Caryologia. International Journal of Cytology, Cytosystematics and Cytogenetics 74(1): 117-126, 2021 Firenze University Press www.fupress.com/caryologia ISSN 0008-7114 (print) | ISSN 2165-5391 (online) | DOI: 10.36253/caryologia-1035 Caryologia International Journal of Cytology, Cytosystematics and Cytogenetics Citation: A. Çetinbaş-Genç, C. Bayam, F. Vardar (2021) Morphological, biochemi- cal and molecular hallmarks of pro- grammed cell death in stigmatic papil- lae of Brassica oleracea L.. Caryologia 74(1): 117-126. doi: 10.36253/caryolo- gia-1035 Received: July 28, 2020 Accepted: April 26, 2021 Published: July 20, 2021 Copyright: © 2021 A. Çetinbaş-Genç, C. Bayam, F. Vardar. This is an open access, peer-reviewed article pub- lished by Firenze University Press (http://www.fupress.com/caryologia) and distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distri- bution, and reproduction in any medi- um, provided the original author and source are credited. Data Availability Statement: All rel- evant data are within the paper and its Supporting Information files. Competing Interests: The Author(s) declare(s) no conflict of interest. ORCID AÇG: 0000-0001-5125-9395 Morphological, biochemical and molecular hallmarks of programmed cell death in stigmatic papillae of Brassica oleracea L. Aslıhan Çetinbaş-Genç*, Cansu Bayam, Filiz Vardar Department of Biology, Marmara University, Kadıköy, 34722, Istanbul, Turkey *Corresponding author. E-mail: aslihan.cetinbas@marmara.edu.tr Abstract. The aim of this study is to determine the programmed cell death hallmarks in the stigmatic papillae of Brassica oleracea L. The flower development was divided in two main stages; pre-anthesis and post-anthesis. Programmed cell death hallmarks were examined in parallel to these stages. At pre-anthesis, the stigmatic papillae were ovoid and their dense cytoplasm were rich in insoluble polysaccharide and protein. At post-anthesis, vacuolization and enlargement were quite evident in papillae. Besides, the protein content decreased, but reactive oxygen species increased in comparison to the pre-anthesis stage. Although no significant change in superoxide dismutase activity was detected, catalase activity decreased and hydrogen peroxide content increased at post-anthesis. DAPI stained nuclei appeared rounded and smooth appearance at pre- anthesis, however, some invaginations and fragmentation in nuclei were observed at post-anthesis. Although, TUNEL staining was negative at pre-anthesis, while TUNEL positive reaction was significant in the nuclei of papillae at post-anthesis. In compari- son to the pre-anthesis, the number of fragmented nuclei monitored by DAPI and TUNEL staining increased at post-anthesis. Keywords: programmed cell death, papillae, reactive oxygen species, sexual plant reproduction, TUNEL. 1. INTRODUCTION Brassica oleracea is a member of Brassicaceae family consisting of 4060 species (Bayer et al. 2019). It is an important agronomic plant due to its con- sumption as a vegetable (Neik et al. 2017). Flowers of B. oleracea have 4 sepals, 4 petals, 2 short and 4 long anthers, and one pistil (Arın 2005). Stigma is the pollen receptive surface of the pistil (Edlun et al. 2004). There are two types of stigmas in angiosperms; wet and dry type. Wet type stigmas produce stig- matic secretions while the dry typed stigmas are devoid of stigmatic secretion. In dry typed stigmas, a protein-based pellicle layer covers the papillae cuticle. Despite this distinction between wet and dry stigmas, stigmatic papillae are characterized by the expression of various biomolecules such as the various organic matters such as insoluble polysaccharide and protein, enzymes, and 118 Aslıhan Çetinbaş-Genç, Cansu Bayam, Filiz Vardar reactive oxygen species (ROS) in both types. (McIn- nis et al. 2006). For instance, stigmatic papillae contain proteins, lipids, carbohydrates that are necessary for pollen germination (Edlund et al. 2004). Also, stigmatic enzymes are necessary for stigma receptivity and func- tion (Souza et al. 2016). Besides, ROS regulates the stigma receptivity and plays as a signal molecule in the pollen germination process (Zafra et al. 2010). During develop- ment, the expression of these biomolecules shows vari- ous changes due to several processes such as organ aging, pollination, cell death and etc. Programmed cell death (PCD) is a genetically regu- lated complex process for plant lifespan. It has been proved that PCD is a necessary process both in devel- opmental and defense processes for plants (Serrano et al. 2010). So, it is investigated in two types; environ- mentally induced (ePCD) and developmentally regulated (dPCD) (van Hautegem et al. 2015). While both types are significant processes, dPCD particularly has an impor- tant function during plant life. dPCD occurs in various cells, tissues, or organs for various purposes. However, reproductive development is a rich arena as a showcase for dPCD in plants.  