Development of Intraxylary Phloem in the Stem of Combretum Rotundifolium (Combretaceae)

IAWA14 Journal, Vol. 32 (1), 2011: 14–24 IAWA Journal, Vol. 32 (1), 2011

Development of intraxylary phloem in the stem of Combretum rotundifolium (Combretaceae)

Elaine Zózimo1, Neusa Tamaio1* and Ricardo Cardoso Vieira2

SUMMARY The present study focuses on the origin, secondary growth, and anatomical characteristics of the intraxylary phloem compared to the regular phloem in Combretum rotundifolium Rich. The intraxylary phloem first originates in the primary stem from the perimedullary procambium and, later, from the internal cambium that develops by differentiation of the perimedullary procambium and dedifferentiation of perimedullary parenchyma cells. Tangential sclerenchyma bands, irregular stratification of the cellular elements, and uniseriate rays are present in the regular phloem, but not in the intraxylary phloem. We are the first to report the presence of an unusual bidirectional internal cambium that produces pa- renchymatous cells centrifugally and intraxylary phloem centripetally. Key words: Cambial variants, intraxylary phloem, internal phloem, internal cambium, Combretum rotundifolium, regular phloem.

INTRODUCTION

The intraxylary phloem involves the production of phloem in the pith’s periphery. In Combretum Loefl. (Combretaceae) intraxylary phloem (internal phloem) and/or interxylary phloem (included phloem) may be present (e.g. Solereder 1908; Metcalfe & Chalk 1950; Verhoeven & Van der Schijff 1974; Van Vliet 1979). The occurrence of intraxylary phloem is a synapomorphy of the order Myrtales, to which the Combretaceae belong (Stevens 2001 onwards; Judd et al. 2009). However, many aspects of intraxylary phloem development in the family and order remain unknown, as Quisqualis indica L. is the only species for which some stages of intraxylary phloem development have been described, including its origin from the procambium and its secondary growth resulting from the development of a unilaterally active internal cambium (Baranetzky 1900). Some data on intraxylary phloem for Combretum are available, including its ap- pearance as a ring or group arrangement and the occurrence of an internal cambium (Metcalfe & Chalk 1950; Verhoeven & Van der Schijff 1974; Tilney 2002). The regular phloem shares some characters with other genera of the Combretaceae, such as storied cellular elements, crystals and narrow rays (Den Outer & Fundter 1976).

1) Instituto de Pesquisas Jardim Botânico do Rio de Janeiro, Diretoria de Pesquisa Científica, Rua Pacheco Leão 915, 22460-030 Rio de Janeiro, RJ, Brazil. 2) Laboratório de Morfologia Vegetal da Universidade Federal do Rio de Janeiro, Depto de Botânica, IB, CCS, BL A, Sala A1-108, Av. Brigadeiro Trompowsky s.n., 21941-590 Ilha do Fundão, Rio de Janeiro, RJ, Brazil. *) Corresponding author [[email protected]].

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In the present study, we describe the ontogeny of intraxylary phloem, thus provid- ing information on 1) primary and secondary origin of intraxylary phloem, and 2) the origin and activity of the internal cambium. Furthermore, we compare the intraxylary with the regular secondary phloem.

MATERIAL AND METHODS

Fresh material of the scandent shrub Combretum rotundifolium Rich. was collected at the Instituto de Pesquisas Jardim Botânico do Rio de Janeiro (RB 464426, RBw 8978). Two dried samples of the same species were obtained from the herbarium of the same institution (RB 204242, RB 25014). Samples of the shoot apex and stem segments at different developmental stages were fixed in FAA 70 (formaldehyde, acetic acid, 70% alcohol; 1:1:18) (Johansen 1940) and glutaraldehyde 2.5% in a phosphate buffer solution (Gabriel 1982). After dehydration in alcohol, the samples of the shoot apex and younger internodes were embedded in historesin (Gerrits & Smid 1983). Sections 3–5 µm thick were cut on a rotary microtome, and stained with toluidine blue 0.05% (O’Brien et al. 1964). Sections 18–20 µm thick of the more mature portions, some of which were embedded in polyethylene glycol (Rupp 1964), were cut transversally and longitudinally on a rotary microtome, and stained with astra blue and safranin (Kraus & Arduin 1997). After the staining process, the sections were mounted in synthetic resin.

