Meristematic Activity of the Endodermis and the Pericycle in the Primary Thickening in Monocotyledons

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Anais da Academia Brasileira de Ciências (2005) 77(2): 259-274 (Annals of the Brazilian Academy of Sciences) ISSN 0001-3765 www.scielo.br/aabc

Meristematic activity of the Endodermis and the Pericycle in the primary thickening in monocotyledons. Considerations on the “PTM”

NANUZA L. DE MENEZES1, DELMIRA C. SILVA2, ROSANI C.O. ARRUDA3, GLADYS F. MELO-DE-PINNA1, VANESSA A. CARDOSO1, NEUZA M. CASTRO4, VERA L. SCATENA5 and EDNA SCREMIN-DIAS6 1Universidade de São Paulo, Instituto de Biociências Rua do Matão 277, Travessa 14, Cx. Postal 11461, Cidade Universitária, 05422-970 São Paulo, SP, Brasil 2Universidade Estadual de Santa Cruz, Departamento de Biologia Campus Soane Nazaré de Andrade, Km16, Rodovia Ilhéus-Itabuna, 45662-000 Ilhéus, BA, Brasil 3Universidade Federal do Estado do Rio de Janeiro, Centro de Ciências Biológicas e da Saúde Av. Pasteur 458, 22290-240 Rio de Janeiro, RJ, Brasil 4Universidade Federal de Uberlândia, Instituto de Biologia Campus Umuarama, Bloco 2D, Sala 28, 3840-902 Uberlândia, MG, Brasil 5Universidade Estadual de São Paulo, Instituto de Biociências de Rio Claro, Departamento de Botânica Av. 24A, 1515, Bela Vista, 13506-900 Rio Claro, SP, Brasil 6Universidade Federal de Mato Grosso do Sul, Centro de Ciências Biológicas e da Saúde(CCBS, DBI) Laboratório de Botânica, Cx. Postal 649, 74070-900 Campo Grande, MS, Brasil

Manuscript received on February 11, 2005; accepted for publication on February 16, 2005; contributed by Nanuza L. de Menezes*

ABSTRACT This paper proposes a new interpretation for primary thickening in monocotyledons. The anatomy of the vegetative organs of the following species was examined: Cephalostemon riedelianus (Rapataceae), Cyperus papyrus (Cyperaceae), Lagenocarpus rigidus, L. junciformis (Cyperaceae), Echinodorus paniculatus (Alis- mataceae) and Zingiber ofﬁcinale (Zingiberaceae). The endodermis with meristematic activity was observed in the root of all the species, in the stem of Cyperus, Cephalostemum and Lagenocarpus rigidus, and in the leaf trace of Cyperus and leaf of Echinodorus. Considering the continuity of tissues through the root, stem and leaf, the authors conclude that in the stem the pericycle remains active throughout the life of the plant as the generator of the vascular tissue. The “Primary Thickening Meristem” is in fact the pericycle plus the endodermis and its derivatives (or only the pericycle). Close to the stem apex, the assemblage of seems to be a unique meristem, giving rise to the inner cortex and vascular tissues. Key words: Cephalostemon, Cyperus, Echinodorus, Lagenocarpus, Zingiber, meristematic endodermis, pericycle, primary thickening.

INTRODUCTION

Since the 19th Century, researchers have presented *Member Academia Brasileira de Ciências Correspondence to: Nanuza L. de Menezes proposals in an attempt to understand the tissues that E-mail: [email protected] form the primary body of a monocotyledon stem,