Because dPCD can take place in sex determination, anther tapetum, megaspore, synergid, and antipodal cells, nucellus, endosperm, stylar trans- mitting tissue, stigmatic papillae or etc. (Brighigna et al. 2006; Vardar and Ünal 2012; Papini et al. 2011). dPCD is accompanied by various developmental stages at stig- ma during female reproductive organ development. For instance, stigma no longer required for a flower after pol- lination and it is eliminated by PCD (Rogers 2006). The stigmatic branches of Actinidia chinensis are degenerated by dPCD after pollination (Ferradas et al. 2014). Also, Stigma goes PCD when incompatible pollen lands on the stigma. Thus, PCD is involved in the pollen selection process of stigma (Wu and Cheung 2000). For instance, the stigmatic papillae undergo dPCD after incompatible pollination in Olea europaea (Serrano et al. 2010). Characteristic hallmarks of dPCD in plants can be observed by various morphological, biological, and molecular methods. The fundamental descriptive hall- marks are cell shrinkage, cytoplasmic and nuclear break- down, and DNA fragmentation (Serrano et al. 2010). Also, ROS accumulation is among the hallmarks of PCD causing harmful chain effects in the cell. Since the accu- mulation of ROS causes oxidative stress, they are bal- anced by scavenging mechanisms including antioxidants such as superoxide dismutase (SOD) and catalase (CAT) (Apel and Hirt 2004; Pandhair and Sekhon 2006). SOD accelerates the conversion of superoxide, which is one of the reactive and toxic ROS, to hydrogen peroxide (H2O2). CAT catalyzes the deterioration of H2O2 thereby over- coming oxidative stress. So, changes in SOD and CAT enzyme activity and H2O2 content can give hint about the level of oxidative stress (Wang et al. 2010). The aim of the present study is to investigate the morphological, biochemical, and molecular hallmarks of dPCD in stigmatic papillae of Brassica oleracea L. The obtained results may provide new insights into the role of dPCD in stigma development and help to improve the knowledge on dPCD hallmarks in reproductive organs. To this end, we assayed different dPCD markers in stig- matic papillae excised from flowers at pre-anthesis and post-anthesis. 2. MATERIAL AND METHODS 2.1 Determination of flower development stage Flowers of B. oleracea L. were collected from the vicinity of Akçakoca/Düzce (Turkey) in 2019. The stig- ma development was divided into 2 main stages (pre- anthesis and post-anthesis) correlated with some mor- phological markers of the flower such as the position of calyx and corolla, anther dehiscence, and the absence or presence of pollen on it. The flower buds with calyx covering half of the bud and collected 2-3 days before anthesis were accepted at pre-anthesis. At this stage, there were no pollen grains on the stigma, because the anthers were still indehiscent. The flowers with senescent petals and collected 2-3 days after anthesis were accepted at post-anthesis. At this stage, a lot of pollen grains were visible on the stigma due to anther dehiscence. 2.2 Morphological and biochemical changes After fixation in acetic acid:alcohol solution (1:3, v/v), pistils were dehydrated and embedded in paraffin blocks. To investigate the morphological and biochemi- cal changes, sections (8-10 μm) were stained with Peri- odic Acid-Schiff (PAS) (Feder and O’Brien 1968) for insoluble polysaccharides and, stained with Coomassie Brilliant Blue (CBB) (Fisher 1968) for proteins. Images were captured using Olympus BX-51 microscope and KAMER AM software. The optical density (OD) of insoluble polysaccharide and protein contents of papil- lae were computed using Image J software (Rodrigo et al. 1997). 20 papillae were used for each group. 2.3 ROS accumulation and antioxidant enzyme activity ROS accumulation of papillae was determined according to previous studies (Fabian et al. 2019). Fresh 119Morphological, biochemical and molecular hallmarks of programmed cell death in stigmatic papillae of Brassica oleracea L. tissues were labeled by 20 μM 2’,7’-dichlorodihydro- fluorescein diacetate (H2DCFDA) and images were cap- tured using Olympus BX-51 f luorescence microscope and KAMER AM software. Fluorescence intensities of 20 papillae were measured using the Image J soft- ware. Superoxide dismutase (SOD) and catalase (CAT) activities were detected according to Li et al. (2000) and Prochazkova et al. (2001), respectively. After homogeni- zation of 0.03 g tissue in 1500 μl 50 mM PBS (pH 7.8) and centrifugation at 12,000×g for 15 min, supernatants were used as SOD and CAT enzyme source. To meas- ure the spectrophotometric SOD activity, 300 μl super- natant (same volume of 50 mM PBS for control) was added to 2400 μl measurement buffer containing 1500 μl of 50 mM PBS (pH 7.8), 300 μl of 130mM L-methio- nine, 300μl of 750μM nitro blue tetrazolium, 300 μl of 100 μM EDTA-Na2. After incubation under light (50 μmol m-2 s-1) for 3 minutes, SOD activity was measured at 560 nm. A non-incubated mixture was used as the blank. To measure the spectrophotometric CAT activity, 200 μl supernatant was added to 2400 μl measurement buffer containing 1500 μl of 0.2 M PBS (pH 7.0) with 1% (w/v) PVP and 1000 μl of H2O2. CAT activity was meas- ured by the decrease in absorbance for 2 min at 240 nm. H2O2 content was measured according to Junglee et al. (2014). After homogenization of 0.03 g tissue in 2000 μl buffer containing 0.1% trichloroacetic acid, 1 M KI, 10 mM phosphate saline buffer, and centrifugation at 12,000×g for 15 min, the supernatant was incubated in dark for 20 minutes. Afterward, H2O2 content was meas- ured at 390 nm, spectrophotometrically. 