Flora 230 (2017) 66-74 Contents lists available at ScienceDirect Ě FLORA] Flora ELSEVIER journal homepage: www.elsevier.com/locate/flora The velamen of epiphytic orchids: Variation in structure and (ft\ correlations with nutrient absorption Thais Arruda Costa Jocaa, Denis Coelho de Oliveira3, Gerhard Zotzbc, Uwe Winklerbt, Ana Silvia Franco Pinheiro Moreira 3* a Universidade Federal de Uberländia, Institute de Biologia, Rua Ceavä s/n, Bloco 2D, Campus Umuarama, CEP 38400-902, Uberländia, MG, Brazil b Universität Oldenburg, Institute for Biology and Environmental Sciences, Oldenburg, Germany c Smithsonian Tropical Research Institute, Balboa, Panama CrossMark ARTICLE INFO ABSTRACT Article history: Received 6 October 2016 Received in revised form 7 March 2017 Accepted 13 March 2017 Edited by Hermann Heilmeier Available online 16 March 2017 Keywords: Nutrient uptake Orchidaceae Passage cells Pectins Root anatomy Velamen structure Roots of epiphytic orchids typically possess a velamen radicum, a multiple epidermis composed of dead cells. It is largely unexplored how the structure of the velamen, the cortex and vascular cylinder facilitate the flow of water and nutrients towards the plant. Up to now, structural root features were rarely correlated with functional attributes. In this study, we compare anatomical features with nutrient uptake rates (specifically of phosphorus and rubidium) of roots of 18 taxa of epiphytic orchids. The relative proportion of the velamen of the cross-sectional area varied from 11 to 97%. Species with the largest relative velamen area facilitate the flow of water and nutrients, and species with larger relative proportion of vascular cylinder and cortex have a greater number of protoxylem strands and passage cells of the endo-dermis. The number of passage cells in the endodermis and of differential wall thickenings in cortical cells may play an important role in apoplastic and symplastic flow. The velamen pectic matrix, with negative charge, can retain cations (like Rb+), while a higher number of strands in the xylem, aligned with passage cells of the endodermis, could facilitate the entry of these ions into the vascular cylinder. © 2017 Elsevier GmbH. All rights reserved. 1. Introduction The epiphytic environment imposes a number of restrictions to plants. Many of the anatomical features commonly found among epiphytes are related to the uptake of water and nutrients, such as absorbing scales in the Bromeliaceae or the velamen radicum in Orchidaceae (Withner, 1959; Benzing, 1990; Zotz and Hietz, 2001; Laube and Zotz, 2003). Although the availability of water and nutrients is sporadic and depends on atmospheric sources (Benzing, 1990), growth in tree crowns ensures an environment with an improved supply of light (Tsavkelova et al„ 2001). It is known that approximately 10% of all vascular plants are epiphytes, the Orchidaceae being particularly noteworthy because around 70% of all species are epiphytic (Zotz, 2013). This success in tree crowns is arguably due to anatomical and physiological strategies that compensate for the scarcity of water and nutrients, promoting in the * Corresponding author. E-mail addresses: thais_arrudacj@hotmail.com (T.AC. Joca), denisoliveira@inbio.ufu.br (D.C. de Oliveira), gerhard.zotz@uni-oldenburg.de (G. Zotz), anasilviamoreira@gmail.com (AS.F.P. Moreira). 1 In memorian http://dx.doi.Org/10.1016/j.flora.2017.03.009 0367-2530/© 2017 Elsevier GmbH. All rights reserved. effective absorption and use of these resources when intermittently available (Scatena and Nunes, 1996). Among the adaptive features of orchid roots, the presence of velamen stands out (Zotz and Hietz, 2001). The velamen is located externally to the exodermis, the outer layer of the cortex, and constitutes a multiple epidermis composed of dead cells (Pridgeon, 1987). Initially, Went (1940) proposed an important role of the velamen in capturing and immobilizing solutions that arrive at the root via stem flow, and recent experimental evidence provides support for this notion (Zotz and Winkler, 2013). In addition to the function of water absorption, the velamen also reduces the loss of water at times of low availability of water and confers mechanical protection for the root (Pridgeon, 1987; Benzing, 1996). Internally to the velamen, the cortex consists of parenchymatic cells. In Orchidaceae there are different types of parietal thickenings, i.e. reticulated, uniform or phi forms (Stern, 1999; Stern andjudd, 2001; Moreira et al., 2013). The exodermis and endodermis (outermost and innermost layers of the cortex, respectively) delineate the cortical parenchyma in these roots, and the presence of an endodermis acts as boundary between the cortex and the vascular cylinder. Exodermis and endodermis are formed by cells that can have Casparian strips, be highly lignified and sometimes dead at maturity, alternating with passage cells which remain alive T.ACJoca et al. / Flora 230 (2017) 66-74 67 (Pridgeon, 1987; Trepanier et al., 2008). The lignified cells (or with Casparian strips) in these layers act as a barrier for the movement of ions and other substances through the apoplast (Esnault et al., 1994; Ma and Peterson, 2003), while the passage cells are the main cells capable of symplastic movement (Peterson and Enstone, 1996). These cells allow water and nutrient absorption (Peterson and Enstone, 1996), and are responsible for the selectivity of solutes to be transported to the vascular cylinder (Peterson, 1988; Trepanier et al., 2008). This study wants to relate the investment of Orchidaceae roots in velamen and/or in other internal structures to the functions assigned to the passage cells and velamen found in the cited literature, specifically the absorption of nutrients. We focus on the relationship of the thickness of the velamen and the number of passage cells (of both the endodermis and exodermis) as structural characteristics on the one hand and the rate of absorption of phosphorus and rubidium on the other hand for a set of 18 taxa of epiphytic orchids. 2. Material and methods Individuals of Bifrenaria harrisoniae (Hook.) Rchb.f, Catasetum planiceps Lindl., Cattleya skinneri Bateman alba, Caularthron bil-amellatum (Rchb.f.) R.E. Schult, Dendrobium flmbriatum Hooker, Dendrobium nobile Lindl., Dendrobium nobile hybrid, Dimerandra emarginata (G. Mey.) Hoehne, Doritis pulcherrima Lindl., Ency-clia ghillanyi Pabst, Epidendrum ciliare L, Epidendrum noctumum Jacq., Gongora unicolor Schltr., Miltonia bluntii Rchb.f., Phalaenopsis comu-cervie Blume & Rchb.f, Phalaenopsis hybrid, Oncidium sp. and Trichoglottis bipunctata (C.S.PP. Parish & Rchb.f.) Tang & F.T. Wang were obtained from the Botanical Garden of the University of Marburg (Germany) or from collections in Panama, and acclimated in a greenhouse of the University of Oldenburg (Germany), at a temperature of26/20 °C (day/night) and a relative humidity of ca. 65%. Fragments of aerial roots were submitted to tests of nutrients absorption (phosphorus and rubidium) in the Radionuclide Laboratory of the University of Oldenburg and parallel samples were fixed in 4% paraformaldehyde (Roland and Vian, 1991). The anatomical analyses were performed in the Laboratory of Plant Anatomy and Development of the Universidade Federal de Uberlandia, Brazil. 2.1. Experiments of nutrient absorption Nutrient absorption was assayed similar to the procedure described in Zotz and Winkler (2013) using the following radioisotopes: carrier free 32P phosphoric acid (Hartmann Analytic, Germany), containing 37MBq in IOOjjlI water (initial specific activity: 5.5 MBqnmol_132P); aquatic solutions of 86RbCl (Hartmann Analytic, Germany) with a specific activity of 720 MBq mg-1 Rb+ and aquatic solutions of [14C(U)-D-glucose] with a specific activity of 13.3 GBqmmol-1. The radioisotope of rubidium is a well-established analytical analogue for potassium, because the uptake kinetics of K+ and Rb+ are generally comparable (Lauchli and Epstein, 1970). Aliquots of labelled substances with an initial activity of 0.15 mBq in 5 ml incubation solution were used in uptake experiments with the 18 different taxa. The uptake of phosphate was measured in solutions containing radioactive labelled 32 P phosphate and unlabelled phosphate to give a final substrate concentration of 15 jjlM. Radioactive, labelled 86Rb and unlabelled RbCl were mixed to obtain a similar final concentration of 15 jjlM. Such concentrations are within the range of concentrations naturally occurring in rainwater (e.g. Benzing, 2000). Four root segments of each taxon, excised at 4 cm from the root tip, were submerged in solutions of incubation in test tubes, and absorption rates were determined by the decrease of the radioac- tivity in aliquots of 10 jjlI of the solutions of incubation (with pH 6.1) after standardizing the volume of liquid, adding the solution not labelled to the initial volume of incubation, mixing with Pasteur pipettes. The velamen was removed just above the submerged portion to prevent the passive diffusion in this tissue. The cumulative absorption of phosphate and rubidium was calculated from the decrease of the radioactivity in the solution of incubation with a constant volume, according to the following equation: Absorption(nmol) = PS-(dpmt0/dpmn xPj), where P2 is the initial quantity of substrate in the solution (nmol); dpmt0 is the counting rate in 10 jjlI at beginning of absorption and DPMrt the counting rate in 10 jjlI at the end of the period of absorption. The rates were expressed against root length (cm). 