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in particular, the limit zone between the cortex and histochemical system, and all these cells develop or the vascular cylinder. According to Mangin (1882), are induced to develop Casparian strips. Van Fleet this zone has been given different denomina- (1961) admits, as a sine qua non condition, the en- tions, such as the “pericambium”, “perimeristem”, dodermis as the inner cortex layer in the root, stem “properimeristem”, “generating zone”, “couche and petiole; and the inner layer of the mesophyll. dyctiogène”, and “thickening ring”. However, there The author draws attention to the fact that many au- has general agreement that this zone is made up of thors denominate the endodermis as “endodermoid adventitious roots. layer” when it presents Casparian strips in the stem More recently, authors attempting to un- and leaves, a term with which he does not agree. derstand this region have admitted the presence of a Another aspect which has been somewhat ne- primary thickening meristem (PTM), which is re- glected, is the radial disposition of the cortex cells sponsible for the primary thickening of the stem of the roots of mono and dicotyledons, a radiation in virtually all monocotyledons, as Rudall (1991) which originates in the cells of the endodermis. Ac- clearly demonstrates in her revision work on the cording to Mangin (1882), the root cortex of mono- PTM. For the same author (Rudall 1991) and oth- cotyledons consists of two regions: the external re- ers (especially Cheadle 1937, DeMason 1979a, b, gion, with disorganized parenchymatous cells, and 1980, 1983, Stevenson and Fisher 1980, DeMason the internal region, with cells organized in radial and Wilson 1985, Gifford and Bayer 1995), the rows. Of those who attempted to interpret these function of promoting the formation of adventitious radial rows of cells, the pioneers were Williams roots has also been attributed to the PTM. (1947), Hurst (1956 apud Van Fleet 1961) and Van Also according to Rudall (1991), some authors Fleet (1961). admit that the PTM corresponds to the pericyclic Williams (1947), while studying the apical region of the stem. Tomlinson and Zimmermann meristem, as well as the primary tissues in 74 species (1969, p. 174) states, literally, that “The problem of monocotyledons and 105 species of dicotyledons, was to decide whether there was a region in the demonstrated the presence of a layer of cells, which monocotyledonous stem, to which the term “peri- he denominated the plerome, a layer which would cycle” could be given. This is an entirely artiﬁcial later differentiate into the endodermis. Cortical cells concept, since in most monocotyledonous stems, the in all the roots investigated were produced by the cortex and central cylinder each end where the other rapid division of this layer of cells, and the arrange- begins”. ment of the cortical cells in relationship to the en- Another theme which has generated much dodermal layer, suggests the meristematic nature of polemic debate is the presence of the endodermis this layer, functioning as a true cambium. Following in the stem and leaves. In his extraordinary revi- the work of Williams (1947), Hurst (1956 apud Van sion work, Van Fleet (1961) demonstrates, giving a Fleet 1961) describes the same type of division in wealth of data and details, the presence of the en- Smilax as the results of a pro-endodermis, and hence dodermis in these organs. According to this author, Van Fleet (1961) refer to the endodermis as meris- although in the root (and underground stems) it can tematic. Heimsch (1951), studying the development be characterized by the presence of the Casparian of vascular tissue in the roots of barley, mentions strips, suberized walls or unilaterial deposits of cel- the works of Williams (1947), as being in agree- lulose in the aerial stems and leaves, it may or may ment with his own. However, in the roots of Pisum not present Casparian strips and other characteris- sativum, this same layer was referred to as a pro- tics, denominated “starch sheath”, “bundle sheath”, endodermal layer (Popham 1955), and again, more “border parenchyma” and “mestome sheath”. How- recently, in Trifolium (Mueller 1991), Hydrocharis ever, these varied types of cells produce the same morsus-ranae (Seago et al. 1999), and in Typha

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glaucai (Seago and Scholey 1999). the vouchers were deposited in the departmental Urano and Menezes (1996), with Cephaloste- herbarium (SPF). mon riedelianus, and Conceição and Menezes Rhizomes of Cephalostemon riedelianus Ko- (1996), with Nymphoides indica, refer to these ra- ern (Rapateaceae), N. Menezes 1347 b (SPF), and of diate layers as the result of “mother cells of the en- Lagenocarpus rigidus (Kunth.) Nees (Cyperaceae), dodermis”. On the other hand, Melo-de-Pinna and Vitta 690 (UEC), L. junciformis (Kunth) O. Kunth, Menezes (2003) refer to these layers as being the re- Vitta 540 (UEC) were collected at Serra do Cipó sults of meristematic activity of the endodermis, fol- (MG). Leaves of Echinodorus paniculatus Micheli lowingWilliams (1947) andVan Fleet (1961). Chea- (Alismataceae) were collected in Mato Grosso do dle (1937), observes the presence of radiate growth Sul, E. Scremin-Dias s/n (SPF 136.463). in the cortex of the root in Dracaena goldieana, The rhizomes of Cephalostemon and Lageno- external to the intact endodermis, and names this ra- carpus, as well as the leaves of Echinodorus, were diate region a “secondary cambial zone”. Guillaud fixed in FAA (Johansen 1940); following dehydra- (1878), referring to the same region also mentions a tion in ethanol series, the material was conserved “meristem external to the endodermis”, in reference in ethanol 70%. After inclusion in paraffin, histo- to the same region of radiate cortical cells. logical sections varying from 10 to 15 µm in thick- According to Cheadle (1937), there is no clear ness were prepared according to standard techniques differentiation of tissues on the cauline apex, and (Sass 1951, Johansen 1940). Later, the sections it therefore seems impossible to detect which layer were stained with astra blue and safranin (Bukatsch originates what he denominates as a “thickening 1972), concomitant with a double staining of crys- ring”. This could be the pericycle, which is gener- tal violet and Orange G (Purvis et al. 1964). The ally accepted as being outermost layer of the stele, slides were secured by sealing the cover slips with or possibly the endodermis, the innermost layer of Canadian balsam. the cortex. The rhizomes of Cyperus and Zingiber were The aims of this paper are to demonstrate: hand cut using a razor blade, and the cuts, after staining with astra blue and safranin (Kraus et al. 1998), (1) the continuity of tissues between the root, stem were mounted in 33% glycerin. For suberin, Sudan and leaf in monocotyledons; IV was used. (2) the pericycle as a generating layer of vascular tissues; RESULTS (3) the endodermis as having a meristematic func- The cortex in the root of Cyperus papyrus (Fig. 1-3) tion in the root, stem and leaf, combining to was found to be composed mostly of radiate rows form part of the stem and root cortex and part of cells originating from the endodermis. After the of the leaf mesophyll, and divisions, which give rise to the derivatives of the (4) to present a new hypothesis to explain growth meristematic endodermis (DME), (Fig. 3), thicken- in thickness in monocotyledons. ing and lignification of the internal cortex cells occurs, and the endodermis acquires Casparian strips MATERIALS AND METHODS (Fig. 1 and 2). There is a complete continuity be- Part of the material studied was collected in the tween the tissues of the root and those of the rhizome gardens of the Department of Botany of the IB- that gave rise to it (Fig. 4 and 5), and it can be ob- USP: rhizomes of Cyperus papyrus L. (Cyper- served that the layers of DME are also found to be aceae), N. Menezes 1416, rhizome of Zingiber of- thickened in the rhizome (Fig. 4). The pericycle is ficinale Roscoe (Zingiberaceae), N. Menezes 1417; uniseriate. Continuity of the tissues between the root

