Pulvinus Functional Traits in Relation to Leaf Movements: a Light and Transmission Electron Microscopy Study of the Vascular System§ Tatiane M

Micron 39 (2008) 7–16 www.elsevier.com/locate/micron

Pulvinus functional traits in relation to leaf movements: A light and transmission electron microscopy study of the vascular system§ Tatiane M. Rodrigues *, Silvia R. Machado Sa˜o Paulo State University – UNESP, Institute of Biosciences, Department of Botany, PO Box 510, 18618-000 Botucatu, SP, Brazil Received 11 June 2007; received in revised form 4 September 2007; accepted 6 September 2007

Abstract Previous studies on legume pulvini suggest that the vascular system plays an important role in the redistribution of ions and transmission of stimuli during leaf’s movements. However, the number of anatomical and ultrastructural studies is limited to few species. The aim of this paper is to investigate the structure and cellular features of the pulvinus vascular system of nine legume species from Brazilian cerrado, looking for structural traits pointing to its participation in the leaf’s movements. Samples were excised from the medial region of opened pulvinus of Bauhinia rufa, Copaifera langsdorffii, Senna rugosa (Caesalpinioideae), Andira humilis, Dalbergia miscolobium, Zornia diphylla (Faboideae), Mimosa rixosa, Mimosa flexuosa and Stryphnodendron polyphyllum (Mimosoideae), and were prepared following light microscopy, transmission electron microscopy and histochemical standard techniques. The vascular system occupies a central position, comprises phloem and xylem and is delimited by a living sheath of septate fibers in all the species studied. This living cells sheath connects the cortex to the vascular tissues via numerous plasmodesmata. The absence of fibers and sclereids, the presence of phenolic idioblasts and the abundance and diversity of protein inclusions in the sieve tube members are remarkable features of the phloem. Pitted vessel elements, parenchyma cells with abundant cytoplasm and living fibriform elements characterize the xylem. The lack of lignified tissues and extensive symplastic continuity by plasmodesmata are remarkable features of the vascular system of pulvini of the all studied species. # 2007 Elsevier Ltd. All rights reserved.

Keywords: Fabaceae; Leaf movement; Pulvinus; Structure; Vascular system

1. Introduction The majority of pulvinus’s movement information is based on changes in the shape and size of cortical parenchyma cells Pulvini are organs that regulate leaf position and thus (called motor cells) caused by alterations in their turgor in intervene in photosynthetic activity in many angiosperms response to K+ and Cl fluxes, as well as other ions (Satter and families, mainly in Fabaceae species those are of considerable Galston, 1981; Moran et al., 1990). On this view, leaf interest in agriculture in tropical and subtropical areas. Due movements are similar in many points to stomata movements, their activity, pulvini are involved in their adaptation to the widely studied and well documented at molecular level. environment. In natural habitats, pulvinus movements benefit Researches indicate that besides the turgor changes of motor the plant by maximizing the radiation absorbed in situations of cells, the pulvini movements are due to alterations in the limited light and, on the other hand, by reducing the deficit of configuration of the actin microfilaments (Fleurat-Lessard, irradiance in situations where the photosynthesis may be 1988; Kameyama et al., 2000; Yamashiro et al., 2001). limited, thereby reducing transpiration, leaf temperature and However, there is evidence suggesting that not only the cortical photoinhibition (Ehleringer and Forseth, 1980; Koller, 1990; cells but also the vascular apparatus of the pulvinus participates Caldas et al., 1997). in the redistribution of ions and in the transmission of stimuli during the leaf’s movements (Pfeffer, 1907; Toriyama, 1954; Satter and Galston, 1981; Fleurat-Lessard and Bonnemain, § Part of T.M. Rodrigues’ Master’s thesis in connection with the Post- 1978; Moysset and Simo´n, 1991). graduate Program in Biological Sciences (Botany) of the Institute of Bios- ciences, UNESP, Botucatu, SP, Brazil. The little information available on the ultrastructure of * Corresponding author. Tel.: +55 14 38116053; fax: +55 14 38153744. pulvinus vascular tissues of legumes is limited to a few E-mail address: [email protected] (T.M. Rodrigues). species of Mimosoideae, especially Mimosa pudica, which is

