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 sporogenous 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 tonoplast 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, proteins, 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 disappearance 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 androgenesis 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 gametophyte 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 the development of pollen grains: 1. Premeiotic stage 2. Tetrad stage 3. Young microspore stage 4. Vacuolated microspore stage (Late microspore) 5. Bicellular pollen grain stage.

Anther development

In L. undulatus the anther wall is consisted of epidermis, endothecium, middle layer and tapetum (Fig. 1A). The single-layered epidermis remained intact up to anthesis. Endothecial cells acquired thickenings beginning from the vacuolated microspore stage (Fig. 1F). These thickenings aroused along the inner tangential walls extended outward and upwards terminating near the outer tangential wall. Middle layer was ephemeral and crushed at young microspore stage (Fig. 1C).

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Table 1 Widths, lengths and areas of tapetal cells of Lathyrus undulatus in different developmental stages

Width (μm) Length (μm) Area (μm2) Premeiotic stage 16.1±0.5 10.6±0.3 170.6±8.3 Tetrad stage 28.0±1 30.7±0.8 833.2±27.5 Young microspore stage 26.4±2.7 43.2±2.3 1146.4±115.3 Vacuolated pollen stage 25.2±1.2 20.0±0.8 491.8±28.4

The width, length and area at each stage are presented as a mean ± standard error calculated from measurements of 50 cells.

The secretory tapetum is composed of a single layer of glandular cells and sur- rounds the sporogenous tissue. At early developmental stages, tapetal cells appeared similar to pollen mother cells (PMCs) with prominent and spherical nuclei (Fig. 1A). Concurrent with meiosis in PMCs, tapetum expanded both tangentially and radially, as vacuoles increased in size. Small vacuoles fused to form a large central one occu- pying a major portion of the cells up to tetrad stage (Fig. 1B). The central vacuole displaced the cytoplasm to the periphery of the cell. The tapetal cells expanded more in the radial direction than in the tangential direction at the tetrad stage. Measurements of the lengths of the tangential and radial walls in cross sections also provided quan- titative evidences that the tapetal cells most fully developed at young microspore stage (Table 1). During this period, the tapetum underwent substantial changes in cell organization including nucleus morphology typical for PCD. The concentrated cytoplasm densely stained and the central vacuole was dominant. Nucleus periphery appeared undulated and nucleolus was not obvious due to dense staining (Fig. 1C). After lysis of the cell walls, which takes place at the young microspore stage, tapetal cells became naked protoplasts and retain their position at the region of the anther loculus. At vacuolated microspore stage, the volume of tapetal cells were reduced drastically and these cells became ﬂattened accompanied by the central vacuole collapse

Fig. 2. Nucleus degeneration in the tapetum of L. undulatus at different developmental stages stained with DAPI. (A) Tapetal nuclei at tetrad stage, note the evenly dispersed chromatin in whole nucleus. (B) Tapetal nuclei at young microspore stage, starting degeneration. (C–E) Tapetal nuclei at vacuolated microspore stage, showing further degeneration. Scale bar = 10 μm

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(Fig. 1D). Subsequently, the cells underwent progressive disintegration and addition- ally, nucleus lost its spherical shape and nucleolus (Fig. 1E). After degradation of the tapetum, epidermis and single row U-shaped endothecium existed in mature anther wall, and pollen grains remained in the loculus (Fig. 1F).

Nuclear degeneration and PCD

To elaborate the morphological changes in the nuclear degeneration of tapetal cells, the nuclear DNA was labeled by DNA fluorochrome DAPI. At tetrad stage, the spherical tapetal nuclei emitted bright blue fluorescence and the chromatin was dispersed evenly over the whole nucleus (Fig. 2A). At the young microspore stage, some subtle structures, such as granules or flocculi appeared (Fig. 2B). At vacuolated pollen stage, tapetal nuclei lost their shape (Fig. 2C–E). The chromatin, instead of being evenly dispersed, became partially condensed into a compact mass with fluorescence increasing in intensity. The pycnotic nuclear degeneration was characterized by nuclear deformation, distinct shrinkage in volume, formation of an irregular mass, absence of nucleolus prior to the degradation of chromatin. As the area of the condensed chromatin expanded, the whole nucleus became pycnotic and assumed a very bright, compact and slender appearance. The nucleolus disappeared prior to the degradation of chromatin (chromatolysis). The damage of nDNA in tapetal cells firstly appeared at the young microspore stage and increased during vacuolated pollen stage. 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 iden- tify the apoptotic tapetal cells. TUNEL staining was negative prior to young microspore stage at tapetal cells except for a few nuclei; however it was positive in the epidermal nuclei. The TUNEL reaction sharply stained the nuclei of the cells in the anther wall (epidermis, endothecium, middle lamella and tapetum) at young microspore stage (Fig. 3A). At vacuolated pollen stage, as distinct from epidermis, endothecium, middle lamella and tapetal nuclei were still TUNEL positive although they lost its intensity (Fig. 3B). The negative control without any staining clearly established autofluorescence of the anther cell walls and exine but no fluorescence in the nuclei of cells (Fig. 3C).

