Development and Programmed Cell Death in the Filament Cells of ...

CARYOLOGIA

Vol. 64, no. 2: 164-172, 2011

Development and Programmed Cell Death in the Filament Cells of Lathyrus undulatus Boiss. VARDAR* FILIZ and ÜNAL MERAL Marmara University, Science and Art Faculty, Department of Biology, Göztepe, 34722, İstanbul, TURKEY.

Abstract — The androecium in Lathyrus undulatus Boiss. consisted of 10 stamens. The anther was joined to the filament on the outward facing surface (dorsifixed). After dehiscence anther and filament separate from connecting edge and anther falls off. In the present study development and programmed cell death process in the filament cells of Lathyrus undulatus Boiss. (Fabaceae) were analyzed by light, fluorescence and electron microcopy, during pollen development. The observations indicated that filament cells most fully developed at tetrad stage. The cytochemical identifications of proteins, insoluble polysaccharides and lipids within and around the vascular bundle suggest that filament cells are involved in the transport of the organic materials. Moreover, filament cells subject to programmed cell death confirmed with TUNEL from young to mature pollen stage including structural alterations such as nucleus and chromatin condensation, vacuolization, concentric circle of membranes and disintegrated plastids. Granular materials and electron lucent areas in the cytoplasm were common at mature pollen stage. The attained data suggests that degenerated anther separate from filament by programmed cell death of the connecting edge. Key words: cytochemistry, Fabaceae, filament, Lathyrus undulatus Boiss., programmed cell death.

INTRODUCTION In higher plants, male reproductive organs – the stamens – consist of two parts, anther and filament. The anther is specialized to produce pollen and must dehisce at the right time to release the mature pollen grains. The anther thecae were joined together by connective tissue connecting anther to filament. The filament which is generally long, thin hair-like structure supplies nutrient to the anther and elongates to facilitate the delivery of pollen grains (GOLDBERG et al. 1993). In autogamous (self-fertilization) plants, three processes occurring late in stamen development contribute to a successful pollination at

*Corresponding author: phone: +90 216 345 11 86-1177; fax: +90 216 347 87 83; e-mail: [email protected] Co-author: phone: +90 216 345 11 86-1356; fax: +90 216 347 87 83; e-mail: [email protected]

anthesis: (1) elongation of the filament at preanthesis, bringing the anthers close to the stigma; (2) pollen maturation, allowing deposition of fertilizing pollen grains on the stigma and thus reaching the ovules; and (3) anther dehiscence through programmed cell death, to release pollen on the stigma upon filament elongation (CECCHETTI et al. 2008). In the model plant Arabidopsis, for self-pollination occurrence the anther must dehisce at the right time to release the mature pollen grains when the female organ pistil is ready to receive and the filament must reach the right height to deliver the released pollen grains to the pistil. Male fertility in the self-fertilizing Arabidopsis requires not only normal pollen development, but also correct timing of anther dehiscence and appropriate filament elongation to release them to the stigma of the pistil at the right time (ZHAO and MA 2000). Studies (STINTZI and BROWSE 2000; SANDERS et al. 2000) revealed that the male sterile mutants of Arabidopsis the filament did not elongate sufficiently to allow the anther to reach the height

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PCD IN THE FILAMENT CELLS OF L . UNDULATUS

of the stigma. During pollen maturation and pollen release through anther dehiscence, tissue degeneration and cell death take place in male reproductive organ (GOLDBERG et al. 1993). In course of degeneration, dehisced anther and filament separates from connecting edge and anther falls off, similarly leaf senescence. Plant senescence is a developmentally regulated and genetically programmed process that ultimately leads to death of a particular cell, organ or whole plant. The senescence involves structural, biochemical and molecular changes, which are also the hallmarks of programmed cell death (PCD). Generally in plants, senescence can also be considered as one of the examples for PCD (VAN DOORN and WOLTERING 2004; TRIPATHI and TUTEJA 2007). It is an important and final event of plant development and usually observed in many different tissues such as leaves, petals, reproductive organs (stamens and style), root cap, and endosperma. Reproductive organs provide an excellent model for irreversible PCD in which failure to properly enter compromises reproductive unsuccess, sometimes even resulting in sterility. The mechanism by which the hormones or their precursors are rapidly transmitted and sensed to bring about the changes in different tissues