0968-4328/$ – see front matter # 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.micron.2007.09.001 8 T.M. Rodrigues, S.R. Machado / Micron 39 (2008) 7–16 characterized by rapid leaf movements. Detailed information continuum of savanna formations lies between these two on the pulvinus vascular system of Brazilian cerrado legumes is extremes and spans the entire range of woody plant density; the restricted to Pterodon pubescens (Machado and Rodrigues, whole biome is collectively referred to as the cerrado 2004; Rodrigues and Machado, 2004), a tree with slow leaf (Oliveira-Filho and Ratter, 2002). Most trees and shrubs have movement. In this species, we verified the occurrence of a a thick bark, twisted trunks and scleromorphic leaves (Franco sheath composed of septate fibers, originated from the et al., 2005). Soils are deep, strongly acid dystrophic latosols, pericycle, with living protoplast around the phloem. This with high Al contend. The climate is Cwb (mesothermal with finding is a novelty, since the sheath around the phloem of other dry winters) by Ko¨ppen (1931) classification, in which the legume specie has been shown a collenchymatous sheath (Esau, warmest month presents an average temperature no higher than 1970; Fleurat-Lessard and Bonnemain, 1978; Moysset and 22 8C, and the month of July is the coldest and driest of the Simo´n, 1991). year. The annual rainfall is 1534 mm, with a distinct dry season In this study, we investigated the anatomical and histological from May to September (81–89 mm, respectively). Average characteristics of the pulvinus central cylinder of nine legumes (diurnal) relative humidity is around 80% during the rainy of the Brazilian cerrado, looking for structural and cellular season and drops to 55% during the dry season when daily traits that might intervene in mechanical properties of pulvini, minimum relative humidity reaches values around 15%. Mean and in lateral transport of stimuli and nutrients between central annual temperature is about 20.3 8C. Climatic data were cylinder and cortex. This paper also addresses the question collected from Meteorological Station of the Prataˆnia about the nature of the cell sheath around the phloem. municipality. Nine legume species belonging to the three subfamilies and 2. Materials and methods with different type and velocity of leaf movement were selected as indicated in Table 1. For all the species, samples were 2.1. Plants excised from the median region of the full opened primary pulvinus from mature leaves located at the fifth node from the This study involved legume species occurring in a particular apex. Three individuals were sampled from each species, area of cerrado vegetation (S22848050.200 and W48844035.800) yielding 10 leaves for each individual. located on the Palmeira da Serra ranch in the municipality of Prataˆnia, state of Sa˜o Paulo, Brazil. The Brazilian cerrado 2.2. Light microscopy (LM) covers nearly 2 million km2, representing ca. 22% of the country’s land surface (Oliveira and Marquis, 2002). The Pulvinus samples were fixed in FAA 50 (Johansen, 1940), biome is extremely variable in physiognomy and ranges from dehydrated in an alcohol series and embedded in methacrylate open grassland to forest with a discontinuous grass layer. A resin (Gerrits, 1991). The 8 mm thick sections, cut with a rotary