2.4 Analysis of DNA Fragmentation To determine the DNA fragmentation, 4’,6-Diami- dine-2’-phenylindole dihydrochloride (DAPI) (Schweizer 1976) and terminal deoxynucleotidyl transferase  dUTP nick end labeling  (TUNEL)  (O’Brien et al. 1997) test were performed. After fixation in PBS containing 4% paraformaldehyde, pistils were dehydrated and embed- ded in paraffin blocks. Sections (8-10 μm) were stained with DAPI and, TUNEL assay was conducted using the ApopTag®  Plus Fluorescein  In situ  Apoptosis Detection kit (Chemicon, Temecula, CA, USA). To avoid false-posi- tive TUNEL results, TUNEL results were evaluated con- sidering the control slides included in the kit supplied by the company were used. Images were captured using Olympus BX-51 f luorescence microscope and KAM- ERAM software. To evaluate the significant differences in nuclei undergoing PCD, percentages of DAPI stained and TUNEL positive nuclei were presented counting approximately 300 nuclei for each treatment. 2.5 Statistical analysis Statistical analyses were performed by IBM SPSS 16.0 software and data were subjected to one-way anal- ysis of variance (ANOVA) with a threshold P value of 0.05. 3. RESULTS 3.1 Morphological and biochemical changes The morphological and biochemical features of papillae were investigated to analyze their main differ- ences at pre-anthesis and post-anthesis. Papillae were ovoid and tightly packed cells at pre-anthesis. They had a thin wall, small vacuole (arrows, Fig. 1c, e), and dense cytoplasm (dots, Fig. 1c, e). Papillae cells lost their tight alignment with the increase of their diameters during the development. In comparison with the pre-anthesis, the lengths of papillae were significantly increased by 84.12% at post-anthesis (Fig. 1a). Also, the widths of papillae were significantly increased by 42.31%, in com- parison with the pre-anthesis (Fig. 1b). At post-anthesis, it was remarkable that the vacuole was quite enlarged and covered a large part of the cell (arrows, Fig. 1d, f ). Moreover, organic matter contents of papillae such as insoluble polysaccharide and protein were changed at post-anthesis (Fig. 1c-f ). Cytoplasmic content was rich in insoluble polysaccharide and protein contents at pre- anthesis stage (Fig. 1c, e). According to the OD results of PAS stained papillae, no significant change in insol- uble polysaccharide content of papillae was detected between pre-anthesis and post-anthesis (Fig. 1g). Howev- er, according to the OD results of CBB stained papillae, protein contents of papillae were significantly decreased by 33.68% at post-anthesis, when compared with the pre-anthesis (Fig. 1h). 3.2 ROS accumulation and antioxidant enzyme activity To determine the ROS accumulation difference of papillae at two development stages, stigmatic tissues were stained by H2DCFDA. While ROS accumulation was poor at pre-anthesis, an increase in ROS accumula- tion was quite remarkable at post-anthesis (arrows, Fig. 2a, b). To present the subtler differences, fluorescence intensities of H2DCFDA labeled papillae were measured. When compared with the pre-anthesis, the fluorescence intensity of H2DCFDA was significantly increased by % 31.69 at post-anthesis (Fig. 2c). To reveal the effects of ROS accumulation on the antioxidant system, changes 120 Aslıhan Çetinbaş-Genç, Cansu Bayam, Filiz Vardar in SOD-CAT activity and H2O2 content were investi- gated. When compared to pre-anthesis, no significant change in SOD activity was detected at post-anthe- sis (Fig. 2d). However, H2O2 content was significantly increased by %23.21 (Fig. 2e) and CAT activity was sig- nificantly decreased by %37.5, at post-anthesis (Fig. 2f). 3.3 Analysis of Nuclear DNA Fragmentation To determinate the nuclear morphology and DNA fragmentation, DAPI staining and TUNEL tests were performed. DAPI stained nuclei were showed rounded and smooth appearance at pre-anthesis. The spherical nuclei of papillae emitted bright blue fluorescence and the chromatin was dispersed regularly (arrows, Fig. 3a). However, nuclei lost their rounded appearance and some invaginations and fragmentation were observed at post- anthesis (arrows, Fig. 3b). In comparison with the pre- anthesis, the number of fragmented nuclei monitored by DAPI staining was significantly increased about 11-fold at post-anthesis (Fig. 3e). TUNEL assay results were in parallel with the DAPI results. Although TUNEL stain- ing was negative at pre-anthesis, the TUNEL positive reaction was significant in the nuclei of papillae at post- anthesis (arrows, Fig. 3c, d). The number of fragmented nuclei monitored by TUNEL staining was significantly increased about 6-fold at post-anthesis (Fig. 3f). 