Ultrastructure

Considering DNA fragmentation indicating PCD, the changes in the ultrastructure of tapetal cells were manifested by TEM analysis. At premeiotic stage, cell organelles were difﬁcultly recognized, only nucleus, mitochondria (spherical), plastids and short endoplasmic reticulum (ER) cisternae could be identiﬁed in the tapetal cytoplasm. A large number of small vacuoles dis-

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Fig. 3. TUNEL staining of L. undulatus anthers at different developmental stages. (A) Young microspore stage, anther wall and ﬁlament cells show TUNEL positive reaction. (B) Vacuolated microspore stage, only tapetal cells show TUNEL positive reaction (arrows). (C) Negative control and autoﬂuorescence of pollen grains (arrows). Scale bar = 100 μm

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Fig. 4. Ultrastructure of a tapetal cell at tetrad stage. (A) Tapetal cell with spherical nucleus. (B) Tapetal cell is in contact with callose wall (*) of tetrad. Double arrows in (A) and (B) show interruptions of tapetal cell walls. cw: cell wall; m: mitochondria; v: vacuole. Scale bar = 1 μm persed in the cytoplasm. The tapetal cell wall was thin and consisted of fine cellulose fibrils. At tetrad stage, the number of mitochondria, plastids and short ER cisternae increased and appeared irregularly in the cytoplasm presenting a denser appearance. Mitochondria are circular or elliptical with very poorly developed cristae. The nucleus is prominent, spherical and chromatin evenly dispersed (Fig. 4A). During the early tetrad stage, large central vacuole occupied a major portion of the cells. The tapetal wall structure was consistent and it was in contact with the adjacent tetrads (Fig. 4B). At late tetrad stage, the wall became thinner and showed interruptions or breakdown at some points (Fig. 4A). At young microspore stage, the ER developed in discrete areas of the cytoplasm and compacted into groups of long, parallel strands (Fig. 5A, B). In the cytoplasm numerous small vacuoles appeared in addition to large central vacuole (Fig. 5C). Although the plasma membrane was still continuous, the cell wall was no longer distinct. Some plastids contained starch deposits, and internal membrane development was visible (Fig. 5C). Morphological changes of nucleus which were the first signs of degeneration indicating PCD were observed as membrane invaginations (Fig. 5D). Close to microspore vacuolization, the chromatin was strongly condensed, and the nuclei were highly fragmented. Tonoplast indentations were realized at central vacuole, as well (Fig. 5A). Any degradative changes in the structure of other organelles are manifested at this stage of tapetal development. At the vacuolated microspore stage, the tapetal cells showed definite signs of degeneration characterized of PCD. All tapetal organelles showed conspicuous changes in the ultrastructure compared to the previous stages. Fusion shapes between remaining small vacuoles were evident. Some of the small vacuoles contained electron dense deposits, fibrillar materials and osmiophilic material of cytoplasmic origin. Tonoplast could pouch inward the vacuole and form a membrane loop bounding a mass of cytoplasm which was to be taken into the vacuole (Fig. 6A, B). When the large central vacuole degenerated drastically, tapetal cells lost their geometrical

