This article was downloaded by: [B-on Consortium - 2007] On: 14 November 2008 Access details: Access Details: [subscription number 778384761] Publisher Taylor & Francis Informa Ltd Registered in England and Wales Registered Number: 1072954 Registered office: Mortimer House, 37-41 Mortimer Street, London W1T 3JH, UK Grana Publication details, including instructions for authors and subscription information: http://www.informaworld.com/smpp/title~content=t713648917 Ultrastructure and cytochemistry of Eucalyptus globulus (Myrtaceae) pollen grain Susana A. Eliseu a; Augusto M. Dinis a a Laboratory of Electron Microscopy and Palynology, Department of Botany, University of Coimbra, Coimbra, Portugal Online Publication Date: 01 March 2008 To cite this Article Eliseu, Susana A. and Dinis, Augusto M.(2008)'Ultrastructure and cytochemistry of Eucalyptus globulus (Myrtaceae) pollen grain',Grana,47:1,39 — 51 To link to this Article: DOI: 10.1080/00173130801923364 URL: http://dx.doi.org/10.1080/00173130801923364 PLEASE SCROLL DOWN FOR ARTICLE Full terms and conditions of use: http://www.informaworld.com/terms-and-conditions-of-access.pdf This article may be used for research, teaching and private study purposes. Any substantial or systematic reproduction, re-distribution, re-selling, loan or sub-licensing, systematic supply or distribution in any form to anyone is expressly forbidden. The publisher does not give any warranty express or implied or make any representation that the contents will be complete or accurate or up to date. The accuracy of any instructions, formulae and drug doses should be independently verified with primary sources. The publisher shall not be liable for any loss, actions, claims, proceedings, demand or costs or damages whatsoever or howsoever caused arising directly or indirectly in connection with or arising out of the use of this material. Grana, 2008; 47: 39–51 Ultrastructure and cytochemistry of Eucalyptus globulus (Myrtaceae) pollen grain SUSANA A. ELISEU & AUGUSTO M. DINIS Laboratory of Electron Microscopy and Palynology, Department of Botany, University of Coimbra, Coimbra, Portugal Abstract The morphology, ultrastructure and cytochemistry of Eucalyptus globulus mature pollen were investigated using light (LM), scanning electron (SEM) and transmission electron microscopy (TEM). The pollen morphology is typically myrtaceous with a suite of characters that allows its distinction from the pollen of other Eucalyptus species. The exine consists of a thick endexine and a massive ectexine with hardly distinguishable columellae. The endexine is 2-layered towards the apertural regions where the inner and spongy-granulate layer forms a continuous colpus membrane and a thinner pore membrane. Under the pores the intine is 3-layered forming complex onci. The spindle-shaped generative cell (GC) is deeply undulated and located in a cup-shaped depression of the vegetative cell (VC) nucleus. In the dense VC cytoplasm the main storage reserves are lipid bodies and insoluble carbohydrates in the cytosol, although proteins are also present. The most characteristic feature of the VC cytoplasm is the extremely well-developed rough endoplasmic reticulum (RER), which forms extensive stacks filling large areas of the central cytoplasm. Single RER cisternae are also scattered throughout the cytoplasm, most of them establishing an intimate association with lipid bodies, storage vacuoles, the VC plasmalemma and a few proplastids. The physiological significance of the RER stacks and of the RER cisternae association with other cell components, as well as the structure and function of an endomembrane compartment, found only in the freeze-fixed pollen, are discussed. Keywords: Pollen grain, cell structure, vegetative cell, endoplasmic reticulum, storage reserves, Eucalyptus, Myrtaceae Eucalyptus globulus Labill. (Myrtaceae) is one of the with a distinctive apocolpial field. A suite of char- world’s most widespread hardwood trees. In acters, including grain size, types of apocolpial field Portugal it occupies ca. 20% of the total forest area and apocolpial edges, surface patterning and its being the second most abundant, and one of the distribution on the mesocolpia and margo colpi, Downloaded By: [B-on Consortium - 2007] At: 10:55 14 November 2008 most important tree species (DGRF, 2006). Due to may be used to define and separate a number of its social, economical and environmental impacts, Eucalyptus pollen types (Pickett & Newsome, 1997). Eucalyptus globulus has been the object of several So far, no detailed ultrastructural and cytochem- genetic, ecological and physiological studies. The ical studies on the cellular organization of the pollen biology of Eucalyptus has mainly focussed on Eucalyptus mature pollen have been