Anatomical terminology Six anatomical terms are adopted here, as follows: “perimedullary procambium” and “internal cambium” for the meristems in an atypical position, e.g. the peripheral area of the pith, and “regular procambium” and “regular cambium” for meristems in a typical position. Phloem produced externally and in the peripheral area of the pith is termed here as “regular phloem” and “intraxylary phloem”, respectively. The secondary phloem description follows the methodology of Richter et al. (1996).

RESULTS

First vascular tissue developmental stage (Fig. 1) A transverse section of the first internode shows that the regular procambium is initiated by an almost continuous band of cells with dense cytoplasm and conspicuous nuclei. At this stage, protophloem cannot be distinguished, but the first protoxylem elements are visible, bordered by peripheral pith parenchyma cells.

Origin of the intraxylary phloem In the third internode, the vascular bundles consist of protophloem and protoxylem already differentiated in a collateral arrangement. The perimedullary procambium, which consists of 2–3 cell layers, becomes evident right below the protophloem and protoxylem, with the exception of four leaf trace regions (Fig. 2–4). Initially, the perimedullary procambium shows 1) segments that are connected to the regular pro-

Downloaded from Brill.com09/25/2021 09:14:58PM via free access 16 IAWA Journal, Vol. 32 (1), 2011 cambium, and 2) segments that start actively generating phloem vascular cells (Fig. 4). Subsequently, all of these arcs differentiate into isolated groups of intraxylary phloem in the pith’s periphery immediately below the primary xylem (Fig. 5).

Figures 1–5. Transverse sections of young stem showing development of perimedullary procambium and intraxylary phloem in Combretum rotundifolium. – 1: First visible protoxylem elements (arrow) and parenchyma cells bordering the protoxylem. – 2: Overview of third internode with depressed areas lacking perimedullary procambium (arrows) in the vascular system. – 3: Detail of figure 2, showing a depressed area without perimedullary procambium, with protoxylem (arrows) and parenchyma cells right below. – 4: Detail of figure 2, with collateral bundles and perimedullary procambium (arrows) with low activity, producing the first phloem cells (circle). – 5: Isolated strands of intraxylary phloem (circles) from perimedullary procambium. — Scale bar for 1, 3 & 5 = 25 µm; for 2 & 4 = 50 µm.

→ Figures 6–11. Transverse sections of young stem showing the development of the regular and internal cambium, and of the intraxylary secondary phloem in Combretum rotundifolium. – 6: Well- developed regular cambium (large arrow), and intraxylary primary phloem with medullar ray (small arrow) in the pith’s periphery. – 7: Development of internal cambium (large arrow) from the perimedullary procambium. Small arrow indicates vascular ray of the intraxylary phloem. – 8: Areas of the pith’s periphery (arrows) lacking intraxylary phloem from the beginning of vascular differentiation. – 9: Development of internal cambium (large arrow) from parenchyma cell

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dedifferentiation on the pith’s periphery. Note the periclinal divisions in some of the cells (small arrow), and cells with obvious nuclei. – 10: Completely established single internal cambium with intraxylary secondary phloem (arrows). – 11: Bidirectional internal cambium activity with centrifugal axial and radial parenchyma cells (large arrow) and centripetal intraxylary phloem (small arrow). — Scale bar = 50 µm.

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Asynchronous development of the regular and intraxylary phloem (Fig. 6) In the sixth internode, development of the regular cambium begins with regular procambium differentiation. During secondary growth, the intraxylary phloem is even more confined to the perimedullary region. The intraxylary phloem basically consists of sieve tube elements, companion cells, axial parenchyma cells and stretched medullary cells constituting the medullary rays. Vascular rays are absent, further characterizing the intraxylary phloem as still in its primary growth phase.

