BIOLOGIA PLANTARUM 57 (2): 298-304, 2013 DOI: 10.1007/s10535-012-0295-8 Multilamellar bodies linked to two active plasmalemma regions in the pollen grains of Sarcocapnos pulcherrima M.C. FERNÁNDEZ1*, M.A. PÉREZ-GUTIERREZ2, V.N. SUAREZ-SANTIAGO2, M.J. SALINAS-BONILLO3, and A.T. ROMERO-GARCÍA2 Department of Cell Biology, Faculty of Sciences, University of Granada, E-18071 Granada, Spain1 Department of Botany, Faculty of Sciences, University of Granada, E-18071 Granada, Spain2 Department of Plant Biology and Ecology, University of Almeria, E-04071 Almeria, Spain3 Abstract The presence of visible multilamellar bodies in the cytoplasm of pollen grains of at least seven species of the family Papaveraceae has led us to study the behaviour of these bodies during pollen-grain ontogeny and in growing pollen tubes of Sarcocapnos pulcherrima C. Morales & R. Garcia germinated in vitro. Our transmission-electron-microscope (TEM) studies in pollen grains show that the multilamellar bodies may be classified as: 1) small, isolated and placed in the region of apertures in the cytoplasm; and 2) large, in clusters and in contact with the active plasmalemma apertures only when tubules are being formed in the apertural intine. Similar types of multilamellar bodies to those observed in the pollen apertures can be seen near the apex of the growing pollen tube (small and isolated) and in contact with the apex plasmalemma (large and clustered). Our results support the hypothesis that the multilamellar bodies are functionally linked to moments when the cytoplasmic membrane is very active. We have also linked the multilamellar bodies to Golgi vesicles as they both react positively to acid-phosphatase (AP) staining and also to the plasmalemma by the thiocarbohydrazide-silver proteinate-staining (TCH-Sp) electron-contrasting technique. Additional key words: ontogeny, Papaveraceae, pollen apertures, pollen tubes, TEM, ultrastructure. Introduction Intracellular membrane inclusions have been frequently of Papaveraceae and also within the apex of the pollen described. Due to the numerous locations and conditions tube in Sarcocapnos pulcherrima, germinated in vitro. in which these structures may be found, they have We also found lamellar structures in the form of myelin received various names and have been endowed with bodies in Sarcocapnos pulcherrima pollen tubes. The diverse functions. myelin and multilamellar bodies described here are not In plant cells, multivesicular bodies have been related either in their structure or location. associated with the proteolytic processes in Arabidopsis Pollen apertures play a crucial role in the sexual (Otegui et al. 2006) and the protein bodies of mung bean reproduction of plants and have a very complex structure cotyledons (Van der Wilden et al. 1980). Arabino- which undergoes significant changes during its onto- galactan proteins have been found in Nicotiana tabacum genesis (Fernández and Rodríguez-García 1989, 1995). both in plasmalemmasomes and in multi-lamellar bodies One of the clearest apertural changes observed in some in the pollen tubes as well as in other sub-cellular sites taxa is the differentiation of the apertural intine, involving (Ferguson et al. 1999). a lens-shaped thickening known as the intinous oncus We describe here multilamellar bodies observed in the which plays an important role in germination. The vicinity of apertures in the pollen grains of seven species cytoplasmic membrane below the intinous oncus is very ⎯⎯⎯⎯ Received 31 May 2012, accepted 4 September 2012. Abbreviations: AP - acid-phosphatase; TCH-Sp - thiocarbohydrazide-silver proteinate; TEM - transmission electron microscopy. Acknowledgements: This research was funded by the Spanish Ministry of Science and Innovation, CGL2008-01554/BOS. We thank Dr. Dirk de Meyere of the National Botanical Garden of Belgium and Henrik Zutterlung of the Botanical Garden of Göteborg (Sweden) for their help in the collection of material for analysis in our work. We are grateful for the support of Concepción Hernández Castillo, María José Martínez Guerrero and David Porcel Muñoz of the Scientific Instrumentation Centre of the University of Granada for preparing our samples and TEM visualization. We also thank Angela L. Tate for translating and editing our text. * Author for correspondence: fax: (+ 34) 958 243258, e-mail: [email protected] 298 MULTILAMELLAR BODIES LINKED TO PLASMALEMMA active producing tubules that extend towards the intine The aim of our study was to detect multilamellar and generate a layer of membranous tubules in the bodies in the pollen grains of seven species of the intinous oncus. Initially, the tubules are in contact with Papaveraceae family and in the pollen tubes of the plasmalemma and finally they separate from the