4. DISCUSSION dPCD is a major process during reproductive devel- opment in plants and occurs at various developmental stages (Wang et al. 2020). Atrophy of tapetum, non- Figure 2. ROS accumulation and antioxidant enzyme activity of papillae at pre-anthesis and post-anthesis. a ROS accumulation at pre-anthesis. b ROS accumulation at post-anthesis. c Fluorescence intensity of H2DCFDA at pre-anthesis and post-anthesis. d Change in SOD activity. e Change in H2O2 content. f Change in CAT activ- ity. White arrows indicate the low ROS accumulation and red arrows indicate the high ROS accumulation. Bar: 20 μm. Figure 1. Morphological and biochemical changes of papillae at pre-anthesis and post-anthesis. a Length of papillae. b Width of papillae. c PAS stained papillae at pre-anthesis. d PAS stained papil- lae at post-anthesis. e CBB stained papillae at pre-anthesis. f CBB stained papillae at post-anthesis. g OD of PAS stained papillae. h OD of CBB stained papillae. Black arrows indicate the vacuoles, red arrows indicate the cell wall and points indicate the cytoplasms. Bar: 20 μm. 121Morphological, biochemical and molecular hallmarks of programmed cell death in stigmatic papillae of Brassica oleracea L. functional megaspores, nucellus, synergids, antipo- dal, and suspensor cells are some examples of dPCD in the development of reproductive organs (Kurusu and Kuchitsu et al. 2017; Buono et al. 2019). Also, dPCD may take place at stigmatic branches or stigmatic papillae during female reproductive organ development. Espe- cially papilla cell death is a good model for studying on PCD process (Ye et al. 2020). Serrano et al. (2010) have been reported that stigmatic papillae degenerated by dPCD after the pollination process in Olea europaea. Ferradas et al. (2014) have been described stigmatic branches of Actinidia chinensis degenerated after polli- nation. Besides, Huang et al. (2020) have been reported the PCD in stigmatic papillae of Raphanus sativus and Brassica napa after pollination. According to our results, stigmatic papillae degenerated by dPCD after the anthe- sis stage. Balk and Leaver (2001) have been indicated the alterations in tapetal cell morphology of Helianthus annuus, during dPCD. Also, Qiu et al. (2008) have been reported structural disintegration in non-functional megaspores of Lactuca sativa during dPCD. Serrano et al. (2010) have been reported the plasma membrane damage and alterations of cell morphology in stigmatic papillae during PCD. Also, Ferradas et al. (2014) have been indicated the progressive vacuolization and orga- nelle disintegration in stigmatic branches during PCD. Similarly, we detected some alterations in the shapes of papillae cells at post-anthesis. These alterations were probably related to both the increase of their diameters and dPCD process. During dPCD processes, researchers have been reported the vacuolization in the inner integument of Brassica napus (Wan et al. 2002), in tapetal cells of Ory- za sativa (Ku et al. 2003) and in tapetum and filament of Lathyrus undulatus (Vardar and Ünal 2012). Besides, vacuolization was reported during dPCD processes ofstyle tissue of Ficus carica (Aytürk and Ünal 2018), stigma of Arabidopsis thaliana (Gao et al. 2018), and anther and ovule of Opuntia robusta (HernandezCruz et al. 2019). Parallel to these literatures, it was remarkable that the vacuoles were large and covered the large part of the cell at the post-anthesis stage which we detected the dPCD. Researchers have been reported the decrease in the protein content of cytoplasm in petals of Nicotia- na tabacum (Serafini-Fracassini et al. 2002), in tepals of Iris and Alstroemeria (Wagstaff et al. 2005) and Lilium candidum (Mochizuki-Kawai et al. 2015) during dPCD. Similar to these findings, we detected the decrease in the protein content of papillae at the post-anthesis stage which we detected the dPCD. ROS is the major regulator of plant growth and development due to its interaction capability with all cellular substances such as protein, lipid, signaling molecules, hormones and etc. (Waszczak et al. 2018; Sankaranarayanan et al. 2020). ROS acts as cellular sign- aling molecules in lower doses. However excessive ROS production leads to PCD (Oracz and Karpinsky 2016). Yadegari and Drews (2004) have been specified that ROS plays vital role in the control and implementation of dPCD of aleurone and endosperm cells. Also, Hayashi et al. (2001) have indicated that ROS accumulation in the central cell of the embryo sac acted as a signal molecule in dPCD of antipodal cells. Besides, Tripathi and Tuteja (2007) have been reported that ROS is accompanied to dPCD process in sepals, petals, and ovules. Duan et al. (2014) have been specified that ROS production caused cell wall alteration for the reception of pollen tubes in synergid cells of Arabidopsis thaliana by causing dPCD in them. Also, van Durme and Nowack (2016) have been implicated that ROS signal regulates the dPCD of tapetal cells at the right time. ROS also has a role in signal- ing networks promoting pollen germination and pollen tube growth on stigma. Thus, the concentration of ROS on the stigma affects the stigma receptivity and germi- Figure 3. Analysis of nuclear DNA fragmentation. a DAPI stained spherical nuclei at pre-anthesis. b DAPI stained degenerated nuclei at post-anthesis. c TUNEL negative reaction at pre-anthesis. d TUNEL positive reaction at post-anthesis. e DAPI stained frag- mented nuclei rate. f TUNEL