Internal cambium development and activity In the eighth internode the start of secondary intraxylary phloem growth can be observed (Fig. 7). The internal cambium develops through differentiation of the perimedullary procambium in the areas with intraxylary phloem. At this stage, the areas corresponding to the remains of foliar trace outputs remain without intraxylary phloem differentiation (Fig. 2, 3 & 8). In the tenth internode, new internal cambium segments are formed from a cellular dedifferentiation process whereby parenchyma cells located in the periphery of the pith undergo radial and tangential expansion followed by divisions, as evidenced by prominent nuclei and thin periclinal walls (Fig. 9). Subsequently, a complete cylinder of internal cambium is established, and intraxylary phloem production begins throughout the pith’s periphery (Fig. 10). The internal cambium functions bidirectionally, but with an unusual production of parenchyma cells centrifugally and intraxylary phloem cells centripetally. The paren- chymatous band formed centrifugally is composed of three layers of cells, some of which form short radial series that are continuous with the rays of the intraxylary phloem (Fig. 11).

Comparison of the secondary intraxylary and regular phloem In the mature stem segments, the intraxylary phloem shows conspicuous secondary growth towards the pith, which is compressed (Fig. 12). The general arrangement of the intraxylary phloem is characterized by non-collapsed and collapsed portions with a poor delimitation, with some collapsed cell portions occurring very close to the internal cambium (Fig. 13). Ray dilatation occurs through cell expansion and division, resulting in multiseriate rays (Fig. 14). On the other hand, the regular phloem presents a general arrangement characterized by non-collapsed and collapsed phloem that are well delimited by the presence of sclerenchyma bands. The ray cells only dilatate slightly, and do not contribute to the distinction between the collapsed and not collapsed phloem (Fig. 15). Further differences can be found in the arrangement of phloem cells in the non-collapsed portions of the intraxylary and regular phloem (Fig. 14 & 16). In the intraxylary phloem, phloem cells are diffusely distributed (Fig. 14), while in the regular phloem, an irregular stratification of tangential bands occurs. Each band consists of a single cell type, as follows: crystalliferous idioblasts, parenchyma cells, sieve tube elements and companion cells, and parenchyma cells (Fig. 16). Fiber-sclereids are present at the edge of the intraxylary phloem, while the regular phloem shows tangential bands of fibers (Fig. 13 & 15).

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Figures 12–16. Transverse sections of mature stem showing regular secondary phloem and intraxylary secondary phloem of Combretum rotundifolium. – 12: Conspicuous intraxylary secondary phloem (arrows) compressing the pith. – 13: Intraxylary secondary phloem with collapsed cells (large arrow) near the cambial zone (black arrow). Fiber-sclereids presents in the internal margin of the intraxylary phloem (small arrows). – 14: Diffuse arrangement of phloem cells with sieve tube elements in groups (circle) and multiseriate ray (arrow) in intraxylary phloem. – 15: Regular phloem with non-collapsed (Ncp) and collapsed phloem (Cp), well-delimited by tangential sclerenchyma bands (large arrow), and uniseriate rays (small arrow). – 16: Irregular stratified arrangement of phloem cells in the regular phloem, with sequential bands of crystalliferous idioblasts (C), parenchyma cells (P), sieve tube elements (STE) and parenchyma cells (P). — Scale bar for 12, 13 & 15 = 50 µm; for 14 & 16 = 25 µm.

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In both the intraxylary and regular phloem calcium oxalate crystals are present, and sieve tube elements are predominantly arranged in groups (Fig. 14 & 16). Table 1 shows the anatomical similarities and differences of the intraxylary and regular phloem.

Table 1. Qualitative comparison between intraxylary and regular phloem in Combretum rotundifolium.