published. pollination and pollen grain germination (e.g. These studies are important for a better under- Heslop-Harrison & Heslop-Harrison, 1985; standing of the reproductive biology of a species and Patterson et al., 2004), although some studies on for identification of characteristics that may be of pollen morphology and pollen wall organization have taxonomic and (or) phylogenetic interest. Studies on been done in a few species (e.g. Gadek & Martin, other plants have shown that the composition and 1982; Patel et al., 1984; Heslop-Harrison & Heslop- organization of the pollen grains are different among Harrison, 1985; Zhou & Heusser, 1996; Pickett & species (e.g. Jensen et al., 1974; Cresti et al., 1975, Newsome, 1997). Typically, Eucalyptus pollen is 1985, 1988, 1990; Van Aelst & Van Went, 1991; triangular, isopolar, radiosymmetric, parasyncolpate, Van Aelst et al., 1993; Hess, 1995; Dinis et al., Correspondence: Augusto M. Dinis, Laboratory of Electron Microscopy and Palynology, Department of Botany, Faculty of Sciences and Technology, University of Coimbra, 3001-455 Coimbra, Portugal. E-mail: [email protected] (Received 17 October 2007; accepted 15 January 2008) ISSN 0017-3134 print/ISSN 1651-2049 online # 2008 Taylor & Francis DOI: 10.1080/00173130801923364 40 S. A. Eliseu and A. M. Dinis 2000). The main differences are in respect to the Freeze-fixation and freeze-substitution. Mature pollen pollen wall architecture, the number and degree of was attached to agar-coated wire loops and freeze- development of the organelles, the amount and fixed and freeze-substituted according to the nature of reserve substances, and the generative cell procedure of Dinis et al. (2000). After substitution (GC) composition and its association with the has been completed the samples were rinsed in pure nucleus of the vegetative cell (VC). In the present acetone and embedded in Spurr’s resin. study, the morphology, ultrastucture and cytochem- For all fixations, sections were cut on a LKB istry of the mature pollen of Eucalyptus globulus were Ultratome NOVA ultramicrotome equipped with a investigated using both chemical fixation and rapid diamond knife, conventionally stained with uranyl freeze-fixation. Our goal is to gain a detailed acetate and lead citrate, and observed in a JEOL knowledge of the structural organization of the JEM-100 SX at 80 kV. pollen of this species to better understand its physiology and its performance during shedding, Cytochemistry storage and germination. Insoluble polysaccharides were detected in 2 mm thick sections subjected to the periodic acid-Schiff Material and methods (PAS) reaction. At the ultrastructural level, detec- tion of neutral polysaccharides and other carbohy- Plant material and pollen description drates was performed according to the method of Anthers of Eucalyptus globulus from the Coimbra Thie´ry (1967). For both tests, controls consisted of Botanical Garden (Coimbra, Portugal) were used. sections treated in the same way but without The terminology for pollen description follows Punt periodic acid oxidation. Acidic polysaccharides and et al. (2007). glycoproteins were detected with 1% phosphotungs- tic acid (PTA) in 10% chromic acid (Farrigiana & Marinozzi, 1979), the control consisting of the same Scanning Electron Microscopy (SEM) treatment but with pH 7.3. Proteins were detected Mature pollen grains were fixed in 2.5% buffered in semithin sections stained with Coomassie brilliant glutaraldehyde (see below), dehydrated in a graded blue, and unsaturated lipids were detected in 0.5 mm ethanol series and critical point dried. Then, the thick sections stained with Sudan black B (Bhandari, grains were mounted on aluminium stubs and 1997). At the ultrastructural level, proteins were sputter coated with a 20 nm layer of gold-palladium detected in sections subjected to enzymatic extrac- prior to examination with a JEOL JSM-5400 at tion with protease (Dinis et al., 2000). DNA was 15 kV. detected in fresh pollen permeabilized with the agent Triton X-100, which was added to the staining solution (1:10, v/v) consisting of 0.01% DAPI (4,6- Transmission Electron Microscopy (TEM) diamino-2-phenylindole) in 0.1 mol/l sodium caco- Chemical fixation. Due to difficulties in preserving the dylate buffer, pH 6.8; observations were made under Downloaded By: [B-on Consortium - 2007] At: 10:55 14 November 2008 pollen grain ultrastructure, different protocols were ultraviolet irradiation in an epifluorescence micro- used for fixation of dehiscing anthers and isolated scope (Nikon Optiphot model XF-EF). mature pollen: i) 2.5% glutaraldehyde in 0.1 mol/l sodium cacodylate buffer (pH 6.8) supplemented Results with 1 mmol/l calcium chloride. After rinsing with the same buffer, samples were post-fixed in 1%