such as petals, stamens and ovary are not yet well known (WU and CHEUNG 2000; ROGERS 2006). Although filament cells undertake very important roles during anther development and pollination, the available data are very few and incomplete concerning cytochemical and structural development, and PCD process in the connecting edge of dehisced and degenerated anther. However cell death signs in filament cells were only mentioned in a limited number of anther development and PCD studies, such as in Hordeum vulgare (WANG et al. 1999) and Lilium (VARNIER et al. 2005). Therefore to better understand, we undertook a detailed analysis of the development and PCD process in the filament cells of Lathyrus undulatus Boiss. (Fabaceae) by light, fluorescence and electron microcopy. The behavior of filament cells during development makes the L. undulatus a promising system for study of cellular events that appear as a consequence of PCD in stamens. MATERIAL AND METHODS Flower buds of Lathyrus undulatus Boiss. (Fabaceae) growing in natural habitats in the vi-

Fig. 1 — SEM micrographs of stamens of L. undulatus. (a) The anther was joined to the filament on the outward facing surface (dorsifixed). (b) After dehiscence, degenerated anther and filament separate from connecting edge and anther falls off.

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cinity of Beykoz-İstanbul (Turkey) were collected in March-April. Flower buds were fixed in 3% glutaraldehyde in 0.05 M cacodylate buffer 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 dehydrated in ethanol series, and embedded in Epoxy resin using propylene oxide. All of the sections were collected from the anther-filament connecting edge. Semithin sections (1µm) 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). The fixed materials for scanning electron microscopy (SEM) dried with hexamethyldisilazane (HMDS). For cytochemical observations, the osmication step was omitted from the fixation. Semithin sections (1 µm) were stained with periodic acid-Schiff (PAS) (FEDER and O’BRIEN 1968) for insoluble polysaccharides, Coomassie Brilliant Blue (FISHER 1968) for proteins, Sudan Black B for lipids (PEARSE 1961). The TUNEL (terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end labeling) technique is the labelling of free 3’OH

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termini with modified nucleotides in an enzymatic reaction that identifies the DNA strand breaks. For this reaction, separated flower buds were fixed in 4% paraformaldehyde in phosphate-buffered saline (PBS, pH 7.0) for 4 hours at room temperature and embedded in paraffin. Cross sections were placed on poly-L-lysine coated slides and incubated in reagents from ApopTag® Plus Fluorescein In situ Apoptosis Detection kit (Chemicon) following 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. RESULTS The androecium in Lathyrus undulatus Boiss. consisted of 10 stamens, and the filaments united to form two bundles of (9)+l (diadelphous) arrangement. The anther was joined to the filament on the outward facing surface (dorsifixed) (Figure 1a). The filament was terete and threadlike. After dehiscence anther and filament separate from connecting edge and anther falls off (Figure 1b).

Fig. 2 — Semi-thin sections of L. undulatus filament cells at different developmental stages stained with Toluidine blue. (a) Xylem (x), phloem (ph) parenchymatic (p) and connective (c) tissue cells at premeiotic stage. (b) Tetrad stage. (c) Cell shrinkage (in circles), vacuolization (v) and morphologic nucleus disorder (arrows) at young pollen stage. (d) Mature pollen stage. x: xylem. Bar in (d) = 20 µm and also applies to (a-c).

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In order to elucidate the structural and cytochemical features of filament cells of Lathyrus undulatus Boiss., the sections were collected from the anther-filament connecting edge and analyzed in 4 stages correlated with the development of pollen grains: 1. Premeiotic stage 2. Tetrad stage 3. Young pollen stage 4. Mature pollen grain stage. At premeiotic stage, the filament was composed of parenchyma surrounding amphicribral vascular bundle (concentric vascular bundle that has the phloem surrounding the xylem). The single vascular bundle traversed the filament and prolonged into the connective tissue between the anther lobes. The parenchymatic cells with spherical nuclei did not contain a prominent intercellular space system (Figure 2a). At the indicated stage, cytochemical reactions revealed that cytoplasm of filament cells presented weak reaction with regard to insoluble polysaccharides,

protein, and lipids as carried out by PAS (Figure 3a), Coomassie brilliant blue (Figure 4a), and Sudan black B (Figure 5a), respectively. At tetrad stage, the cytoplasm of filament cells, particularly the parenchymatic cells, became denser as distinct from premeiotic stage (Figure 2b). The cytochemical analysis distinguished that the dense cytoplasm of parenchymatic cells started to accumulate insoluble polysaccharides (Figure 3b), protein (Figure 4b) and lipids (Figure 5b), along with development. At young pollen stage, the parenchymatic cells of filament underwent a number of changes in cell organization including cell shrinkage, vacuolization and nucleus disorder. The cytoplasm displaced to the periphery of the cell in the vacuolated cells. Some of the nuclei showed morphological changes and lost their spherical shape which is the one of the signs of degeneration indicating PCD (Figure 2c). At this stage vascular bundle cells were conspicuous with thickened secondary walls. Besides the filament cells gave intense reaction with regard to insolu-