positive nuclei rate. White arrows indicate the nuclear morphology and red arrows indicate the TUNEL positive nuclei. Bar: 20 μm. 122 Aslıhan Çetinbaş-Genç, Cansu Bayam, Filiz Vardar nation capability of pollen (Zafra et al. 2010). Breygina et al. (2020) and Zhang et al. (2020) have been reported that proper doses of ROS in stigma exudate are impor- tant for the communication between the pollen/pol- len tube and female tissues at various stages. However, excessive ROS accumulation may induce the dPCD of papillae. Researchers have been reported ROS-medi- ated dPCD occurred in papillae during incompatible pollination in the Olea europaea (Serrano et al. 2015). According to our results, the intense ROS signal was quite remarkable in papillae cells at the post-anthesis stage that we detected the dPCD. One of the most com- monly occurring and most stable ROS is H2O2. It gen- erated by the reduction of superoxide anions via SOD. Also, CAT breakdown H2O2 to H2O and O2 (Yanık et al. 2018). Enzymes such as SOD and CAT that regu- late the H2O2 content show differential expression dur- ing dPCD (Singh et al. 2016). According to our results, no significant change in SOD activity was detected at post-anthesis when compared to pre-anthesis. However, H2O2 content was significantly increased at post-anthe- sis. Also, CAT activity was significantly decreased at post-anthesis. Researchers have been reported that the high H2O2 content of stigmatic papillae may be related to the stigmatic receptivity or dPCD process (Serrano et al. 2012; Xie et al. 2014). Therefore, high H2O2 content in the post-anthesis indicates that the stigma is recep- tive at this stage. However, since dPCD occurs in papil- lae at post-anthesis, high H2O2 content is more likely to be related to PCD. Besides, researchers have been indi- cated that high H2O2 content is involved dPCD process of petal and tapetal cells (Tripathi and Tuteja 2007; Xie et al. 2014). Also, researchers have been reported that ROS increased due to the increased SOD and decreased CAT activities in sepals of daylily that undergoing dPCD (Panavas and Rubinstein 1998). DAPI staining is one of the most commonly used methods for check the nuclei morphology. Research- ers have been reported the various alterations by DAPI staining in chromatin, DNA, and nucleus during dPCD; such as nuclei shrinkage and chromatin condensa- tion in tapetal cells of Lobivia rauschii and Tillandsia albida (Papini et al. 1999), chromosomal degradation in suspensor and endosperm of Vicia faba (Wredle et al. 2001) and, nuclear deformation and volume chang- es in synergid and antipodal nuclei of T. aestivum (An and You 2004). Also, researchers have been reported the nucleus and DNA degradation in suspensor and endosperm of Phaseolus coccineus (Lombardi et al. 2007), nucleus degeneration and chromatin fragmenta- tion in synergids of Malus domestica (Tagliasacchi et al. 2007) and, chromatin condensation in non-functional megaspores of Lactuca sativa (Qiu et al. 2008) during dPCD. Besides, nucleus and chromatin deformations in anther wall cells of Lathyrus undulatus (Vardar and Ünal 2012) and, chromatin condensation in stamen pri- mordia of Cucumis sativus (Pawełkowicz et al. 2019) were indicated as doped hallmarks by various research- ers. Also, Shi et al. (2020) have been reported chroma- tin fragmentation in suspensor cells undergoing PCD of Nicotiana tabacum. In parallel to these literatures, we detected the various invaginations and fragmenta- tion in papillae nuclei at the post-anthesis stage which we detected the dPCD. Similarly, Ferradas et al. (2014) have been reported that dPCD occurs in stigmatic branches and papillae of Actinidia chinensis after polli- nation, and chromatin condensation and nucleus degra- dation are quite remarkable during this dPCD process. Besides, Gao et al. (2018) have been shown the nucleus degradation in stigmatic papillae of Arabidopsis thali- ana during dPCD. Also, TUNEL method is one of the most common and definitive methods used to determine PCD. It allows the determine PCD by marking the free 3 ’OH ends of DNA formed by endonucleases in cells. Researchers have been detected the TUNEL positive reaction in cells undergoing PCD such as in filament of Hordeum vulgare (Wang et al. 1999), in tapetal cells of Zea mays (González-Sanchez et al. 2004) and, in suspen- sor and endosperm of Phaseolus coccineus (Lombardi et al. 2007). Also, TUNEL positive nuclei were detected in anther wall cells and filament of Lathyrus undulatus (Vardar and Ünal 2012), in style of Ficus carica (Aytürk and Ünal 2018), in anther of Opuntia robusta’s female flower (HernándezCruz et al. 2019) and in stamen pri- mordia of Cucumis sativus’s female flower (Pawelkowicz et al. 2019). Shi et al. (2020) have been reported TUNEL positive reaction in suspensor undergoing PCD of Nico- tiana tabacum. Jimenez-Duran et al. (2021) have been detected DNA fragmentation by TUNEL assay during the dPCD process of Marathrum schiedeanum’s cen- tral cell. At the post-anthesis stage, we also detected the TUNEL positive nuclei in papillae. Similarly, Ferradas et al. (2014) have been detected TUNEL positive nuclei in Actinidia chinensis’s