Characteristics Intraxylary phloem Regular phloem Tangential sclerenchyma bands Absent Present Irregular stratification of cell elements Absent Present Ray dilation More pronounced formed by Less pronounced formed by the expansion and division cell expansion of cells Delimitation between collapsed Not clear Distinct and non-collapsed area

DISCUSSION

Development of the perimedullary procambium, vascular bundles and intraxylary phloem In Combretum rotundifolium, the vascular system is initially composed of vascular collateral bundles from the regular procambium. The perimedullary procambium, when installing, connects to the regular procambium, producing a sinuous pattern in the early developmental stages. It seems likely that both procambia have a common origin through promeristem differentiation, despite the fact that the regular procambium starts its activity first. However, since the perimedullary procambium may have distinct origins, future studies employing techniques that use specific markers to determine its origin are necessary. Perimedullary procambium activity forms the primary intraxylary phloem in C. rotundifolium. However, intraxylary phloem can originate from various tissues: 1) procam- bial cells (Loganiaceae, Scott & Brebner 1889; Solanaceae, Cucurbitaceae, Asclepiada- ceae, Combretaceae, Apocynaceae, Campanulaceae, Convolvulacae, Araliaceae, Polygonaceae and Myrtaceae, Baranetzky 1900), 2) procambially derived cells (Con- volvulacae, Mikesell & Schroeder 1984; Patil et al. 2009), 3) meristem ground cells (Convolvulaceae, Scott 1891; Solanaceae, Esau 1938) and 4) dedifferentiation of parenchyma cells (Solanaceae, Esau 1938; Convolvulaceae, Patil et al. 2009). The later origin of intraxylary phloem compared to the vascular bundles is common in other families (Solanaceae, Esau 1938; Fabaceae, Kuo & Pate 1981; Convolvulaceae, Mikesell & Schroeder 1984), but in Combretaceae there is one report of a simultaneous or prior origin of intraxylary phloem compared to the vascular bundle in Quisqualis indica (Baranetzky 1900). Ontogenetic studies in plants with intraxylary phloem are

Downloaded from Brill.com09/25/2021 09:14:58PM via free access Zózimo, Tamaio & Vieira — Intraxylary phloem in Combretum 21 rare and usually do not discuss the nature of the vascular bundles bordered by internal phloem (Scott 1891; Baranetzky 1900; Esau 1938; Mikesell & Schroeder 1984; Patil et al. 2009), except Scott and Brebner (1889) who found a simultaneous origin of regular and intraxylary phloem in Strychnos (Loganiaceae), both having a common origin through the procambium, which means that the vascular bundles can be classified as bicollateral. In contrast, our ontogenetic analysis allows us to conclude that the primary intraxylary phloem in C. rotundifolium does not form a fascicular unit with these vascular tissues, meaning the vascular bundles can be classified as collateral. Our findings of a later origin of the intraxylary primary phloem confirms the incon- sistent terminology commonly applied to vascular bundles in Combretaceae. Phloem bordering the inner edge of xylem is described as either bicollateral bundles or intraxylary phloem (e.g., Solereder 1908; Verhoeven & Van der Schijff 1974; Van Vliet & Baas 1984). According to Metcalfe (1983), bicollateral bundles and intraxylary phloem are distinct cambial variations, but he does not report the developmental aspects of these variations. However, accuracy in classifying vascular bundles is only possible through ontogenetic studies.