Table 1 Summary of the legume studied species, their habits, type and velocity of leaf movement, pulvinus width and percentage occupied by the pulvinus regions Subfamily Species Habit Leaf movement Total width Percentage Percentage Percentage Vascular of pulvinus occupied occupied by occupied by system (mm) by cortex vascular pith in the shape cylinder vascular cylinder Caesalpinioideae Bauhinia rufa Shrub Slow nyctinastic 4.48 48.44 51.56 43.72 Opened ring (Bong.) Steud and heliotropic with pith vascular bundles Copaifera Tree Slow nyctinastic 2.66 48.50 51.5 46.32 Ring langsdorffii Desf. and heliotropic Senna rugosa (G. Don.) Shrub Slow nyctinastic 3.64 44.23 55.77 29.06 Ring H.S. Irwin & Barneby and heliotropic Faboideae Andira humilis Shrub Slow nyctinastic 2.97 61.30 38.70 26.96 Arc Mart. Ex Benth. and heliotropic Dalbergia Tree Slow nyctinastic 3.53 60.42 39.58 28.12 Ring miscolobium Benth. and heliotropic Zornia diphylla Pers. Herbaceous Slow heliotropic 1.92 61.98 38.02 38.36 Semi-arc Mimosoideae Mimosa rixosa Mart. Herbaceous Slow nyctinastic 1.85 50.81 49.19 40.66 Continuous and heliotropic cylinder and fast seismonastic Mimosa flexuosa Mart. Herbaceous Slow nyctinastic 1.14 53.50 46.50 46.15 Continuous and heliotropic cylinder and fast seismonastic Stryphnodendron Tree Slow nyctinastic 3.11 57.23 42.77 28.57 Continuous polyphyllum Mart. and heliotropic cylinder T.M. Rodrigues, S.R. Machado / Micron 39 (2008) 7–16 9 microtome, were stained with 0.05% Toluidine blue (O’Brien juxtaposed extremities, and two collateral bundles in the pith. et al., 1964), pH 4.7. Freehand sections taken from samples The vascular system of Andira humilis (Fig. 1D) is arc-shaped stored in 70% alcohol were sliced with shaving blades and with free outward curved extremities. In Zornia diphylla stained with Safranin and Astra blue stain (Burger and Richter, (Fig. 1F), five vascular bundles separated by interfascicular 1991). To detect the presence of lignin and pectin, freehand parenchyma are organized in a semi-arc. In M. rixosa (Fig. 1G), fresh sections were treated, respectively, with acid phloroglucin M. flexuosa (Fig. 1H) and Stryphnodendron polyphyllum (Sass, 1951) and ruthenium red solution 0.02% (Jensen, 1962). (Fig. 1I), the vascular tissues comprise a continuous cylinder. Lipids, starch grains, phenolic compounds and oxalate calcium The outer portion of the vascular system of the studied crystals were identified using Sudan IV (Johansen, 1940), species is composed of a sheath of five to seven small cell layers Lugol reagent (Johansen, 1940), 10% ferric chloride (Johansen, with a very thick non-lignified primary cell wall (Fig. 2A, C and 1940) and 10% chloride acid (Chamberlain, 1932), respec- E). These cells have dense cytoplasm and voluminous and tively. clearly visible nucleus; they are able to divide periclinally, Measurement of pulvinus and central cylinder width was giving rise to septa (Fig. 2C). The longitudinal sections done in transversal sections using a millimeter ruler. All (Fig. 2D) and the dissociated material revealed that these cells samples were obtained from the pulvinus median region. The are elongated with thin ends and their walls have conspicuous total width of pulvinus is expressed in mm and the region primary pit fields, each with three or more septa. Thus, they are occupied of cortex, vascular cylinder and pith are expressed as characterized as septate fibers, which substitute the fibers with percentages. thick and lignified cell walls surrounding the phloem of the petiole (Fig. 2B) in the same species. 2.3. Transmission electron microscopy (TEM) In all the species of this study, the primary pulvinus phloem is composed of sieve tube members associated with one or more For ultrastructural studies, the samples were fixed in 2.5% conspicuous companion cells and parenchyma cells (Fig. 2E). glutaraldehyde solution in 0.1 M phosphate buffer, pH 7.3, for Fibers and sclereids are absent. The phloem shows phenolic 24 h at 5 8C, post-fixed with 1% osmium tetroxide in the same idioblasts (Fig. 2A) in all the species except Z. diphylla.InB. buffer for 1 h, dehydrated in acetone series and embedded in rufa (Fig. 2A), A. humilis, D. miscolobium and M. rixosa, these Araldite (Machado and Rodrigues, 2004). Ultra-thin sections phenolic idioblasts are more abundant and voluminous. In B. were stained with uranyl acetate and lead citrate (Reynolds, rufa, S. rugosa and A. humilis, the phloem has idioblasts with 1963) and then examined in a Philips EM 301 electron prismatic calcium oxalate crystals. Mucilaginous idioblasts that microscope. stain as intense pink under Toluidine blue occur in M. rixosa (Fig. 2E). 