Ultrastructural Aspects and Programmed Cell Death in the Tapetal Cells of Lathyrus Undulatus Boiss

Acta Biologica Hungarica 63(1), pp. 52–66 (2012) DOI: 10.1556/ABiol.63.2012.1.5 ULTRASTRUCTURAL ASPECTS AND PROGRAMMED CELL DEATH IN THE TAPETAL CELLS OF LATHYRUS UNDULATUS BOISS FILIZ VARDAR * and MERAL ÜNAL Science and Art Faculty, Department of Biology, Marmara University, Göztepe, 34722, İstanbul, Turkey (Received: October 5, 2010; accepted: March 21, 2011) Programmed cell death (PCD) in the tapetum of Lathyrus undulatus L. was analyzed based on light, fluorescence and electron microscopy to characterize its spatial and temporal occurrence. Development and processes of PCD in secretory tapetal cells of Lathyrus undulatus L. were correlated with the sporog- enous cells and pollen grains. At early stages of development the tapetal cells appeared similar to pollen mother cells, structurally. Concurrent with meiosis, tapetum expanded both tangentially and radially as vacuoles increased in size. Tapetal cells most fully developed at young microspore stage. However, tapetum underwent substantial changes in cell organization including nucleus morphology monitored by DAPI. The TUNEL staining confirmed the occurrence of intra-nucleosomal DNA cleavage. In addition to nuclear degeneration which is the first hallmark of PCD other diagnostic features were observed at vacuolated microspore stage intensely; such as chromatin condensation at the periphery of the nucleus, nuclear membrane degeneration, chromatin release to the cytoplasm, vacuole collapse according to tono- plast rupture, shrinkage of the cytoplasm, the increase and enlargement of the endoplasmic reticulum cisternae and disruption of the plasma membrane. After vacuole collapse due to possible release of hydrolytic enzymes the cell components degraded. Tapetal cells completely degenerated at bicellular pollen stage. Keywords: Lathyrus undulatus Boiss. – programmed cell death – tapetum – TUNEL – vacuole collapse INTRODUCTION Programmed cell death (PCD) in plants has attracted much attention recently, since it plays an important role in maintaining normal reproductive development. In angiosperms, PCD has been found in a variety of cells in reproductive organs including reproductive primordium abortion, style transmitting tissue, non-functional * Corresponding author; e-mail: fi[email protected] Abbreviations: DAPI: 4’,6-diamidino-2-phenylindole; ER: Endoplasmic reticulum; nDNA: nuclear deoxyribonucleic acid; PCD: Programmed Cell Death; TEM: Transmission electron microscopy; TUNEL: Terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end labelling; VPE: Vacuolar processing enzyme. 0236-5383/$ 20.00 © 2012 Akadémiai Kiadó, Budapest Programmed cell death in tapetum 53 megaspores, synergids, antipodals, nucellar cells, endosperm, anther tapetum and abortive pollen in male sterility [8, 23, 38]. PCD is characterized by some typical morphological characteristics such as pycnotic nucleus, DNA fragmentation, shrinkage of cytoplasm, vacuolization and breakdown of cellular contents [4, 21, 22, 36]. Although tapetal PCD involves the common morphological characteristics, vacuolization and vacuole collapse are not mentioned before. The anther tapetum of angiosperms which is a specialized secretory tissue sur- rounds meiocytes/pollen grains and undergoes structural and biochemical changes during the final phase of cell differentiation and death [14]. Tapetal death is essential to provide many molecules required for pollen development including nutrients, pro- teins, lipids and polysaccharides. The molecules contribute to microspore release and pollen wall formation [22]. Death of tapetum in normal anther development is not an uncontrolled event, but a PCD process [21, 40]. In normal development, soon after microspores are released from the tetrad and before pollen mitosis, it has been shown that PCD takes place in some anther sporophytic tissues, resulting in tapetum disap- pearance and development of the dehiscence zone [3, 32]. Premature or delayed degradation of the tapetum results in male sterility. The death of tapetum, coordinated with death of anther wall cells, is indispensable to realization of anther dehiscence and the release of mature pollen grains [14]. Papini et al. [21] researched on tapetal alterations of Lobivia rauschii and Tillandsia albida during PCD. The researchers discussed animal apoptosis and tapetal PCD ultrastructurally and concluded that they shared some common signs such as; shrinkage of the whole cell and nuclei, condensation of chromatin, enlargement of ER and persistence of mitochondria. Wang et al. [37] described massive PCD in tapetum cells by TUNEL staining in response to osmotic and starvation stresses during androgen- esis induction in barley microspores. Futhermore, Leśniewska et al. [15] analyzed