Fig. 3 — Semi-thin sections of L. undulatus filament cells at different developmental stages stained with Periodic acid-Schiff. (a) Premeiotic stage. (b) Tetrad stage. (c) Young pollen stage. (d) Mature pollen stage. Bar in (d) = 20 µm and also applies to (a-c).

Fig. 4 — Semi-thin sections of L. undulatus filament cells at different developmental stages stained with Coomassie Brilliant Blue. (a) Premeiotic stage. (b) Tetrad stage. (c) Young pollen stage. (d) Mature pollen stage. Bar in (d) = 20 µm and also applies to (a-c).

Fig. 5 — Semi-thin sections of L. undulatus filament cells at different developmental stages stained with Sudan Black B. (a) Premeiotic stage. (b) Tetrad stage. (c) Young pollen stage. d. Mature pollen stage. Bar in (d) = 10 µm and also applies to (a-c).

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ble polysaccharides (Figure 3c), protein (Figure 4c) and lipids (Figure 5c). Additionally, lipid droplets were observed occasionally. With regard to morphological changes of nuclei, TUNEL procedure was applied. The TUNEL reaction, which labels the 3’ OH ends of DNA strand breaks, was used in order to confirm the occurrence of intra-nucleosomal DNA cleavage and to identify the apoptotic filament cells. Although TUNEL staining was negative at meiotic stages (Figure 6a), the filament cells indicated TUNEL positive reaction at young microspore stage (Figure 6b). Ultrastructural studies displayed that chromatin lost its normal interphase appearance (chromation disperses throughout the nucleus), and condensed into many aggregates. Patches of condensed chromatin often lined the periphery of the nuclei (Figure 7a). Signals of filament cell degeneration were distinct nucleus disorder, vacuolization, concentric circle of membranes, disintegrated plastids, compact mitochondria and lipid droplets were also monitored. Besides, the cytoplasm diluted, granular materials and electron lucent areas appeared. Intercellular space occurred between the parenchymatic cells (Figure 7b).

At mature pollen stage, concurrent with progressive vacuolization, compact cytoplasmic substances and nucleus located at the cell periphery (Figure 2d). The cytoplasm exhibited positive reaction for mentioned organic substances currently (Figure 3d, 4d, 5d), though many parenchymatic vascular cells lost their cellular contents and nuclei. Similarly vascular bundle cells lost their shapes. At mature pollen stage, filament cells were still TUNEL positive however they lost their intensity (Figure 6c). Furthermore TUNEL staining was negative just before anther dehiscence (Figure 6d). Ultrastructural results revealed that plastids remained in the cytoplasm of filament cells which lost the most of their organelles. No nuclei were observed in the degenerated cells and they were crashed between the vacuolated cells. Granular materials and electron lucent areas were common at this stage (Figure 7c-e). DISCUSSION In the present study, the development and PCD events in the filament cells of L. undulatus were elucidated during pollen development. Pre-

Fig. 6 — TUNEL staining of L. undulatus filament cells at different developmental stages. (a) TUNEL negative reaction in filament cells (in circle) at meiotic stage. (b) TUNEL positive reaction in filament cells (in circle) at young pollen stage. (c) TUNEL reaction in filament cells (in circle) lost their intensity at the beginning of mature pollen stage. Mature pollen grains (arrows) in the anther lobes show autofluorescence. (d) TUNEL negative reaction in filament cells (in circle) just before anther dehishence. Mature pollen grains (arrows) are in the anther lobes. Bar in (d) = 10 µm and also applies to (a-c).

