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 ClÀ 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