma cell (*), and periclinal (arrowhead) and anticlinal (arrow) divisions of collenchyma cells. – E: First periderm formation in the depressions. – F: Actively dividing rows of cells. — Scale bars = 500 μm in A; 100 μm in B; 40 μm in C; 18 μm in D & E; 150 μm in F.



←Figure 3. Cross sections of young stem branches of Erythroxylum tortuosum. – A: Radial rows of cells around the circumference of the stem. – B: Detail of the previous figure showing the first phellogen (arrowhead). – C: First phellogen (arrowheads) and second phellogen (arrow). – D: Detail of the previous figure showing first phellogen (arrowhead) and second phellogen (ar-row). – E: Development of successive periderms. – F: Detail of previous figure showing cork cells (PH) developed from second phellogen (arrow). – G: Detail of previous figure showing collen-chyma cells (CO) in second periderm. — Scale bars = 500 μm in A & C; 25 μm in B; 50 μm in D; 2 mm in E; 100 μm in F; 150 μm in G.

Figure 4. Cross sections of the stem of Erythroxylum tortuosum. – A: Rhytidome (RT), cortex (CT) and secondary phloem (SP). – B: Sclereids (arrow) and cork cells (*) filled with phenolic sub-stances in rhytidome. – C: Rhytidome showing sinuous blackish lines delimiting successive peri-derms (arrowheads) in the basal part of older stems (see figure 1B). — Scale bars = 500 μm in A; 80 μm in B; 150 μm in C.



DISCUSSION

The protective tissue of the stem of Erythroxylum tortuosum is composed by a rhytid-ome. In the periderm of Erythroxylum suberosum (Beiguelman 1962; Massa & Arens 1971), Byrsonima coccolobifolia, Annona coriacea and Ouratea spectabilis (Beiguel-man 1962a, 1962b, 1962c), all of which are woody cerrado species, the cork is differ-entiated directly from the continuous subepidermal phellogen, as commonly described for the stem of woody species (Eames & MacDaniels 1947; Esau 1960; Fahn 1990; Mazzoni-Viveiros & Costa 2006). Unlike these species, the first phellogen in the stem of E. tortuosum is discernible only after the differentiation of the several radial rows of cells. Preparatory divisions before the phellogen is defined are common (Esau 1960). In E. tortuosum the early formed cork cells are wider and have thinner walls than those formed later. This characteristic suggests the occurrence of phellogen periodicity in this species. However, studies on the seasonal activity of phellogen and production of cork are scarce in the tropics. Studies on the phellogen activity and rhythm of peri-derm production in relation to phenology and climatic factors in Brazilian cerrado are currently being carried out. It is remarkable that the formation of the first periderm in E. tortuosum occurs prior to the differentiation of the cambium. The ability to form periderms in the young parts of the stem provides protection of dormant meristems (bud banks) that can be potentially used for vegetative regeneration after injury, seasonal regrowth, vegetative growth and flowering (Klimesová & Klimes 2007). Periderms in buds (terminal and axillary), cataphylls, leaf petiole and midrib have been found in different shrubby and sub-shrubby cerrado species (Machado et al. 2006). In E. tortuosum the occurrence of a rhytidome with a massive cork provides thermal isolation of the living inner tissues of its stems against the action of fire, particularly in the dry season. The occurrence of the periderms in the young parts of the stem and of rhytidome in the older parts represents pyrophytic characteristics and may explain, in part, the fire tolerance of this species.

ACKNOWLEDGEMENTS

The authors are indebted to the FAPESP (Fundação de Amparo à Pesquisa do Estado de São Paulo, Process no. 03/00957-1) for a doctoral grant and financial support (Process no. 00/12469-3) awarded to the first author, and CNPq (Conselho Nacional de Desenvolvimento Científico e Tecnológico) for a productivity grant awarded to S.R. Machado.

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IAWA Journal, Vol. 29 (1), 2008: 69–77

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