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viously, the occurrence of PCD in the anthers was reported primarily during tapetal degeneration (PAPINI et al. 1999; WU and CHEUNG 2000; VARNIER et al. 2005; VARDAR and ÜNAL 2012), epidermal cells adjacent to the stomium, endothecium and the connective tissue (BONNER and DICKINSON 1989; BEALS and GOLDBERG 1997). The best of our knowledge, no report has been published of development and PCD events in the filament cells, although detailed studies were performed in anther wall cells. In this context the developmental events in the filament cells of L. undulatus were analyzed in integrity based on light, fluorescence, electron microscopy, taking PCD

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into consideration. The results exhibited that the filament cells of L. undulatus underwent PCD throughout pollen development, from young microspore to mature pollen grain. GOLDBERG et al. (1993) explained that the filament is a tube of vascular tissue that anchors the stamen and serves as a conduit for water and nutrients. At mature pollen stage the anther enlarges and pushes upward in the flower by filament extension, and tissue degeneration, dehiscence and pollen grain release occur. As the researchers’ indication, together with the pollen maturation and anther dehishence filament cells degenerate as well the anther wall cells. In L.

Fig. 7 — Ultrastructure of L. undulatus filament cells at different developmental stages. (a) Nucleus disorder, chromatin condensation (arrow heads), vacuolization (v), concentric circle of membranes (arrow), disintegrated plastids (p), compact mitochondria (m), intercellular space between the parenchymatic cells (double arrows) at young pollen stage. (b) Higher magnification of (a); intercellular space between the parenchymatic cells (double arrows), disintegrated plastids (p), lipid droplet (arrow). (c) Degenerated and crashed cells (arrow) between vacuolated (v) cells at mature pollen stage. Plastids (p) are still remained. (d, e) Granular materials (asterisk) in cytoplasm, electron lucent areas (double asterisks) and vacuoles (v) are common in the filament cells at mature pollen stage. n: nucleus, p: plastid. Bars = 1 µm. Bar in (e) also applies to (c, d).

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undulatus, filament cells developed and started to accumulate insoluble polysaccharide, protein and lipid as from tetrad stage providing its nutrient conduction. Besides, the cells indicated degeneration signs including nucleus invaginations signalling PCD. As previously reported in tapetum and middle layer of L. undulatus degenerated simultaneously (VARDAR and ÜNAL 2011a; 2011b; 2012). Ultrastructural changes during PCD such as vacuolization, concentric circle of membranes, chromatin condensation and nucleus invaginations observed in the filament cells of L. undulatus were reported in aerenchyma formation of Sagittaria lancifolia (SCHUSSLER and LONGSTRETH 2000), endosperm of Oryza sativa (WEI et al. 2002), nucellus of Ginkgo biloba (LI et al. 2003), tapetal cells of L. undulatus (VARDAR and ÜNAL 2012) and trichome (PAPINI et al. 2010) and nucellus of Tillandsia (BRIGHIGNA et al. 2006). In PCD, biochemical cascades activate specific proteolytic enzymes that cleave DNA into small, oligonucleosomal fragments (FRIEDLANDER 2003; VILA and PRZEDBORSKI 2003). DNA fragmentation was assessed by TUNEL in the filament cells of L. undulatus. Although TUNEL positive reaction was intensive at young microspore stage, the positive reaction was less intensive at the beginning of mature pollen stage. WANG et al. (1999) reported TUNEL positive reaction in the anther wall and filament cells of Hordeum vulgare during anther development. VIZCAY-BARRENA and WILSON (2006) observed TUNEL-positive staining in the whole connective tissue of both wild type and mutant Arabidopsis. This is also the case in L. undulatus. VARNIER et al. (2005) indicated that just before microspore mitosis, the signs of PCD simultaneously reached the endothecium, the epidermis and the connective tissue. In these cell types, the nucleus exhibited the first invaginations and some mitochondria were affected simultaneously by the development of internal vacuoles and dark vesicles. The researchers indicated that the features were maintained until pollen maturation and anther dehiscence, but neither endothecial nor epidermal nor connective cells degenerated. In conclusion, we provide the first detailed cytological description of filament cells in the course of the anther development, based on structural and cytochemical features. The observations indicated that filament cells most fully developed at tetrad stage. The histochemical identifications of insoluble polysaccharides,

proteins and lipids within and around the vascular bundle suggest that they are involved in the transport of these organic materials. Moreover filament cells subject to PCD represented with TUNEL from young to mature pollen grain stage including structural alterations such as nucleus and chromatin condensation, vacuolization, concentric circle of membranes and disintegrated plastids. These results suggest that after dehiscence anther degenerate and separate from filament by PCD of the connecting edge. Our data providing a new look at the timing of filament development and degeneration will be the object of further PCD investigations. Acknowledgement — This work was supported by the Research Foundation of Marmara University (BAPKO no. FEN-DKR 151105-0227).

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