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Fig. 1-5 – Cyperus papyrus. Root in transverse (1-3) and longitudinal (4 and 5) sections showing almost all the cortex with the placing of its radiate cells, originated by endodermal (En) activity, constituting the DME (Derivatives of Meristematic Endodermis). Note in Fig. 4, the tissue continuity between the root and the rhizome. The inner cortex is shown thickened at the root (Fig. 1, 2 and 4) and in the rhizome (Fig. 4). The pericycle (Pr) is uniseriate. Rc – rhizome cortex. The bars correspond respectively to: 400µm, 100µm, 100µm, 600µm and 40µm. and the rhizome can be better observed in Fig. 6-8. where the DME cells in the stem possess the pri- The layers of the DME which constitute the inter- mary walls, the leaf trace in the cortex also presents nal cortex of the rhizome appear with primary walls DME layers without lignification (Fig. 12), how- (Fig. 6-9). The pericycle was found to be adjacent ever when the former become lignified, the DME of and internal to the endodermis, and is multiseriate the trace also appears lignified (Fig. 15). In those (Fig. 9). New vascular bundles can also be observed. regions closer to the apex (Fig. 16 and 17), it is pos- Initially, the endodermis exhibits Casparian sible to verify endodermal initials dividing, and the strips, and later, the walls of the endodermal cells beginning of the formation of DME layers. It is in contact with the pericycle (Fig. 9) can be seen to also possible to see the beginning of division of the be suberized. Suberization extends to all the walls pericycle (Fig. 16), placed between the endodermal of the endodermal cells (Fig. 10, 11 and 13). In initials and the procambium strands. In Fig. 17 it is Fig. 10 the DME cells still show only primary walls, possible to see the organization of a vascular bun- but, further away from the rhizome apex, secondary dle, which already (Fig. 18), exhibits differentiation walls are acquired, which become lignified in a cen- of tracheal elements (although with only primary tripetal pattern. This lignification finally reaches the walls) and the endodermis, with imperceptible Cas- endodermal cells themselves (Fig. 14). At the point parian strips (indicated by arrows). In regions of