2.2. Structural analysis For anatomical comparisons of the roots, histological slides of transverse hand cuts were prepared 3 cm from the apex. The material was clarified in 50% sodium hypochlorite (Kraus and Arduin, 1997) and stained with 1% alcoholic solution of safranin and 0.5% Astra blue (l:9v/v) (Bukatsch, 1972). The slides were assembled with gelatin glycerinated of Kaiser (Johansen, 1940) and the material photographed under light microscope Leica" DM500 equipped with digital camera Leica" ICC50 HD. For each species, four individuals were analyzed when available (see analysis of results). For each individual, 15 transverse cuts were performed. The diameter, the number of layers, the thickness and the cross-sectional area occupied by the velamen, and the cross-sectional areas of vascular cylinder and the cortex were determined at three different points of a given cut. The cross-sectional areas of different tissue types were expressed relative to the total area. The total number of cells of the exodermis and endodermis, the thickness of the cell walls of the exodermic cells and the number of passage cells were quantified as were the ratios of passage cells to the other cell components of the endodermis and exodermis. In addition, we counted the number of strands of xylem and phloem. For all counts and measurements, IMAGE J 1.x software (National Institute of Health, USA) was used. 2.3. Histochemical analysis The presence of proteins was used to identify living cells and tissues. Transverse sections were incubated in 1% bromophe-nol blue solution (w/v) for 10-15 min (Durrum, 1950), washed in 50% ethanol (v/v) and assembled in distilled and deionized water. Pectins were detected incubating transverse sections in 50% ethanol, and staining with 0.2% ruthenium red (w/v) for 10 min (Chamberlain, 1932; modified). 2.4. Data analysis Structural data were analyzed with the averages of the individuals in each species. For Caularthron bilamellatum, Cattleya skinneri alba, Dendrobium flmbriatum, Epidendrum ciliare and Miltonia bluntii, there was only material available from a single individual. For Phalaenopsis hybrid, Gongora unicolor and Trichoglottis bipunctata there were only two available individuals of each taxon. In all other cases, four individuals were used. We evaluated the correlation between variables using Pearson test for parametric data. The same test was used to compare the structural characteristics with the rates of absorption of rubidium and phosphorus. All tests were performed using SYSTAT 10.2 soft- 68 T.A.C. ]ocaet al. / Flora 230 (2017) 66- 74 ware (SPSS, 2000, San Francisco). A level of P< 0.05 was interpreted as significant. 3. Results OOnOOOOOO^OOOn 3.1. Root morphology and anatomy The structure of the roots of the 18 analyzed taxa was qualitatively similar (for a typical example see Fig. 1A) although the velamen differed fourfold in the number of cell layers (3-11 layers of dead cells, Table 1) and the patterns of parietal thickenings varied as well. Parietal thickenings in the cortical parenchyma were lacking in most species (Table 2), while eight species had some parenchymatic cells with conspicuously cell wall-thickenings of three different types; either reticulated (Fig. IB), uniform (Fig. 1C) or phi (Fig. ID and E). Noteworthy, in a few cases we found variation even within a species. Cells with reticulated thickenings were found in Bifrenaria harrisoniae in one of the roots of a single individual, while cells with uniform thickenings were found in all the other roots of this species. Cattleya skinneri alba showed cells with prominent phi-thickening throughout the cortex in all the roots, while in Catasetumplaniceps, this type of thickening was only found in three roots out of seven analyzed. In this species, some roots showed phi-thickening encircling the entire cortex, while in other roots, the phi-thickening was interrupted. In Encycliaghillanyi, phi-thickening was slightly prominent in only one of the roots. In Trichoglottis bipunctata, the reticulated thickening was found in all the roots. Most cells of the exodermis and endodermis, which delimit the cortical parenchyma, are dead, with walls showing different degrees of thickening, interspersed by passage cells. The strands of xylem and phloem are intercalated in the vascular cylinder and immersed in parenchymatic cells that show centripetal sclerification (Fig. 1F and G). Histochemical tests showed that only the passage cells in the exodermis (Fig. 2A) and endodermis are alive, and the velamen maintains the same pectic structure even at maturity (Fig. 2B). Some species had a very well developed velamen (Fig. 3A), while in others cortex and vascular cylinder dominated the transversal area of the root. There was a continuous variation from species with very large to species with very small proportions of the velamen, the extreme values being 97% (Catasetum planiceps, Table 2) and 11 % (Phalaenopsis comu-cervie, Table 2, Fig. 3B) of the area of a cross section of the roots occupied by this tissue. In relation to the ratio of the number of passage cells with the number of the thickened cells of the exodermis (Table 1), interspecific differences were relatively small, with values between 0.1 and 0.3. The same was observed for the endodermis, which presented values between 0.2 and 0.6. The smallest ratios were found in Phalaenopsis hybrid, Doritis pulcherrima and Phalaenopsis comu-cervie. Endodermis and exodermis had the same type of "O" or "U"-thickenings for all the analyzed sections of a given taxon. The endodermis had "O" thickening for all the taxa evaluated (Fig. 3C), and this was also true for the exodermis of most species (Fig. 3D). "U"-thickening of the cell walls in the exodermis were observed only for Phalaenopsis hybrid, Epidendrum ciliare (Fig. 3E), E. noc-turnum and Doritis pulcherrima. 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