stigmatic branches and papil- lae undergoing dPCD. Also, Gao et al. (2018) have been reported the TUNEL positive nuclei in Arabidopsis thali- ana’s stigmatic papillae undergoing dPCD. 5. CONCLUSION In conclusion, our results including vacuoliza- tion, decreased protein content, increased ROS con- tent, increased H2O2 content and decreased CAT activ- 123Morphological, biochemical and molecular hallmarks of programmed cell death in stigmatic papillae of Brassica oleracea L. ity, and also nuclear fragmentation marked by DAPI and TUNEL positive nuclei at the post-anthesis stage revealed that papillae cells undergo dPCD at the post- anthesis stage. We think that our results will contribute to a clear understanding of dPCD in plants, especially during reproductive development. ACKNOWLEDGEMENTS We thank Prof Meral Ünal for her support. This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sec- tors. REFERENCES An LH, You RL (2004) Studies on nuclear degeneration during programmed cell death of synergid and antip- odal cells in Triticum aestivum.  Sex Plant Reprod 17(4):195-201. https://doi.org/10.1007/s00497-004- 0220-1 Apel K, Hirt H (2004) Reactive oxygen species: metabo- lism, oxidative stress, and signal transduction. Annu Rev Plant Biol 55:373-399. https://doi.org/10.1146/ annurev.arplant.55.031903.141701 Arın L (2005) Alabaş (Brassica oleraceae var. gongylodes L.) Yetiştiriciliği. Alatarım 4(2):13-17. Aytürk Ö, Ünal M (2018) Reorganization of microtubules in pistil cells undergoing programmed cell death in Ficus carica L.  Caryologia 71(4):482-488. https://doi. org/10.1080/00087114.2018.1505238 Balk J, Leaver CJ (2001) The PET1-CMS mitochon- drial mutation in sunflower is associated with pre- mature programmed cell death and cytochrome c release. The Plant Cell 13(8):1803-1818. https://doi. org/10.1105/TPC.010116 Bayer PE, Golicz AA, Tirnaz S, Chan CKK, Edwards D, Batley J (2019) Variation in abundance of predict- ed resistance genes in the Brassica oleracea pange- nome.  Plant Biotech J 17(4):789-800. https://doi. org/10.1111/pbi.13015 Buono RA, Hudecek R, Nowack MK (2019) Plant pro- teases during developmental programmed cell death. J Exp Bot 70(7):2097-2112. https://doi.org/10.1093/ jxb/erz072 Breygina M, Klimenko E, Shilov E, Mamaeva A, Zgoda V, Fesenko I (2020) ROS in tobacco stigma exu- date affect pollen proteome and provoke mem- brane hyperpolarization. bioRxiv. https://doi. org/10.1101/2020.04.28.065706 Brighigna L, Papini A, Milocani E, Vesprini JL (2006) Programmed cell death in the nucellus of Tillandsia (Bromeliaceae).  Caryologia  59(4):334-339. https:// doi.org/10.1080/00087114.2006.10797935 Duan Q, Kita D, Johnson EA, Aggarwal M, Gates L, Wu HM, Cheung AY (2014) Reactive oxygen species mediate pollen tube rupture to release sperm for fer- tilization in Arabidopsis.  Nat Commun 5:3129. htt- ps://doi:10.1038/ncomms4129 Edlund AF, Swanson R, Preuss D (2004) Pollen and stig- ma structure and function: the role of diversity in pollination.  The Plant Cell 6(suppl 1):84-S97. https:// doi.org/10.1105/tpc.015800 Fabian A, Safran E, Szabo-Eitel G, Barnabas B, Jager K (2019) Stigma functionality and fertility are reduced by heat and drought co-stress in wheat.  Front Plant Sci 10:244. https://doi.org/10.3389/fpls.2019.00244 Feder N, O’Brien TP (1968) Plant microtechnique: Some principles and new methods. Am J Bot 55(1):123– 142. https://doi.org/10.1002/j.1537-2197.1968. tb06952.x Ferradas Y, Lopez M, Rey M, Gonzalez MV (2014) Pro- grammed cell death in kiwifruit stigmatic arms and its relationship to the effective pollination period and the progamic phase.  Ann Bot  114(1):35-45. https:// doi.org/10.1093/aob/mcu073 Fisher DB, Jensen WA, Ashton ME (1968) Histochemical studies of pollen: Storage pockets in the endoplas- mic reticulum. Histochemie 13(2):169-182. https:// doi:10.1007/BF00266578 Gao Z, Daneva A, Salanenka Y, Van Durme M, Huys- mans M, Lin Z, Winter FD, Vanneste S, Karimi M, Van de Velde J, Vandepoele K, Van de Walle D, Dewettinck K, Lambrecht BN, Nowack MK (2018) KIRA1 and ORESARA1 terminate flower receptivity by promoting cell death in the stigma of Arabidop- sis.  Nat Plant  4(6):365. https://doi:10.1038/s41477- 018-0160-7 Gonzalez-Sanchez M, Rosato M, Chiavarino M, Puer- tas MJ (2004) Chromosome instabilities and pro- grammed cell death in tapetal cells of maize with B chromosomes and effects on pollen viability. Genet- ics 166(2):999-1009. https://doi.org/10.1534/genet- ics.166.2.999 Hayashi Y, Yamada K, Shimada T, Matsushima R, Nishi- zawa NK, Nishimura MA (2001) Proteinase-storing body that prepares for cell death or stresses in the epidermal cells of Arabidopsis. Plant Cell Physiol 42:894-899. https://doi.org/10.1093/pcp/pce144 HernAndez-Cruz R, Silva-Martinez J, Garcia-Campu- sano F, Cruz-Garcia F, Orozco-Arroyo G, Alfaro I, Vázquez-Santana S (2019) Comparative development 124 Aslıhan Çetinbaş-Genç, Cansu Bayam, Filiz Vardar of staminate and pistillate flowers in the dioecious cactus Opuntia robusta. Plant Reprod 1-17. https:// doi.org/10.1007/s00497-019-00365-w Jimenez-Duran K, Perez-Pacheco MK, Wong R, Colla- zo-Ortega M, Marquez-Guzman J (2021) Pro- grammed cell death is the cause of central cell degeneration and single fertilization in Marath- rum schiedeanum (Cham.) Tul (Podostemaceae). Aquat Bot 169: 103345. https://doi.org/10.1016/j. aquabot.2020.103345 Junglee S, Urban L, Sallanon H, Lopez-Lauri F (2014) Optimized assay for hydrogen peroxide determina- tion in plant tissue using potassium iodide. Am J Analyt Chem 5:730–736. https://doi.org/10.4236/ ajac.2014.511081 Ku S, Yoon H, Suh HS, Chung YY (2003) Male-sterility of thermosensitive genic male-sterile rice is asso- ciated with premature programmed cell death of the tapetum.  