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Fig. 6-9 – Cyperus papyrus. Transverse sections of the rhizome. Note (Fig. 6, 7 and 8) continuity of tissues between the root and the rhizome; the endodermis (En) in Fig. 7 appears with Casparian strips (arrow). The pericycle (Pr) appears as generating tissue of vascular tissues, especially in Fig. 9. DME – Derivatives of the Meristematic Endodermis; Rc – rhizome cortex; Vc – vascular cylinder. The bars correspond respectively to: 600µm, 400µm, 400µm and 100µm. the rhizome further from the apex, the inner cor- ceptible (Fig. 23). tex formed by the DME shows cells with thickened In a longitudinal section of the rhizome walls (Fig. 19), the pericycle being the generative of Cephalostemon riedelianus, as already seen in layer. Lagenocarpus, it is observed that the DME layers In a longitudinal section of a bud of the rhizome are thickened at the base of the rhizome (Fig. 24 and of Lagenocarpus rigidus (Fig. 20 and 21), it can be 25), and where they are not thickened, they appear noted that, near the apex (although the section is not translucent under the microscope. The pericyclic median), the walls of the DME layer are not thick- region forms a peripheral plexus (Fig. 25). Conti- ened, while those at the base are. The pericycle of nuity of the DME tissues between the adventitious the DME layers can be perfectly distinguished. In root and the rhizome is perfectly visible (Fig. 26, the region indicated by an arrow in Fig. 21, although 27 and 28), so that in Fig. 28 one can observe the formed by various layers of meristematic cells, it is radial rows of DME cells. possible to distinguish the pericycle from the en- In Zingiber ofﬁcinale the stem endodermis does dodermal initials (Fig. 22), by the lack of corre- not exhibit meristematic activity, and the pericycle, spondence between the cells resulting from the two adjacent to the endodermis with Casparian strips, meristems, and by a slight thickening of the exter- is clearly the generating layer for vascular tissues nal wall of the pericycle cells (arrow). In a region (Fig. 29 and 30). In Lagenocarpus rigidus, the en- further from the apex, this separation is more per- dodermis exhibits meristematic activity in both the

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Fig. 10-15 – Cyperus papyrus. Different levels of transverse sections of the rhizome, one noting the endodermis (En) in Fig. 10, with suberize walls (better observed in Fig. 13). The DME (Derivatives of the Meristematic Endodermis) cells are not thickened (Fig. 10). At the level presented in Fig. 11, the progressive thickening of the walls of DME cells can be seen, and in Fig. 12, a leaf trace in the cortex of rhizome, at the corresponding level in Fig. 10, also presents derivatives of the meristematic endodermis (DME). Fig. 13 shows the result of the reaction with Sudan IV. Fig. 14 – Note the completely thickened DME layers, and at the same level, the leaf trace (Fig. 15) with thickened DME cells. Pr – pericycle. The bars correspond respectively to: 200µm, 100µm, 100µm, 50µm, 50µm and 50µm. adventitious roots and the rhizome, with the same DISCUSSION type of centripetal thickening observed in Cyperus papyrus (Fig. 31). However in L. junciformis, the Although a few authors refer to the presence of endo- endodermis does not present meristematic activity dermis in the stem, almost none refer to the presence in the rhizome (Fig. 32). of the endodermis in the leaf, although Esau (1965), Meristematic activity is also found in the leaf, based on the work of Van Fleet (1961), states “... the as can be seen in Echinodorus paniculatus, where bundle sheath of angiospermous leaves is an endo- endodermal initials can be distinguished (Fig. 33). dermis” (Esau 1965 – pg. 441). Most authors refer These undergo periclinal divisions (Fig. 34) that will only to the “sheath of the leaf bundles”, ignoring its give rise to the DME layers (Fig. 35) which, in origin. As for the meristematic activity of the en- turn, will constitute a large part of the mesophyll dodermis, even Van Fleet (1961), in his admirable (Fig. 36). After periclinal division of the endoder- review of the endodermis, does not refer to meris- mal initial cells ceases, these exhibit anticlinal di- tematic activity in this tissue, either in the stem or visions, which accompany the differentiation of the the leaf. Van Fleet (1961), as well as all the au- vascular system, where a uniseriate pericycle can be thors quoted by him, including Williams (1947), re- recognized. fer only to the “cambial activity of the endodermis”

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Fig. 16-19 – Cyperus papyrus. Longitudinal (16 and 17) and transverse sections of the rhizome (18 and 19). Fig. 16 – The endodermal initial (Ei) is still in the division phase, and the pericycle (Pr) presents divisions in Fig. 17, which evidence differentiation of a vascular bundle (arrow). Fig.18 – Observe the endodermis already in differentiation, and adjacent to it, a new vascular bundle (Nv) originating in the pericycle (Pr). The arrows indicate imperceptible Casparian strips. Fig. 19 – All of the inner cortex is thickened and the pericycle gives origin to bundles. DME – Derivatives of the Meristematic Endodermis. Pc – procambium. Rc – rhizome cortex; Vc – vascular cylinder. The bars correspond respectively to: 100µm, 100µm, 50µm and 400µm. in the root. In this work, we demonstrate the pres- as proposed by Hurst (1956 apud Van Fleet 1961). ence of this activity also in the stem and the leaf. We prefer to adopt the proposal of Williams (1947), Here, we have veriﬁed the presence of radiate even though we recognize that both proposals are layers of cells constituting the cortex in the roots the same. of both Cyperus and Cephalostemon. We agree The number of layers that make up the DME in with the proposal of Williams (1947) and Van Fleet the root varies from species to species, forming only (1961), that the endodermis possesses an activity the internal cortex, as in Richterago (Melo-de-Pinna similar to that of the cambium, since the same layer and Menezes 2003) and Zingiber ofﬁcinale, almost of dividing cells gives rise both to the remaining the whole cortex, as in Cyperus papyrus, Bacopa layer of initial cells, as well as to the derivative (Bona and Morretes 2003) and Iantopappus (Melo- layer, which will constitute the next layer of the in- de-Pinna and Menezes 2002), or the whole cortex, ternal cortex. According to Williams (1947), after as in Nymphoides indica (Conceição and Menezes a certain number of divisions, the initials undergo 1996) and Cephalostemon riedelianus (Urano and differentiation, acquiring Casparian strips. Some Menezes 1996). recent authors (Seago and Scholey 1999), attribute According to Krauss (1948), the layer of cells the origin of the radial rows from a pro-endodermis, in the stem, external to the vascular cylinder, has