3. Results In the pulvinus xylem, vessels of variable sizes and lumen contours are associated with voluminous parenchyma cells and 3.1. Light microscopy non-lignified fibers, called fibriform elements here (Fig. 2A). D. miscologium showed parenchyma cells with prismatic calcium A cross-section of the primary pulvinus of the species under oxalate crystals. Phenolic idioblasts were found in the xylem study showed a uniseriate epidermis, a wide parenchymal parenchyma rays of B. rufa (Fig. 2A), C. langsdorffii, A. humilis cortex and a central vascular system (Fig. 1A). and D. miscolobium. These rays cross the xylem and the phloem Table 1 shows the total width of pulvinus to different species and reach the sheath of septate fibers (Fig. 2A). In M. rixosa and the proportional size (expressed as percentage) that the (Fig. 1G), M. flexuosa (Fig. 1H) and S. polyphyllum (Fig. 1I) the cortex, vascular cylinder and pith occupy in it. The numerical parenchyma rays are little visible. data showed that there is not a relation between the pulvinus As seen in Table 1, the size of the pith varies among the total width and the subfamily to that it belongs. However, we species. The pith, which is reduced (Fig. 1A–I) and almost can note a relation between pulvinus width and the movement absent from M. rixosa (Fig. 1G) and M. flexuosa (Fig. 1H), velocity; so, the smallest pulvini (Mimosa rixosa and Mimosa consists of parenchyma cells that are less voluminous than the flexuosa) exhibit fast movements (seismonastic movements). cortical parenchyma cells. In B. rufa, C. langsdorffii, A. humilis, We observed a consistent relation between cortex and vascular Z. diphylla, M. rixosa and M. flexuosa the parenchyma cells cylinder width and the subfamilies, being the greatest values have thicker walls and thin lumen. Cells with phenolic showed by Faboideae followed by Mimosoideae and than, compounds were observed in B. rufa, A. humilis (Fig. 1D), Z. Caesalpinioideae species. The ratio of cylinder size and cortex diphylla and M. flexuosa. These cells are more numerous in D. size is not related to the type and velocity of movement. No miscologium and more voluminous in A. humilis (Fig. 1D) and relation was observed between the pith size or the cell kinds that Z. diphylla. constitute the pith with movement type and velocity. The vascular system of Senna rugosa (Fig. 1A), Copaifera 3.2. Transmission electron microscopy langsdorffii (Fig. 1C) and Dalbergia miscolobium (Fig. 1E) consists of juxtaposed collateral bundles organized in a ring, 3.2.1. Septate fibers without pith bundles. In Bauhinia rufa (Fig. 1B), the vascular The septate fibers can show very thick walls with irregular system has an outer portion with an open-ring shape with contours, loose and multilayered features as in C. langsdorffii 10 T.M. Rodrigues, S.R. Machado / Micron 39 (2008) 7–16

Fig. 1. Light micrographs of cross-section of legume primary pulvini. (A) General feature of Senna rugosa showing uniseriate epidermis (ep), wide parenchyma cortex (ct) and ring-shaped central vascular system (vs). (B–I) General feature of pulvinus vascular system. (B) Bauhinia rufa. Ring-shaped vascular system and two additional collateral vascular bundles in pith position. (C) Copaifera lagnsdorffii. Ring-shaped vascular system. (D) Andira humilis. Arc-shaped vascular system with free ends curved outward. (E) Dalbergia miscolobium. Ring-shaped vascular system. (F) Zornia diphylla. Semi-arc-shaped vascular system composed of five collateral bundles separated by inter-fascicular parenchyma. (G–I) Ring-shaped vascular system. (G) Mimosa rixosa. (H) Mimosa flexuosa. (I) Stryphnodendron polyphyllum. Scale bars: (A) 200 mm; (B–E) 250 mm; (F) 127 mm; (G, I) 160 mm; (H) 110 mm.