anther tapetum with comet assay for detection of nDNA degradation. In addition to tapetal PCD, it was shown that the epidermis, the endothecium and the middle layer undergo PCD like process in Solanum melongena [41], Zea mays [25] and Solanum lycopersicum [28] during pollen maturation. Recently molecular, genetic, ultrastructural and biochemical approaches have been achieved about PCD during male game- tophyte development and sterility such as in rice [29, 42]. Some evidences for involvement of hydrolytic enzymes in anther PCD has been presented. Several researchers remarked that during anther maturation TA56 thiol proteinase [13], cellulase [2], ubiquitin and/or ubiquitinated protein [16], serine palmitoyltransferase level [31] and vacuolar processing enzyme (VPE) which share several structural properties with animal caspase-1 [10] increased suggesting a role for multiple proteolytic systems during cell death. Lathyrus undulatus Boiss. (Fabaceae), which belongs to the Papilionoideae sub- family, is endemic to northwestern Turkey. Our previous research was the anther cytochemistry during pollen maturation in L. undulatus which was the first research among the Lathyrus genus [33]. Vacuolar alterations during the development attracted attention and therefore L. undulatus anthers became the object of the PCD inves- tigations due to the vacuole disruption which was not mentioned earlier in tapetal Acta Biologica Hungarica 63, 2012 54 FILIZ VARDAR and MERAL ÜNAL cells. We undertook a more detailed analysis of development and PCD process in the tapetum of Lathyrus undulatus Boiss. (Fabaceae) by light, fluorescence and electron microscopy. The structural behaviour of tapetal cells during development makes the L. undulatus a promising system for study of cellular events that appear as a conse- quence of tapetal PCD. MATERIALS AND METHODS Light and fluorescence microscopy Flower buds of Lathyrus undulatus Boiss. (Fabaceae) growing in natural habitats in the vicinity of Beykoz-İstanbul (Turkey) was collected in March–April. One anther from each flower bud was gently dissected and squashed for estimation of the development stage by light microscopy. Separated flower buds were fixed in 4% paraformaldehyde in 0.1 M phosphate- buffered saline (0.26 g NaH2PO4; 1.15 g Na2HPO4; 0.9 g NaCl in 100 ml dH2O) pH 7.0 for 4 hours at room temperature and embedded in paraffin. Cross-sections of Fig. 1. Semi-thin sections of L. undulatus anthers at different developmental stages stained with toluidine blue O. (A) PMCs at premeiotic stage and anther wall is made up of epidermis, endothecium, middle layer and tapetum. (B) Tetrad stage with enlarged tapetal cells which contain large central vacuole. (C) Young microspore stage, with dominant vacuole (v), concentrated cytoplasm and undulated nucleus (arrows) in tapetum. (D, E) Vacuolated microspore stage, with degenerating tapetum. The space (*) retained from degenerated middle layer is visible. Nucleus which lost its spherical shape and nucleoli is shown with double arrow. (F) Bicellular pollen stage, tapetal cells completely disintegrated. U-shape endothecium (arrows) existed in mature anther wall. Ep: Epidermis; En: Endothecium; ML: Middle layer; MP: Mature pollen; PMCs: Pollen mother cells; T: Tetrad; Ta: tapetum; v: vacuole; YP: Young pollen. Scale bar = 10 μm Acta Biologica Hungarica 63, 2012 Programmed cell death in tapetum 55 the buds were cut at 8 μm in thickness and stained immediately with 1 μg/ml DAPI [27]. The TUNEL (terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end labeling) technique is the labelling of free 3’OH termini with modified nucle- otides in an enzymatic reaction that identifies the DNA strand breaks. For this reaction, sections were attached to poly-L-lysine coated slides and incubated in reagents from ApopTag® Plus Fluorescein In situ Apoptosis Detection kit (Chemicon) follow- ing the manufacturer’s instructions. The negative controls were labelled in parallel, except for the absence of the TdT. Samples were examined with Leica DM LB2 fluorescence microscope. Electron microscopy Flower buds were fixed in 3% glutaraldehyde in 0.05 M cacodylate buffer (4.28 g/100 ml Na(CH3)2AsO2 · 3H2O and 0.2 M HCl) at pH 7.4 for 6 h at 4 °C and post-fixed in 1% osmium tetroxide in the same buffer for 4 h at 4 °C. The samples were dehy- drated in graded ethanol series (35, 50, 70, 80, 90, 96, 100%) and embedded in Epoxy resin using propylene oxide. Semi-thin sections (1 μm) were stained with toluidine blue and used as controls of the proper stages. Ultrathin sections (~70 nm) contrasted with uranyl acetate and lead citrate, and examined with a JEOL JEM 1011 transmission electron microscope (TEM). RESULTS The anthers of Lathyrus undulatus Boiss. were analyzed in 5 stages correlated with

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