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Fig. 20-23 – Lagenocarpus rigidus. Longitudinal sections of the rhizome. Note in Fig. 20 and 21 a distinct separation between the pericycle (Pr), which is the generating layer of vascular tissue, and the derivative layers of the meristematic endodermis (DME). In Fig. 21 the arrow indicates the region represented in Fig. 22. In Fig. 22, the arrow indicates the wall that separates the pericycle in its meristematic phase, from the endodermal initial (Ei), this being slightly more thickened. Note a gap between the outermost layers of the pericycle and the endodermal initials. Rc – rhizome córtex; Vc – vascular cylinder. The bars correspond respectively to: 600µm, 400µm, 50µm and 100µm.

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Fig. 24-28 – Cephalostemon riedelianus. Transverse sections of the rhizome, one showing in Fig. 24 the DME (Derivatives of the Meristematic Endodermis) layers presenting a translucid aspect when not thickened. Fig. 25 – Note the totally thickened DME layers, the inner cortex of the rhizome and the pericycle (Pr) comprising the generating layer of vascular tissues. Fig. 26 – Note the presence of an adventious root and the continuity between the DME of the root and the rhizome, better visualized in Fig. 27. Fig. 28 – Very distinct layers of cells that comprise the DME. Pc – procambium. Rc – rhizome cortex. Vc – vascular cylinder. The bars correspond respectively to: 600µm, 100µm, 400µm, 200µm and 100µm. been interpreted in various ways, but most authors potentially meristematic tissue. consider it to be an endodermis, with the cells inter- The occurrence of an endodermis in rhizome nal to it constituting the pericycle. of Cyperaceae has already been mentioned by sev- The same author (Krauss 1948) further states eral authors, including Roseck (1992 apud Gifford “... it is not surprising that so many authors describe and Bayer 1995) in Cyperus esculentus, in which the presence of both the endodermis and the pericy- the author noted Casparian strips; Wills and cle in the stem of vascular plants, ﬁrstly, because it Briscoe (1970) and Wills (1985), in Cyperus ro- has traditionally been considered, in descriptions of tundus; Sharma and Mehra (1972), in Fimbrystilis; monocotyledons, that if the stem is divided into cor- Govindarajalu (1974), in several other species of tex and stele, a pericycle and an endodermis limiting Cyperus; Rodrigues and Estelita (2003), in Cype- the stele at the edge must be present; and secondly, rus giganteus; and Eiten (1969) in Eleocharis,in because on carrying out a superﬁcial examination of which she established that although she did not ob- the radial sections of the stem containing adventitial serve Casparian strips, she considered the endoder- roots, the endodermal cells of these roots appear mis easy to recognize due to the large size of its to be continuous with the endodermal cells of the cells. stem”. According to Krauss (l.c.), the pericycle is a Certain authors do not unequivocally identify