(Fig. 3A and B), or thin walls as in S. polyphyllum (Fig. 3C). electron transparent and can be small, as in C. langsdorffii Primary pit fields with plasmodesmata (Fig. 3B) connect the (Fig. 3A and B) and M. rixosa, or they may occupy almost all septate fibers to each other and to the adjacent tissues. These the cell lumen, as in S. polyphyllum (Fig. 3C). Around the cells have a conspicuous irregular-shaped nucleus with evident septum, the cytoplasm is more abundant and dense (Fig. 3C). nucleolus (Fig. 3A) and dense cytoplasm with free ribosomes, Each septum is composed of middle lamellae with several polyribosomes, voluminous mitochondria with well developed plasmodesmata and two portions of primary cell wall (Fig. 3C). crests, dictyosomes with numerous cisterns and adjacent vesicles, plastids with well-structured grana, electron-dense 3.2.2. Phloem stroma and osmiophilic inclusions, endoplasmic reticulum and The phloem comprises sieve tube members and companion oil droplets dispersed or in vacuoles (Fig. 3B). The vacuoles are cells, besides parenchyma cells with developed vacuome and T.M. Rodrigues, S.R. Machado / Micron 39 (2008) 7–16 11

Fig. 2. Light micrographs of legume primary pulvini. (A) Cross-section showing septate fiber sheath (sf) between endodermis (arrows) and phloem (ph) in Bauhinia rufa. Note the voluminous phenolic idioblasts (*) in the phloem. xy: xylem. (B) Cross-section of Bauhinia rufa petiole showing lignified fiber (lf) sheath around the phloem (ph). The asterisk (*) indicate phenolic idioblasts. (C) Cross-section detail of septate fibers of Senna rugosa pulvinus, showing conspicuous nuclei (arrows) and periclinal cell divisions (arrow heads). (D) Longitudinal section of Senna rugosa showing septate fibers with evident nucleus, thick pectin–cellulose walls with primary pit fields (arrow heads) and septa (arrows). (E) Mimosa rixosa cross-section showing endodermis (en), septate fibers (sf) and phloem with sieve tube members (st), companion cells (cc), parenchyma cells (pc) and mucilaginous idioblasts (id). Scale bars: (A, B) 30 mm; (C–E) 20 mm. electron transparent content (Fig. 3D and E) and phenolic encircled by the sieve tube reticulum (Fig. 4C). Intact plastids, idioblasts. Fibers or sclereids were not observed in the phloem which are circular in transversal sections (Fig. 4A and D) and of all the species. have protein inclusions, are called P-plastids. P-plastids were The sieve tube members are characterized by the presence of found with crystalline protein bodies and fibrillar proteins bright pecto-cellulosic walls with variable thickness and a more (Fig. 4A), and with crystalline protein bodies and fibrillar and electron-dense inner contour (Fig. 3D and E). In Z. diphylla tubular proteins (Fig. 4D). (Fig. 3E), the sieve tube members show nacreous walls Companion cells are characterized by voluminous nucleus identified by the greater thickness, inner irregular contour and and dense cytoplasm (Fig. 3D and E), in which ribosomes, loose structure; these cells may be totally or partially voluminous mitochondria and endoplasmic reticulum are obliterated. The sieve tube members are characterized by the visible; vacuoles and vesicles are small or absent. In the most presence of sieve plate, plastids-P (Fig. 4A) and disperse of the species, were observed ordinary companion cells that are protein material. Callose accumulations of various sizes were characterized by thick cell wall with smooth inner surface visible around the pores of the sieve plate (Fig. 4A) in all the (Fig. 3D, except in Z. diphylla and M. rixosa. In these latest, the species. companion cells show transfer cell features as fingerlike wall In all the analyzed species, the reticulum of the sieve tube ingrowths particularly more developed on the cell walls that member was represented by a net of tubular membranes in face away the sieve elements (Fig. 3E). variable degrees of development. These membranes are The phloem parenchyma cells (Fig. 3D and E) show walls of arranged perpendicularly to the inner surface of the cell wall variable thickness and electron-density, with primary pit fields, (Fig. 4A and B), in some cases extending along the entire cell abundant cytoplasm with ellipsoidal chloroplasts and devel- wall. Globular mitochondria were observed near the plasma- oped vacuome that can be electron translucent. In M. rixosa, the lemma of the sieve tube members and may be totally or partially cytoplasm is dense and has numerous free ribosomes, globular 12 T.M. Rodrigues, S.R. Machado / Micron 39 (2008) 7–16