Internal cambium origin and activity The occurrence of an internal cambium is already known for Combretum (e.g. Sole- reder 1908; Verhoeven & Van der Schijff 1974; Tilney 2002), although ontogenetic information is lacking. Following the onset of secondary growth, C. rotundifolium has a continuous internal cambium, with a double origin through perimedullary procambium differentiation and dedifferentiation of perimedullary parenchyma cells. The bidirectional activity of the cambium is an attribute of most eudicotyledon families, while unidirectional meristems are very rare in vascular plants, according to Carlquist (2004). A unidirectional perimedullary cambium is mentioned for Combre- taceae for Q. indica (Baranetzky 1900), unlike our observations in C. rotundifolium, where it has bidirectional activity, centrifugally producing a few bands of parenchyma cells (2–3 bands) and centripetally producing phloem. A similar centrifugal production of avascular tissue was seen in the regular cambium in Strychnos (Scott & Brebner 1889) while in C. nigricans sieve tubes are rare in the regular secondary phloem (Den Outer & Van Veenendaal 1995).

Comparison between regular and intraxylary phloem The regular and intraxylary phloem of C. rotundifolium differ qualitatively with re- gard to the general organization. Only one study extensively comparing the regular and intraxylary phloem could be found (Mikesell & Schroeder 1984). Further less detailed comparative information is available for Loganiaceae (Van Veenendaal & Den Outer 1993) and Convolvulaceae (Patil et al. 2009). Like our study of C. rotundifolium, these studies reveal that there is a similarity in composition between the regular and intraxylary phloem, but the general organization of the phloem tissues was not assessed. Our study is the first to show general organizational differences between these two phloem tissues in Combretaceae. The regular phloem shows a clear delimitation between the non-collapsed and the collapsed portions, unlike the intraxylary phloem. The arrange-

Downloaded from Brill.com09/25/2021 09:14:58PM via free access 22 IAWA Journal, Vol. 32 (1), 2011 ment of the cells of the non-collapsed portion is diffuse in the intraxylary phloem and irregularly stratified in the regular phloem. The vascular rays are multiseriate in the intraxylary phloem and uniseriate in the regular phloem, and the regular phloem has tangential sclerenchyma bands, while sclerenchyma cells are sparse in the intraxylary phloem. Both regular and intraxylary phloem have sieve tube elements arrayed in groups, simple sieve plates, parenchyma cells, crystals and sclerenchyma tissue. All the characteristics observed in the regular phloem are corroborated by data for Combretum in Den Outer & Fundter (1976), while the morphological data of the intraxylary phloem, as given in this report, have not previously been described for Combretum. Some authors have ascribed various physiological functions to the intraxylary phloem in other families, including: 1) assistance in the maintenance of apical dominance, 2) spatial division in the translocation of photoassimilates in the stem, 3) localized redistribution of photosynthate to branches, thus producing flowers and fruits, as well as 4) a higher amount of photosynthate translocated in the intraxylary phloem (e.g., Bonnenmain 1969; Botha et al. 1975; Zamski & Tsivion 1977; Kuo & Pate 1981). However, in Combretaceae the physiological role of the intraxylary phloem remains to be elucidated, and the anatomical data obtained for C. rotundifolium can be important to support the physiological studies of phloem transport. In the literature, the presence of intraxylary phloem for 38 species of Combretum (e.g., Verhoeven & Van der Schijff 1974; Tilney 2002) has been noted; however, since this genus has 250 species (Maurin et al. 2010), it is not known if the remaining species also have intraxylary phloem. Once intraxylary phloem has been observed in more specialized groups among the eudicotyledonous (Bonnemain 1969; Zamski & Tsivion 1977) and might confirm its sinapomorphic nature in Myrtales (Stevens 2001 onwards), more anatomical studies may emerge aimed at confirming its precise nature. Based on the presence of a single internal cambium, we suggest that the intraxylary phloem in C. rotundifolium should be considered as an individual category, additional to those recognized by Carlquist’s (2001).

ACKNOWLEDGEMENTS

We thank the staff at Laboratório de Botânica Estrutural of the Instituto de Pesquisas Jardim Botânico do Rio de Janeiro for technical support, Dr. Nilda Marquete for taxonomic identification and provid- ing information about the species studied, Daniela Canticas for helping with the English language composition and David Martim for reviewing the translation. Thanks are extended to the anonymous reviewers for their thoughtful reading of the manuscript.

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