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Fig. 29-32 – Transverse sections of the rhizome of Zingiber officinale (Fig. 29 and 30) and longitudinal sections of rhizomes of Lagenocarpus rigidus (Fig. 31) and L. junciformis (Fig. 32). In Zingiber the endodermis (En) has no meristematic activity, the generating layer being the pericycle (Pr). In Lagenocarpus rigidus the rhizome presents DME (Derivatives of the Meristematic Endodermis), whereas in L. junciformis the endodermis does not possess meristematical activity in the rhizome. Rc – rhizome cortex; Vc – vascular cylinder. The bars correspond respectively to: 200µm, 50µm, 400µm and 400µm. the innermost layer of the cortex of the rhizome in In the cortex, in rhizomes of Cyperus papyrus, Cyperaceae as an endodermis, but prefer to call this Lagenocarpus rigidus and Cephalostemon riede- the endodermoid layer (Kukkonen 1967, Metcalfe lianus, we demonstrate not only the presence of the 1971), the endodermal sheath (Plowman 1906), or endodermis, but also that of a stratified region of endodermoid cells (Gifford and Bayer 1995). Met- cells which, originating from this endodermis, con- calfe (1971) makes it clear that he refers to this re- stitutes the internal region of the cortex. In the same gion, in the Cyperaceae, as the endodermoid layer, manner as in the root, we also denominate these because the occurrence of Casparian strips has not stratified layers as DME (Derivatives of Meristem- been confirmed, even though he declares that he sus- atic Endodermis). pects their presence in younger cells. The results presented here for the rhizome of We believe Van Fleet (1961) is correct when Cyperus papyrus demonstrate that after cell division he says that the endodermis is not endodermis for starting from the endodermis has been completed, the reason that it has Casparian strips or suberized the cells acquire Casparian strips. After this phase, cell walls, or any other characteristic, but because the endodermis cells become suberized. As the or- it is the innermost layer of the cortex in the root, gan matures, it is observed that progressive thicken- stem and petiole, and the innermost layer of the leaf ing and lignification of the cell walls of DME takes mesophyll. place (similar to that which occurs in the internal

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Fig. 33-36 – Echinodorus paniculatus. Leaves in differentiation, transversally cut, one showing in Fig. 33, endodermal initials (Ei) around the procambium (Pc) of each future bundle. Fig. 34 – The endodermal initials presents periclinal division and its first derivatives to comprise the DME (Derivatives of the Meristematic Endodermis). Fig. 35 – Endodermal initial presenting anticlinal division. Fig. 36 – Endodermis already undergoing differentiation, the mesophyll aerenchyma cavities being enclosed by derivatives of endodermal activity. Pr – pericycle. The bars correspond to 50µm. cortex of the root), until the point is reached where tissues are meristematic, the pericycle and the en- the endodermis itself becomes thickened, as can be dodermis appear to form a single meristem. It is confirmed in the leaf traces. Thickening of the walls only through serial sections that it was possible to of DME cells in the stem and leaf trace go hand in perceive, as in Lagenocarpus (Silva and Menezes hand. 2000, in Silva 2000), that the endodermis forms lay- This same type of endodermal division, as ob- ers of cells centrifugally, while the pericycle forms served in the leaf traces of Cyperus papyrus, also layers of cells centripetally. According to Williams occurs in leaves of Echinodorus paniculatus, where (1947), in the root “... no mitotic figure has ever the cells, as a result of the meristematic activity of been found that could indicate that the meristematic the endodermis, form a large part of the mesophyll, endodermal layer gives origin to cells internally, or providing evidence of continuity between the tissues that the pericycle produces cells externally (except of the stem and the leaf. in the formation of lateral roots)”. As we demon- It is this meristematic activity of the endoder- strate in this work, this observation applies also to mis in the rhizome which, in our opinion, has con- the stem. founded researchers who describe the primary In roots of Lagenocarpus rigidus and Cype- thickening meristem (PTM) in monocotyledons. In rus papyrus, we have been able to confirm an ob- the very young regions of the organ, in which the servation to which Williams (1947) drew attention:

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a slight thickening of the wall separates the endo- Mangin 1882) are in exact agreement with our re- dermal cell from the pericyclic one. This situation sults presented here, in that the pericycle of the rhi- is also noted in Richterago, by Melo-de-Pinna and zome corresponds to the tissue which he denomi- Menezes (2003). nates the “pericambium”. It is important to point out that in certain Mangin (1882) also states that Falkenberg crit- groups, even though thickening of the organ occurs, icizes the name “generating zone”, which is given cell division of the endodermis does not take place, to this layer in Araceae by Van Tieghem (1898). We as we have confirmed in the rhizome of Zingiber do not agree with this criticism, as we consider the officinale, and in Lagenocarpus junciformis. name “generating zone” to be very appropriate for Both in species in which the presence of the pericycle. a meristematic endodermis has been noted, and in Guillaud (1878) states that “in the stem of all species in which the endodermis does not divide, monocotyledons, when the primitive (apical) meris- the generating layer of vascular tissue (pericycle) tem gives rise to the cortex, the medulla and the is always situated internally to and contiguous with procambium strands, a generating zone appears at the endodermis. In our view, the generating layer of the limits of the cortex and the central body”. The vascular tissue in monocotyledons is the pericycle, author describes this as a zone with light, translucent principally because this layer is always contiguous tissues involving the outermost bundles, and denom- with the endodermis. inates it as the “perimeristem”. In our opinion, he The presence of a pericycle is even less fre- could have been referring to either the pericycle or quently mentioned, a fact which is demonstrated by to the derivatives of the meristematic endodermis, Beiguelmann (1962) in the leaf of Erythroxylum, which appear translucent in section when not thick- and by Menezes (1971) in Velloziaceae, through ened. This has been noted here, in Cyperus papyrus developmental studies and the tissue continuity be- and Cephalostemon riedelianus. tween the stem and the leaf. Evidence that it is possible to confuse the PTM According to Van Tieghem (1898), there is a with the endodermis with meristematic activity can bundle-generating zone on the periphery of the be seen in the statement by Rudall (1991), that “the stems of the Araceae, which he denominates the PTM often becomes lignified in older stems”. The “generating zone”, “occupying a part of the circum- cells of both layers (the pericycle and the layers reference in the genus Monstera, and complete in sulting from the meristematic endodermis - DME), Acorus”. According to this same author, this gener- by showing vacuolate cells at certain levels, exhibit ating zone is always found totally encompassed by cells with a translucent appearance, described by the endodermis. Fisher (1978) as the “light zone” in Musa, and by On the other hand, Falkenberg (1876 apud Rudall (1991) in her review of PTM and Secondary Mangin 1882) established that “the most external Thickening Meristem (STM). This observation cor- layer of the central cylinder of the root, the pericam- responds to that of Krauss (1948) in which, in the bium, with its delicate walls, corresponds to the ex- oldest part of the stem of Ananas, the translucid re- ternal layer of the central body of the stem of mono- gion becomes lignified. In this work, we verified cotyledons, mainly of the rhizome. And just as the that it is really the layers resulting from endodermal pericambium of the root is the center for the forma- activity (translucid layers), which form the internal tion of the lateral roots, in the same way, this layer cortex in stems, that becomes lignified in older rhi- of cells with delicate walls occupies the periphery zome. of the central body of the rhizome, and is the tissue Schat (1852 apud Mangin 1882) state that di- from which the adventitious roots are formed”. viding cells situated between the medulla and the These observations by Falkenberg (1876 apud cortex are encountered in all vascular plants. These

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dividing cells he called the “cambial circle” or these authors make no mention of the pericycle tis- “thickening ring”. This ring is more or less com- sue and do not believe that “cap meristem” gives rise pletely lignified in monocotyledons where the stem to vascular tissues. This is mainly because they not no longer thickens. However, when this activity consider it to be identical to the PTM. On the other continues, as in some monocotyledons, as is the case hand, the authors do not recognize the role of this with the stem of Pandanus and Dracaena, con- meristem in primary thickening in Prionium. siderable thickening can be achieved. Adventitious From the results obtained here, any lingering roots which develop are dependant on this tissue, doubt as to the presence of layers originating from which the author calls the “cambium”. According meristematic activity of the endodermis in the stem to him, it is the same meristem that defines the form must now be dispelled, in light of the evidence of of the secondary body, and influences the develop- the origin of adventitious roots, and the possibility ment of lateral roots, shoots and leaves. We be- of following the continuity of the tissues between lieve that the “cambial circle” of Schat corresponds the stem and the root. In the developing root, the to what we believe to be pericycle + endodermis vascular cylinder is completely continuous with the with meristematic activity, and that the “thickening vascular system of the stem, just as the endoder- ring” corresponds, in fact, to layers resulting from mis is perfectly continuous with the endodermis of the meristematic endodermis (DME), thickened as the stem. The same occurs with its resulting layers we have shown in Cephalostemon and Cyperus. (DME). We also demonstrate here that in Cyperus pa- In Ananas comosus, Krauss (1948) noted the pyrus, Cephalostemon riedelianus and Lageno- continuity of tissues between the root and the stem, carpus rigidus, the DME layers thicken because in without, however, admitting continuity between the these plants, secondary growth does not occur. It is endodermis of the root and that of the stem, through likely that same thing occurs with Xyris (Sajo and the non-detection of Casparian strips. Rudall 1999), as well as Cyperus giganteus (Ro- Although Gifford and Bayer (1995) demon- drigues and Estelita 2003), even though the last au- strate the same configuration in Cyperus esculentus, thors have described secondary growth in Cyperus at no time do they mention the continuity of tis- giganteus. sues between root and stem. Furthermore, based on Falkenberg (1876 apud Mangin 1882), in order the works of Gifford and Bayer (1995), particularly to explain the separation of the stem into cortex and through observing figures 8, 12 and 16, it becomes central cylinder, states: “in the stems of the mono- clear that what these authors called PTM refers to cotyledons, an internal mass of tissue is separated the region here called DME. from an external mass, the cortex, this separation As stated earlier, we have no doubt that pri- being frequently visible in transversal sections, by mary thickening in monocotyledons occurs through the formation of a limiting sheath”. He admits that the activity of the pericycle. Naturally, the first bun- the origin of this sheath may be two-fold, that is dles in the stem apex are of procambial origin. The to say, formed by the outermost cells of the central procambium gives origin to the pericycle, which re- body and the innermost cells of the cortex. Our remains active throughout the life of the plant as the sults lead us to agree with Falkenberg, as this region, generating tissue, as in Cyperus, Lagenocarpus and which separates the cortex from the vascular cylin- Zingiber. It is important to point out that the peri- der, can be formed by the pericycle as well as the cycle forms exclusively primary vascular tissue. endodermis and its derivatives. Through the work of Mangin (1882), it is ob- We believe that our results agree more with served that authors always refer to a generating layer the “cap meristem” of Zimmermann and Tomlinson of vascular bundle in monocotyledons, and that it (1968) than with any other concept, even though is this layer, called variously the “pericambium”,