Fig. 3. TEM micrographs of vascular system of legume primary pulvinus. (A) Septate fiber in Copaifera langsdorffii primary pulvinus in longitudinal section showing multilayered thick pectin–cellulose cell wall (cw), nucleus (n) with evident nucleolus (nu) and dense cytoplasm with mitochondria (mi) and polyribosome. (B) Cross-section of Copaifera langsdorffii septate fiber showing loose multilayered thick cell wall (cw) and primary pit field (ppf) with plasmodesmata. In the dense cytoplasm, oil (ol) droplets are observed. (C) Part of Stryphnodendron polyphyllum septate fiber showing thin cell wall and septum constituted of middle lamellae and two portions of cell wall. (D) Mimosa rixosa phloem showing sieve tube members (st) with wide lumen, companion cells (cc) and parenchyma cells (pc). (E) Zornia diphylla phloem showing sieve tube members (st) with nacreous walls, companion cells (cc) modified in transfer cells and parenchyma cells (pc). The arrow indicates crystalline protein body in the sieve tube member. Scale bars: (A) 0.75 mm; (B, C, E) 1.3 mm; (D) 1.5 mm. mitochondria with prominent crests, rough endoplasmic fibriform elements in most of the studied species. In C. reticulum and plastids with vesicles and plastoglobules. langsdorffii and S. polyphyllum (Fig. 5B), the vessel elements Voluminous idioblasts have thin walls, peripheral nucleus, have vestured pits. reduced cytoplasm and central vacuole filled with very The xylem parenchyma cells have thin walls with numerous electron-dense content, identified as phenolic substances by primary pit fields and wide plasmodesmata connecting these ferric chloride. Small vacuoles with lipid content were also cells to each other and to the fibriform elements; they show visible. abundant cytoplasm with voluminous mitochondria with developed membrane system, chloroplasts with starch grains 3.2.3. Xylem and small vacuoles (Fig. 5D). In D. miscolobium and Z. diphylla The xylem is composed of pitted vessels elements the plastids have developed grana, ellipsoidal starch grains and surrounded by parenchyma cells and fibriform elements with osmiophilic globules (Fig. 5D). living protoplast (Fig. 5A). Simple pits (Fig. 5A) connect the In C. langsdorffii (Fig. 5C), S. polyphyllum and M. rixosa vessel elements to each other and to the parenchyma cells and (Fig. 5E), the transfer cells are characterized by walls with T.M. Rodrigues, S.R. Machado / Micron 39 (2008) 7–16 13

Fig. 4. TEM images of phloem of legume primary pulvinus. (A) Mimosa rixosa sieve tube member displaying numerous plastids (pl) with protein inclusions. Note the presence of sieve plate with callose accumulations and extensive sieve tube reticulum (er). (B) Sieve tube reticulum (arrows) next to the plasmalemmain Stryphnodendron polyphyllum. cw: cell wall. (C) Detail showing mitochondria (mi) surrounded by smooth endoplasmic reticulum (ser) next to the plasmalemma, in sieve tube member of Mimosa rixosa. cw: cell wall. (D) Plastid with crystalline protein body (*) and tubular (tp) and fibrillar (fip) protein in Mimosa rixosa sieve tube member. cw: cell wall. Scale bars: (A) 0.75 mm; (B–D) 0.2 mm.

finger-like invaginations on the face tangential to the vessel (Fleurat-Lessard and Roblin, 1982; Rodrigues and Machado, pits. These cells show conspicuous nucleus and dense and 2004). abundant cytoplasm with polyribosomes, numerous mitochon- In the present work, the vascular cylinder shape of pulvini dria, endoplasmic reticulum, plastids with developed grana and varied from ring, arc, semi-arc and continuous cylinder. There vacuoles of variable sizes (Fig. 5E). M. rixosa has larger is not a relation between the vascular cylinder shape and the mitochondria and plastids with osmiophilic inclusions type or the velocity of leaf movement. In Mimosoideae studied (Fig. 5E). species the vascular cylinder shape is invariable, but only The fibriform elements show very thick multilayered walls Mimosa species exhibit fast seismonastic movements. Thus, the with a highly electron-dense inner layer (Fig. 5A and F); these vascular cylinder shape may have a taxonomic value for this cells have a conspicuous nucleus with evident nucleolus and subfamily. poorly developed vacuome, as well as abundant dense A remarkable characteristic in all the studied species was the cytoplasm with polyribosomes, mitochondria (Fig. 5F) and occurrence of a sheath cells constituted of living septate fibers, endoplasmic reticulum. Primary pit fields connect the fibriform without any sclerified cells surrounds the phloem. This result elements to the adjacent parenchyma cells (Fig. 5F). confirms the observation of the septate fibers around the phloem in pulvius of P. pubescens (Faboideae) by Machado and 4. Discussion Rodrigues (2004). So, septate fibers in pulvini are of common occurrence in the three legume subfamilies. This finding is a