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Fig. 5. Ultrastructure of a tapetal cell at young microspore stage. (A) Single arrow shows intact tonoplast; double arrow shows tonoplast indentation. Arrow heads point intact plasma membrane (pm). (B) Compacted ER (arrows). (C) Tapetal plastids (p) and fusing small vacuoles (v). (D) Tapetal nucleus invaginations (arrows). Arrow heads show intact tonoplast. Scale bar = 1 μm shape, shrunk and became ﬂattened. Through the vacuole collapse with the breakdown of the tonoplast, the cytosol disappeared completely leaving out various organelles which appeared to be structurally degenerated (Fig. 6C). The plasma membrane was no longer recognizable. At this stage endomembranes have been strikingly altered and the extraordinarily extended network of ER dilated. With the PCD progressing abundant membranes tightly coiled after layer around the center to form concentric cycle-like structures, named multicycle-like membranes (Fig. 6C, D). A tiny part of cytoplasm, a small number of cytosolic substances or an organelle like mitochondrium were included ⎯⎯⎯⎯→ Fig. 6. Ultrastructure of a tapetal cell at vacuolated microspore stage. (A) Tonoplast blebbing into vacuole with a mass of cytoplasm (arrow). (B) Osmiophilic and granular materials (arrows) in vacuoles. (C) Tightly coiled membranes forming concentric cycle-like structures in the cytoplasm (double arrow). (D) Concentric cycle-like structure in higher magniﬁcation (double arrow). (E) Dilation of ER cisternae (arrows) and electron lucent areas (asteriks). (F) Nuclear membrane dilation (arrows). (G) Chromatin release into cytoplasm (arrows). (H) A degenerated plastid. n: nucleus. Scale bar = 1 μm

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Acta Biologica Hungarica 63, 2012 62 FILIZ VARDAR and MERAL ÜNAL with in the loops of membranes. Electron lucent areas appeared signiﬁcantly (Fig. 6E). In cytoplasm the nucleus lost its shape and shrunk, the chromatin lost its normal interphase appearance, and condensed into many aggregates. Patches of condensed chromatin often lined the periphery of the nuclei (Fig. 6C). Although most of the nuclear envelope still maintained the bilayer structure, the interval between the two nuclear membranes in some tracts was dilated; thus forming enlargements (Fig. 6F). Some regions either the inner or the outer nuclear membrane or both were destroyed to produce a gap. The condensed chromatin tends to be released into cytoplasm through degrading nuclear envelope. Finally nuclear envelope was completely dis- rupted (Fig. 6G). Some of the plastids had osmiophilic structure, and showed little internal membrane development. The degenerated plastids resemble to concentric membrane skeins whilst the mitochondria seem to be the most persistent organelles (Fig. 6H). Later on the tapetum was disintegrated and its remnants were spread out in the loculus. The TEM results indicated that PCD affected the ultrastructure of the tapetal cells from the young microspore stage to 2-celled pollen grain, extending to ﬁnal cell degradation and lysis. Consequently, the whole protoplast collapsed and the tapetum completely degenerated at the 2-celled pollen grain stage.

DISCUSSION

In the present study, the manner of PCD in the tapetal cells of L. undulatus was elu- cidated during pollen development. Previously, PCD in the anthers was reported to occur primarily during tapetum disappearance by various aspects [21, 37, 40]. In apoptosis of animal cells, changes in nuclear morphology follow a standard sequence, characterized by condensation of chromatin, distribution of condensed chromatin into crescents along the periphery of the nuclear envelope, blebbing of the nuclear membrane and finally, separation of the nucleus into discrete masses of condensed chromatin [12, 18]. Similar morphological changes in nuclei are of great importance in identifying plant PCD. In the tapetal cells of L. undulatus first PCD hallmarks appeared at young microspore stage as nuclear deformation and became more severe at vacuolated microspore stage. Varnier et al. [34] reported similar features of nucleus degeneration in the tapetal cells of Lilium as early as the premeiosis stage distinctively. Several researchers also exhibited nucleus degeneration in various types of cells related to PCD with different nucleus stainings [1, 9]. In PCD, biochemical cascades activate specific nucleolytic enzymes that cleave DNA into small, oligonucleosomal fragments [5, 35]. DNA fragmentation was assessed by TUNEL in the tapetal cells of L. undulatus. Although TUNEL positive reaction was intensive in all anther wall cells and connective tissue at young microspore stage, the positive reaction was only apparent in the tapetal cells at vacuolated microspore stage. Wang et al. [37] reported TUNEL positive reaction in the tapetal