Planta 217(4):559-565. https://doi. org/10.1007/s00425-003-1030-7 Kurusu T, Kuchitsu K (2017) Autophagy, programmed cell death and reactive oxygen species in sexual reproduction in plants.  J Plant Res  130(3):491-499. https://doi.org/10.1007/s10265-017-0934-4 Li HS, Sun Q, Zhao SJ, Zhang WH (2000) Experiment principle and technology of plant physiology and biochemistry. Higher Education Press, Beijing. Lombardi L, Ceccarelli N, Picciarelli P, Lorenzi R (2007) DNA degradation during programmed cell death in Phaseolus coccineus suspensor.  Plant Physiol Bioch 45(3-4):221-227. https://doi.org/10.1016/j. plaphy.2007.01.014 McInnis SM, Desikan R, Hancock JT, Hiscock SJ (2006) Production of reactive oxygen species and reac- tive nitrogen species by angiosperm stigmas and pollen: potential signalling crosstalk?. New Phy- tol 172(2):221–228. https://doi.org/10.1111/j.1469- 8137.2006.01875.x Mochizuki-Kawai H, Niki T, Shibuya K, Ichimura K (2015) Programmed cell death progresses dif- ferentially in epidermal and mesophyll cells of lily petals.  PloS one 10(11):e0143502. https://doi. org/10.1371/journal.pone.0143502 Neik TX, Barbetti MJ, Batley J (2017) Current status and challenges in identifying disease resistance genes in Brassica napus.  Front Plant Sci 8:1788. https://doi. org/10.3389/fpls.2017.01788 O’Brien IE, Reutelingsperger CP, Holdaway KM (1997) Annexin‐V and TUNEL use in monitor- ing the progression of apoptosis in plants.  Cytom- etry 29(1):28-33. https://doi.org/10.1002/(SICI)1097- 0320(19970901)29:1<28::AID-CYTO2>3.0.CO;2-9 Oracz K, Karpinski S (2016) Phytohormones signaling pathways and ROS involvement in seed germina- tion.  Front Plant Sci 7:864. https://doi.org/10.3389/ fpls.2016.00864 Panavas T, Rubinstein B (1998) Oxidative events dur- ing programmed cell death of daylily (Hemerocallis hybrid) petals.  Plant Sci 133(2):125-138. https://doi. org/10.1016/S0168-9452(98)00034-X Pandhair V, Sekhon BS (2006) Reactive oxygen spe- cies and antioxidants in plants: an overview.  J Plant Biochem Biot 15(2):71-78. https://doi.org/10.1007/ BF03321907 Papini A, Mosti S, Brighigna L (1999) Programmed-cell- death events during tapetum development of angio- sperms. Protoplasma 207(3-4):213-221. https://doi. org/10.1007/BF01283002 Papini A, Mosti S, Milocani E, Tani G, Di Falco P, Brighi- gna L (2011) Megasporogenesis and programmed cell death in Tillandsia (Bromeliaceae).  Protoplas- ma  248(4):651-662. https://doi.org/10.1007/s00709- 010-0221-x Pawelkowicz ME, Skarzynska A, Plader W, Przybecki Z (2019) Genetic and molecular bases of cucumber (Cucumis sativus L.) sex determination. Mol Breeding 39(3):50. https://doi.org/10.1007/s11032-019-0959-6 Prochazkova D, Sairam RK, Srivastava GC, Singh DV (2001) Oxidative stress and antioxidant activity as the basis of senescence in maize leaves.  Plant Sci 161(4):765-771. https://doi.org/10.1016/S0168- 9452(01)00462-9 Qiu YL, Liu RS, Xie CT, Russell SD, Tian HQ (2008) Cal- cium changes during megasporogenesis and mega- spore degeneration in lettuce (Lactuca sativa L.).  Sex Plant Reprod 21(3):197-204. https://doi.org/10.1007/ s00497-008-0079-7 Rodrigo J, Rivas E, Herrero M (1997) Starch determi- nation in plant tissues using a computerized image analysis system. Physiol Plant 99(1):105–110. https:// doi.org/10.1111/j.1399-3054.1997.tb03437.x Rogers HJ (2006) Programmed cell death in floral organs: how and why do flowers die?.  Ann Bot 97(3):309- 315. https://doi.org/10.1093/aob/mcj051 Sankaranarayanan, S., Ju, Y., & Kessler, S. A. (2020). Reactive Oxygen Species as Mediators of Game- tophyte Development and Double Fertilization in Flowering Plants. Front Plant Sci 11:1199. https://doi. org/10.3389/fpls.2020.01199 Schweizer D (1976) Reverse fluorescent chromosome banding with chromomycin and DAPI. Chromosoma 58(4):307–324. https://doi.org/10.1007/BF00292840 Serafini-Fracassini D, Del Duca S, Monti F, Poli F, Sac- chetti G, Bregoli AM, Biondi S, Della Mea M (2002) 125Morphological, biochemical and molecular hallmarks of programmed cell death in stigmatic papillae of Brassica oleracea L. Transglutaminase activity during senescence and programmed cell death in the corolla of tobac- co (Nicotiana tabacum) flowers.  