The location and the tissue arrangement of the primary novelty, since the sheath around the phloem of other legume pulvinus vascular system in the studied species are congruent specie has been considered a collenchymatous sheath (Esau, with descriptions of the pulvini of other legumes (Fleurat- 1970; Fleurat-Lessard and Bonnemain, 1978; Moysset and Lessard and Bonnemain, 1978; Fleurat-Lessard and Roblin, Simo´n, 1991). Considering that septate fibers sheath constitute 1982; Moysset and Simo´n, 1991). The histological features a well delimited region between the endodermis and the such as developed cortex and reduced central vascular system phloem, it is possible that in the studied species these fibers with lack of lignifications are common to pulvini and are linked arise from the pericycle, as described for the first time in P. to their greatest flexibility and ability for leaf movements pubescens pulvinus (Machado and Rodrigues, 2004). However, 14 T.M. Rodrigues, S.R. Machado / Micron 39 (2008) 7–16

Fig. 5. TEM images of xylem of legume primary pulvinus. (A) General feature of Mimosa rixosa primary pulvinus xylem showing vessel elements (ve), fibriform elements (ff) and parenchyma cells (pc). (B) Detail of Stryphnodendron polyphyllum vessel elements showing vestured pit. (C) Transfer cell showing finger-like invaginations (*) of the cell wall in contact with vestured pit of vessel elements (ve) in Copaifera langsdorffii. Note the presence of nucleus (n), mitochondria (mi), plastids (pl), polyribosome and vesicles. (D) Zornia diphylla parenchyma cell showing mitochondria, plastids (pl) with starch grains and plastglobules, polyribosome and vacuoles (va). The arrow indicates pit field with plasmodesmata. ve: vessel elements. (E) Mimosa rixosa transfer cell, showing mitochondria (mi), plastids (pl) with plastglobules, rough endoplasmic reticulum (rer), polyribosome, vesicles and vacuoles (va). The asterisk (*) indicates cell wall invagination. (F) Detail of Mimosa rixosa fibriform element showing conspicuous nucleus (n) with evident nucleolus (nu), mitochondria (mi), polyribosome and vacuoles (va). Observe the occurrence of pit field (arrow). Scale bars: (A) 5.5 mm; (B) 1.3 mm; (C, E) 0.6 mm; (D) 1 mm; (F) 0.75 mm. ontogenetic studies are necessary to confirm the origin of these the cortex and the vascular cylinder. The absence of mechanical fibers in the studied species. This is an interesting issue of cell elements can facilitate both the cell deformation and the ion differentiation. fluxes, which together regulate leaflet movement (Fleurat- Ultrastructurally, the septate fibers showed living protoplast Lessard and Bonnemain, 1978; Moysset and Simo´n, 1991). with abundant mitochondria suggesting an active metabolism, Considering the phloem, exist a strong concordance of the besides the synthesis and storage of lipids. In addition, the loose histological and cellular aspects among the nine legume studied aspect of their non-lignified walls, the absence of apoplastic species, including absence of fibers and sclereids, presence of barriers and the presence of plasmodesmata in the cell walls phenolic idioblasts and a well developed sieve tube reticulum. increase the possibility an increase of lateral changes between However, some differences were observed as presence of T.M. Rodrigues, S.R. Machado / Micron 39 (2008) 7–16 15 transfer cells in Z. diphylla and M. rixosa and of sieve tube of the leaves are in course to confirm the distribution of the nacreous walls in Z. diphylla. fibriform elements in the studied species. The absence of fibers and sclereids in the phloem of the The density of the cytoplasm, the prominence of the primary pulvinus of the studied species agrees with the pattern membrane systems and numerous plasmodesmata in the observed in P. pubescens (Machado and Rodrigues, 2004), but vertical xylem parenchyma cells is a constant characteristic differs from that described for M. pudica (Esau, 1970, 1971, of all the studied species. It is noticeable that M. rixosa, C. 1973), where such cells were found. langsdorffii and S. polyphyllum parenchyma cells bordering the The presence of large phenolic idioblasts in the phloem is a xylem vessels bear wall ingrowths, whose disposition common feature of all the studied species. In M. pudica corresponds to that type C transfer cells described by Gunning pulvinus, such cells are called ‘‘secretory cells’’ (Haberlandt, and Pate (1969). Similar cells were described in the pulvinus 1928; Fleurat-Lessard and Bonnemain, 1978), and ‘‘wider xylem of M. pudica (Fleurat-Lessard and Bonnemain, 1978). It tubular cells’’ (Kundu and Saha, 1968), and their role is is remarkable that M. rixosa shows phloem