Acta Biologica Hungarica 63, 2012 Programmed cell death in tapetum 63 cells of Hordeum vulgare supporting to presented results. The researchers also pointed out autofluorescence in barley anther wall cells, as we observed in L. undulatus. O’Brien et al. [20] explained the autofluorescence with increased chlorophyll, phe- nolic substances and flavonoids during PCD. Vizcay-Barrena and Wilson [36] performed an important comparison of tapetal degeneration between wild type and mutant of Arabidopsis anthers. The researchers observed TUNEL-positive reaction in the tapetal cells of wild type after microspore release, as expected. However, there was no TUNEL stained tapetal nuclei in mutant Arabidopsis ms1, but they monitored TUNEL positive reaction in released microspores. This research indicated that PCD is required for normal pollen development. The researchers also declared TUNEL- positive staining in the epidermis, endothecium, middle layer, stomium and also in connective tissue of both wild type and mutant. This is also the case in L. undulatus. Previously the anther cytochemistry during pollen maturation in L. undulatus was performed [33] and vacuolar alterations during the development attracted attention. Therefore L. undulatus anthers became the object of the presented PCD investiga- tions due to the vacuole disruption during the degeneration which was not mentioned earlier in tapetal cells. The tapetal cells of L. undulatus most fully developed at young microspore stage and underwent conspicuous structural changes at vacuolated pollen stage. Ultrastructural results confirmed that mainly extraordinarily extended network of ER, electron dense deposits in small vacuoles and invaginations in nucleus were conspicuous. Increased ER network was also recorded in Avena sativa [30], Ulex europaeus [19], Lycopersicon esculentum [24], Lobivia rauschii and Tillandsia albida [21] during tapetal degeneration. Papini et al. [21] characterized the ultrastructural features of tapetal cells during PCD such as fibrillar materials, myelin-like skeins and osmiophilic materials in vacuoles, compacted groups of long ER profiles, cellular shrinkage and persistence of mitochondria consistent with the results obtained from L. undulatus. Although we presented central vacuole collapse due to the tonoplast rupture distinctively, the researchers emphasized that no tonoplast rupture was evident in L. rauschii and T. albida [21]. It was indicated that vacuole collapse induced plasmolysis and formation of cytoplasmic aggregations [10] which was reported in tapetal cells of L. undulatus. Furthermore, Varnier et al. [34] described mitochondrial vacuolization instead of cytoplasmic vacuolization throughout PCD in Lilium tapetal cells. Previously, Polowick and Sawhney [24] indicated only small vacuoles containing electron dense deposits, but not large central vacuole and tonoplast rupture during tapetal development of tomato. Wu and Yang [39] described the vacuolization differently in the tapetal cells of Arabidopsis thaliana. They reported that the vacuoles in tapetal cells underwent progressive enlargement prior to the separation of tetrads but became drastically reduced when tetrads just separated from one another. They also announced that such a drastic change in vacuolar volume was not observed in later anther development. Even though there is no evidence about central vacuole collapse during

Acta Biologica Hungarica 63, 2012 64 FILIZ VARDAR and MERAL ÜNAL tapetal PCD, tonoplast rupture was reported in tracheal elements of Zinnia elegans [6], aerenchyma formation of Sagittaria lancifolia [26] and nucellus of Ginkgo biloba [17]. Fukuda [7] characterized one of the variants of plant PCD, which occurs in tracheal element differentiation, by the early disruption of the central vacuole, leading to the release of lytic enzymes into the cytoplasm. Hatsuagi et al. [10] indicated that one of the key enzymes existed in vacuole is VPE (vacuolar processing enzyme) which acts as a processing enzyme to activate various vacuolar proteins, and it might also convert the inactive hydrolytic enzymes to the active forms, which then degrade the vacuoles and initiate the proteolytic cascade in plant PCD. The researchers also emphasized that disintegration of the vacuolar membranes culminates in complete vacuolar collapse in association with plasmolysis and formation of cytoplasmic aggregations within the cells as it was observed in L. undulatus. The key question pointed was: when does the severe degeneration start during PCD? It can be estimated that after central vacuole collapse degradation enzymes released into the cytoplasm and autolysis started [11]. In conclusion, the tapetum underwent substantial changes in cell organization including nucleus morphology monitored by DAPI at young microspore stage and 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 tonoplast rupture, shrinkage of the cytoplasm, the increase and enlargement of the endoplasmic reticulum cisternae and disruption of the plasma membrane. Central vacuole collapse which was not reported before came forward among the other tapetal PCD results performed in different species. These results suggested that DNA fragmentation and the collapse of tonoplast are critical steps in the initiation of PCD in tapetal cells. After vacuole collapse due to possible release of hydrolytic enzymes the cell components degraded. The vacuole collapse suggests that different mechanisms may be responsible for tapetal PCD. Subsequently, tapetal cells completely degenerated at bicellular pollen stage.

ACKNOWLEDGEMENT

This work was supported by the Research Foundation of Marmara University (BAPKO no. FEN-DKR 151105-0227).

REFERENCES

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