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“perimeristem”, “properimeristem”, “couche dyc- tributes to adventitious root production and the pri- tiogené”, or other denominations, that generates the mary stem body – both centripetally and centrifu- lateral roots in roots and adventitious roots in stems. gally”. In the present work, this layer is believed to be the We do not agree with these positions, which pericycle. We believe that the plant is a unit, that the we declare to be mistaken, as it is only the funda- endodermis is present in the root, the stem and the mental meristem that forms the cortex, and the pro- leaf, and that the tissue internal to the endodermis cambium which forms the primary vascular system; is the pericycle in the root, the stem and the leaf. It this in agreement with Robbins and Ricket (1939), is the pericycle which, as in the roots of dicotyle- Esau (1965, 1977), Fahn (1989), Mauseth (1995), dons forms the cambium in front of the protoxylem Raven et al. (1999), and the 19th Century European poles, in the stems of dicotyledons, forms the inter- researchers mentioned earlier, among many others. fascicular cambium, in the stems of monocotyledons It is important to understand that the pericy- generates the meristem responsible for secondary cle produce tissues centripetally and that the endo- thickening, i.e. the STM (Secondary Thickening dermis, with meristematic activity, produce tissues Meristem). (DEM) centrifugally. Even authors such as Rudall (1984, 1991), who To summarize, the anatomical data obtained always make reference to the primary thickening here strongly supports: meristem (PTM) in monocotyledons, admit that this (1) the hypothesis that the “PTM” is none other meristem “occurs in the pericyclic region”. than the pericycle itself or the pericycle + en- Some authors, such as Skutch (1932), Helm dodermis + DME; (1936 apud Rudall 1991), Krauss (1948) and Stevenson (1980) have described the PTM as (2) the pericycle (originating from the procam- a meristematic region continuous with the apical bium) is the tissue that remains active through- meristem. Actually, the meristematic pericycle is out the life of the plant, giving origin to the continuous with the apical procambium. vascular tissues; We once again state, from what can be seen in Cyperus, Lagenocarpus and Cephalostemon, that (3) the endodermis can also have meristematic ac- what these authors call the PTM is none other than tivity in the stem and leaf of monocotyledons. the pericycle itself, or the pericycle + endodermis + DME. RESUMO According to Gifford and Bayer (1995), the A proposta deste trabalho é mostrar uma nova interpre- PTM only produces cortical parenchyma and does tação do meristema de espessamento primário em mono- not produce vascular tissue, however, judging from cotiledôneas. Anatomia dos órgãos vegetativos das se- their illustrations, they were referring to what we guintes espécies foi examinada: Cephalostemon riede- here consider the DME. Baranetsky (1807 apud lianus (Rapataceae), Cyperus papyrus (Cyperaceae), Cheadle 1937), on examining the development of Lagenocarpus rigidus, L. Junciformis (Cyperaceae), several types of stem apices, established that “tis- Echinodorus paniculatus (Alismataceae) and Zingiber sues of the cortex and the vascular cylinder could ofﬁcinale (Zingiberaceae). A atividade meristemática da be composed of cells of different origins”, which endoderme foi observada nas raizes de todas as espécies, means that it would not only be the procambium that no caule de Cyperus, Cephalostemum e Lagenocarpus forms the stele, or only the fundamental meristem rigidus, e no traço foliar de Cyperus e folha de Echino- that forms the cortex. dorus. Considerando a continuidade dos tecidos através In her review of PTM, Rudall (1991) states that da raiz, caule e folha, as autoras concluem que no caule o most authors have established that “the PTM con- periciclo permanece ativo durante a vida da planta, como

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