Sarcocapnos pulcherrima using transmission electron cytoplasm membrane (Rodríguez-García and Fernández microscope and to classify them according to their size 1988, Fernández et al. 1992). and location. We used acid-phosphatase (AP) staining Another very active area of the cytoplasmic (Megías and Renau 1998) to establish a possible membrane is the apical zone of the pollen tube. This is relationship between the multilamellar bodies and other one of the areas of rapid growth in eukaryotic cells and neighbouring cytoplasmic organelles. To determine the the cytoplasmic membrane of this zone is very active relationships between the plasmalemma and multi- during exocytosis and endocytosis, although the sub- lamellar bodies we also used thiocarbohydrazide-silver cellular location of this growth is as yet unknown. Zonia proteinate-staining (TCH-Sp), an electron-contrasting and Munnik (2008) found that the apex is the area where technique specific to the plasma membrane of plant cells endocytosis and exocytosis occur but that the membrane (Weber 1992). surface area also increases rapidly. Materials and methods Developing anthers and fresh pollen were collected from: (m/v) glutaraldehyde with 0.025 M cacodylate sodium Sarcocapnos pulcherrima C. Morales & R. Garcia buffer (pH 7.2) at room temperature for 24 h. They were (subfamily Fumarioideae, tribe Fumarieae, wild washed with several changes of cacodylate buffer and population sample number GDAC 22851); Hypecoum post-fixed in 1 % (m/v) OsO4 for 2 h. They were then imberbe Sm. (subfamily Hypecoideae, wild population dehydrated in a graded series of ethanol and embedded in sample number GDAC 22824; Fumaria capreolata L. Epon. Ultra-thin sections were cut on a Ultracut E (Leica (subfamily Fumarioideae, tribe Fumarieae, wild Microsystems, Wetzlar, Germany) and stained with 2 % population sample number GDAC 22941; Platystemon (m/v) uranyl acetate and lead citrate (Reynolds 1963). californicum Benth. (subfamily Papaveroideae, tribe Observations were carried out with a Carl Zeiss (Jena Platystemonoideae, living collection accession number Germany) LIBRA 120 Plus transmission electron 20051151-87, National Botanical Garden of Belgium); microscope (TEM). Pseudofumaria alba (Mill.) Lidén (subfamily For AP cytochemistry conducted in Sarcocapnos Fumarioideae, tribe Fumarieae, living collection pulcherrima, we followed Megías and Renau’s (1998) accession number 1985-0760, Botanical Garden of procedure using samples without post-fixation in osmium Göteborg, Sweden; Euptelea poliandra Siebold & Zucc tetroxide. For TCH-Sp cytochemistry in osmium-fixed (family Eupteleaceae, (living collection accession material from Sarcocapnos pulcherrima, we followed number 19723330, National Botanical Garden of Weber’s procedure (1992). Belgium; and Pteridophyllum racemosum (subfamily For in vitro germination, pollen grains of Pteridophylloideae, living collection accession number Sarcocapnos pulcherrima were kept at 25 ºC in 1982-0246, Botanical Garden of Göteborg, Sweden). Brewbaker and Kwack’s medium (1963) and fixed after Anthers of the 7 species in different phases of pollen 1 and 2 h incubation and germination of the pollen tube. maturation were collected. Samples were prefixed in 3 % Results The multilamellar bodies found in the pollen of the seven the oncus was very active producing a large number of species studied were electron dense, irregular in shape, growing tubules that extend towards the intine (Fig. 1B). and dispersed within the cytoplasm, although clearly The tubules were formed from the plasmalemma and within the region of the pollen wall and most frequently groups of large multilamellar bodies could be seen in below the apertural zones (Fig. 1A). They appeared only contact with it (Fig. 1B). The multilamellar bodies were from the intermediate bicellular stage during pollen composed of various peripheral membranous layers ontogeny (Fig. 1), when the generative cell was arranged more or less compactly around an apparently embedded within the pollen grain, until maturity. During empty electron-transparent core (compare Fig. 2A,B,C,D, the intermediate bicellular stage, the intine of the and E). Some of the multilamellar bodies contained only apertural areas thickens into a lens shape forming the compact lamellars and sometimes had a membranous intinous oncus within which a large number of tubules edge (Fig. 2C). either in the process of formation or growth were seen After Sarcocapnos pulcherrima pollen grains were (Fig. 1A). The cytoplasmic membrane in the areas below treated by TCH-Sp staining, the multilamellar bodies 299 M.C. FERNÁNDEZ et al. appearred to be opaque with much deeper staining than isolated multilamellar bodies dispersed throughout the the cytoplasm itself (Fig. 3A,B).