transfer cells (type obscure. According to Haberlandt (1928), these tubular cells A) and xylem transfer cells (type C). Considering that the constitute a continuous system in which variations of pressure presence of ingrowths produces an increase of wall and are transmitted over long distances. Sibaoka (1953) stated that plasmalemma surfaces (Pate and Gunning, 1969), these cells these cells are involved in the propagation of ‘‘m-waves’’. could be the sites of important exchanges between the symplast However, there are no experimental evidences to confirm these and the apoplast in this species that shows fast leaf movements. probable functions. This observation may indicate that leaf movement in this A remarkable characteristic of the pulvini phloem of the species is related to the presence of transfer cells. However, our studied species was the presence of a net of tubular membranes results do not support this hypothesis. arranged perpendicularly to the inner surface of the cell wall In this work, we identified common histological and cellular called sieve tube endoplasmic reticulum (Fig. 4B). The roles features to the pulvini of all the studied species, such as the suggested for the sieve tube endoplasmic reticulum include the occurrence of a sheath of live septate fibers around the vascular channeling of ATP from the mitochondria to proton-pumping tissues, vascular parenchyma cells with abundant cytoplasm ATPase in the plasmalemma, the increase of surface area for the and extensive symplastic connections and lack of lignifications. entry of substances into the lumen of the sieve-tube member, These characteristics have a functional value since they ensure and the functioning as a calcium accumulation site (Sjolund and lateral exchanges (symplastic and apoplastic) of ions and Shih, 1983; Evert, 1990). stimuli among all the pulvinus tissues. Extensive simplastic Transfer cells were observed in the phloem of M. rixosa and connections from the center of the pulvini to the cortical Z. diphylla and represent modified companion cells and the parenchyma were reported by Moysset and Simo´n (1991) for disposition of the wall ingrowths corresponds to that of type A Robinia pseudoacacia. According to Fleurat-Lessard and transfer cells as described by Gunning and Pate (1969). Bonnemain (1978) this device would allow the passage in Transfer cells are common in tissues which have a special the motor cells, of information propagated, not only in the physiology where intensive lateral transfers occur (Offler et al., phloem, but also in the xylem parenchyma. 2002). The presence of these cells in motor organs was reported We must emphasize that, although many works reporting for M. pudica (Fleurat-Lessard and Bonnemain, 1978), a plant pulvinus features exist in the literature, this is the first one that having rapid leave movements. However, their presence in Z. describes histological and cellular characteristics comparing diphylla phloem, a species with slow movements, does not them in nine species growing in the same environmental allow a linkage between this cell type and the velocity of leaf conditions of the cerrado. movement. Sieve tube members with nacreous walls were observed in Z. Acknowledgements diphylla. The role of the nacreous layer is still unknown, however it can be speculated that it can increase the volume of We thank FAPESP for financial support to this study (T.M. the apoplast (Kursanov, 1984), which would facilitate radial Rodrigues scholarship Process MS 03/11050-7 and Biota transport of ions during the leaf movement. Program Process 00/12469-3) and CNPq (Research grant to Considering the xylem, all the studied species show similar S.R. Machado) and the technical team of the Institute of histological and cellular characteristics as presence of fibriform Biosciences’ Electron Microscopy Center, UNESP Botucatu, elements with living protoplast, pitted vessel elements and SP, Brazil, for their assistance in preparing the samples. voluminous vascular parenchyma cells. The presence of fibriform elements with living protoplast References was described in motor organs of few legume species and it was seen that this kind of cell substitutes the vascular lignified fibers Burger, L.M., Richter, H.G., 1991. Anatomia da Madeira. Livraria Nobel, Sa˜o that occur in other parts of the leaf (Fleurat-Lessard and Roblin, Paulo. 1982; Rodrigues and Machado, 2004). Differing of Fleurat- Caldas, L.S., Lu¨ttge, U., Franco, A.C., Haridansan, M., 1997. Leaf heliotropism in Pterodon pubescens, a woody legume from the Brazilian cerrado. Rev. Lessard and Bonnemain (1978) observations, in this work these Brasil. Fisiol. 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