Cell Death Diff 9(3):309. https://doi.org/10.1038/sj.cdd.4400954 Serrano I, Olmedilla A (2012) Histochemical location of key enzyme activities involved in receptivity and self- incompatibility in the olive tree (Olea europaea L.). Plant Sci 197:40–49. https://doi.org/10.1016/j.plants- ci.2012.07.007 Serrano I, Pellicione S, Olmedilla A (2010) Programmed- cell-death hallmarks in incompatible pollen and pap- illar stigma cells of Olea europaea L. under free pol- lination. Plant Cell Rep 29(6):561-572. https://doi. org/10.1007/s00299-010-0845-5 Serrano I, Romero-Puertas MC, Sandalio LM, Olmedil- la A (2015) The role of reactive oxygen species and nitric oxide in programmed cell death associated with self-incompatibility. J Exp Bot 66(10):2869-2876. https://doi.org/10.1093/jxb/erv083 Shi C, Luo P, Zhao P, Sun MX (2020) Detection of embryonic suspensor cell death by whole-mount TUNEL assay in tobacco. Plants 9(9):1196. https:// doi.org/10.3390/plants9091196 Singh R, Singh S, Parihar P, Mishra RK, Tripathi DK, Sin- gh VP, Chaukan DK, Prasad SM (2016) Reactive oxy- gen species (ROS): beneficial companions of plants’ developmental processes.  Front Plant Sci 7:1299. htt- ps://doi.org/10.3389/fpls.2016.01299 Souza EH, Carmello-Guerreiro SM, Souza FVD, Ros- si ML, Martinelli AP (2016) Stigma structure and receptivity in Bromeliaceae. Sci Hort 203:118–125. https://doi.org/10.1016/j.scienta.2016.03.022 Tagliasacchi AM, Andreucci AC, Giraldi E, Felici C, Ruberti F, Forino LMC (2007) Structure, DNA con- tent and DNA methylation of synergids during ovule development in Malus domestica Borkh.  Caryologia 60(3):290-298. https://doi.org/10.1080/00087114.2007 .10797950 Tripathi SK, Tuteja N (2007) Integrated signaling in flower senescence: an overview.  Plant Signal Behav  2(6):437- 445. https://doi.org/10.4161/psb.2.6.4991 Van Durme M, Nowack MK (2016) Mechanisms of developmentally controlled cell death in plants.  Curr Opin Plant Biol  29:29-37. https://doi.org/10.1016/j. pbi.2015.10.013 Van Hautegem T, Waters AJ, Goodrich J, Nowack MK (2015) Only in dying, life: programmed cell death during plant development.  Trends Plant S ci  20(2):102-113. https://doi.org/10.1016/j. tplants.2014.10.003 Vardar F, Ünal M (2012) Development and programmed cell death in the filament cells of Lathyrus undulatus Boiss.  Caryologia 64(2):164-172. https://doi.org/10.10 80/00087114.2002.589779 Wagstaff C, Chanasut U, Harren FJ, Laarhoven LJ, Thom- as B, Rogers HJ, Stead AD (2005) Ethylene and flow- er longevity in Alstroemeria: relationship between tepal senescence, abscission and ethylene biosyn- thesis.  J Exp Bot 56(413):1007-1016. https://doi. org/10.1093/jxb/eri094 Wan L, Xia Q, Qiu X, Selvaraj G (2002) Early stages of seed development in Brassica napus: a seed coat‐spe- cific cysteine proteinase associated with programmed cell death of the inner integument. The Plant Jour- nal 30(1):1-10. https://doi.org/10.1046/j.1365- 313X.2002.01262.x Wang M, Hoekstra S, van Bergen S, Lamers GE, Oppedi- jk BJ, van der Heijden MW, Priesder W, Schilperoort RA (1999) Apoptosis in developing anthers and the role of ABA in this process during androgenesis in Hordeum vulgare L. Plant Mol Biol 39(3):489-501. https://doi.org/10.1023/A:1006198431596 Wang P, Du Y, Li Y, Ren D, Song CP (2010) Hydrogen peroxide–mediated activation of MAP kinase 6 mod- ulates nitric oxide biosynthesis and signal transduc- tion in Arabidopsis.  The Plant Cell 22(9):2981-2998. https://doi.org/10.1105/tpc.109.072959 Wang Y, Ye H, Bai J, Ren F (2020) The regulatory frame- work of developmentally programmed cell death in floral organs: A review. Plant Physiol Biochem 158:103- 112. https://doi.org/10.1016/j.plaphy.2020.11.052 Waszczak C, Carmody M, Kangasjärvi J (2018) Reactive oxygen species in plant signaling. Annu Rev Plant Biol 69:209-236. https://doi.org/10.1146/annurev- arplant-042817-040322 Wredle U, Walles B, Hakman I (2001) DNA fragmenta- tion and nuclear degradation during programmed cell death in the suspensor and endosperm of Vicia faba.  Int J Plant Sci 162(5):1053-1063. https://doi. org/10.1086/321922 Wu HM, Cheung AY (2000) Programmed cell death in plant reproduction, in: Lam, E.,  Fukuda, H.,  Green- berg, J. (Eds.), In  Programmed Cell Death in Higher Plants, Springer, Dordrecht, pp. 23-37. Xie HT, Wan ZY, Li S, Zhang Y (2014) Spatiotemporal production of reactive oxygen species by NADPH oxidase is critical for tapetal programmed cell death and pollen development in Arabidopsis. Plant Cell 26:2007–2023. https://doi.org/10.1105/tpc.114.125427 Yadegari R, Drews GN (2004) Female gametophyte devel- opment.  The Plant Cell,  16(suppl 1):133-S141. htt- ps://doi.org/10.1105/tpc.018192 Yanik F, Aytürk Ö, Çetinbaş-Genç A, Vardar F (2018) Salicylic acid-induced germination, biochemical and 126 Aslıhan Çetinbaş-Genç, Cansu Bayam, Filiz Vardar developmental alterations in rye (Secale cereale L.). Acta Bot Croat 77(1):45–50. https://doi.org/10.2478/ botcro-2018-0003 Ye H, Ren F, Guo H, Guo L, Bai J, Wang Y (2020) Iden- tification of key genes and transcription factors in ageing Arabidopsis papilla cells by transcriptome analysis. Plant Physiol Biochem 147:1-9. https://doi. org/10.1016/j.plaphy.2019.12.008 Zafra A, Rodriguez-Garcia MI, Alche JD (2010) Cellu- lar localization of ROS and NO in olive reproductive tissues during flower development. BMC Plant Biol. 10(1):36–50. https://doi.org/10.1186/1471-2229-10-36 Zhang MJ, Zhang XS, Gao XQ (2020) ROS in the male– female interactions during pollination: function and regulation. Front Plant Sci 11: 177. https://doi. org/10.3389/fpls.2020.00177