Caffeine-Induced Premature Chromosome Condensation Results in the Apoptosis-Like Programmed Cell Death in Root Meristems of Vicia faba

Chemicals and antibodies

Hydroxyurea (HU, 2.5 mM), pararosaniline, bovine serum albumin (BSA), propidium iodide (PI) and 4',6-diamidino-2-phenylindole (DAPI) were purchased from Sigma. Caffeine (CF, 5 mM) was supplied by Merck, Triton X-100 by Fluka, RNase from SERVA. Other chemicals were obtained from POCH S.A. (if not indicated otherwise in the text).

Immunocytochemical and biochemical detection of PARP-2 was performed using rabbit polyclonal antibodies from Agrisera (Vännas, Sweden; #AS10675). The rabbit monoclonal antibodies specific to phospho-H2AX (Ser139) (20E3) were supplied by Cell Signaling (Danvers, MA, USA; #9718). Bound primary antibodies in all investigated cases were detected with the secondary goat anti-rabbit IgG AlexaFluor^®488 antibody (Agrisera, Vännas, Sweden; ABIN2176504, for immunocytochemistry) and also the secondary anti-rabbit IgG (AP-linked) antibody (Cell Signaling, Danvers, MA, USA; 7054, for immunoblotting and tissue printing). The mouse monoclonal antibody β-actin (A5441) and the secondary goat anti-mouse (AP-linked; A3562) antibody were from Sigma-Aldrich (Saint Quentin, France).

Plant material, growth conditions, HU-treatment and PCC induction

Seeds of Vicia faba var. minor cv. Nadwiślański (Center for Seed Production, Sobiejuchy, Poland) were dark-germinated at room temperature on wet filter paper in Petri dishes. Four days after imbibition, 3 cm seedlings were selected and incubated in (i) water (32 h; negative control); (ii) HU (2.5 mM for 32 h; S-phase synchronization; positive control), or (iii) 2.5 mM HU for 24 h and then transferred into a mixture of 2.5 mM HU and 5 mM caffeine for 8 h (HU/CF; total incubation time: 32 h; PCC induction), as described by Rybaczek [21]. During germination and incubation the roots were oriented horizontally and aerated continuously by gentle rotation of fluids in a water-bath shaker (30 rpm).

Cytology

1.5-cm-long apical fragments of V. faba primary roots (n = 30 for each series) were fixed in cold Clarke’s mixture (absolute ethanol/glacial acetic acid; 3:1, v/v) for 1 h (according to Bruni et al. [22]), washed three times with 96% ethanol/rehydrated (70–30% ethanol) distilled water and subjected to Feulgen staining (according to Rybaczek et al. [23]). For this procedure, the roots were hydrolyzed in 4 M HCl (at room temperature for 2 h), and stained with Schiff’s reagent (pararosaniline). After staining (1 h), root fragments were rinsed three times in SO₂-water and once in distilled water. 1.5-mm-long root sections were cut off and squashed in a drop of 45% acetic acid onto Super-Frost microscope slides (Menzel-Gläser) using the dry ice method. After removing the coverslips, the slides were dehydrated, air dried, and embedded in Canada balsam (Merck, Germany). The quantification of mitotic/PCC/AL-PCD cells and scoring of the micronucleus frequency (MN-test) were determined by counterstaining with Schiff's reagent for 1 h at room temperature. Three parameters were evaluated: (1) mitotic index (i.e. percentage of mitotic cells), (2) PCC index (i.e. percentage of PCC-type mitoses in relation to all mitoses, with the proviso that PCC-type aberrant mitoses were calculated as a sum: S-PCC + G2-PCC + segregation defects) and (3) AL-PCD index (i.e. percentage of Feulgen-stained nuclei showing signs of AL-PCD in relation to all meristem cells, i.e. either interphase or mitotic). The percentages were calculated based on 5,000 cells per treatment (1,000 cells on each of the 5 preparations in each series). The experiments were done in triplicate. Cytological observations were made using an Optiphot-2 microscope (Nikon) and images were recorded with a DXM 1200 CCD camera (Nikon). Macroscopic observations of roots (control vs treated with HU vs treated with HU/CF during PCC induction) were made using Stemi 2000C stereoscopic microscope (Zeiss, Jena, Germany) and images were recorded by AxioCam ERc5s CCD camera (Zeiss, Jena, Germany). Quantitative analyses were performed using AxioVision software, 4.8 version (Zeiss, Jena, Germany). Image processing was done in Adobe Photoshop 7.0 (Adobe Systems) or ImageJ 1.37c (Public Domain by Wayne Rasband) according to Abràmoff et al. [24].

Estimation of cell death in planta: acridine orange and ethidium bromide staining

Fluorescence staining with acridine orange (AO) and ethidium bromide (EB) was used for detection of cell death according to the method described by Byczkowska et al. [8]. This method allows gradual staining of cells depending on their stage: living to dead. AO penetrates all cells both living and dead but EB can only enter a cell after disintegration of the cell’s membrane. Therefore, living cells containing only AO appear green under fluorescent microscopy, cells in early apoptosis appear green-yellow to yellow, cells in late apoptosis appear yellow-orange to bright orange, and dead cells appear as dark orange to bright red [8]. Briefly, 1.5-cm-long apical fragments of living roots (n = 30 for each series) were cut off and washed 2 times in 0.01M phosphate buffer, pH 7.4 (PHB) and stained for 4 min with 1 ml of a mixture containing 100 μg ml^-1 AO and 100 μg ml^-1 EB in PHB. After removing the 'staining mixture' the root fragments were washed 2 times in PHB, fixed with 1% glutardialdehyde (Merck) in PHB for 15 min, and cut with a razor blade along the long axis of the root. The thin sections obtained were washed 2 times for 2 min with PHB and placed onto Super Frost glass slides (Menzel-Gläser) with a drop of PHB. Observations were performed by Optiphot-2 fluorescence microscope (Nikon) equipped with a B-2A filter (blue light; λ = 495 nm). It is worth noting that the images typical of double AO/EB staining described here (distinguishable as green-yellow, yellow, yellow-orange, bright orange and red fluorescence; per Byczkowska et al. [8]) were only visible through a narrow-strip type of fluorescent filter (in a wide-strip all observed nuclei were totally green). Images were recorded at exactly the same time of integration using a DS-Fi1 CCD camera (Nikon, Japan) and Act-1 software (Precoptic, Warsaw, Poland). Quantitative analysis was performed using the basic functions of ImageJ v1.37c software (Public Domain by Wayne Rasband). To this end, we used the selection tool to trace out the area of each root which were then measured in pixels. Then, we used the cut-off threshold (threshold → thresholding method: minimum; cut off color: 125–255): (1) we selected all the points with the following colors: red, yellow and green, and (2) measured their surface areas using analyses in Excel (Microsoft Office, 2003). The whole root area was 100% and respective areas occupied by the three different colors in each of the roots were calculated (indicating green–living cells, yellow–dying cells; red–dead cells). In the next stage, analyses were performed in a similar manner, except that the analyzed areas were limited to each root zone (II—meristematic zone containing apical meristem, III—elongation zone, IV—differentiation zone). Means were calculated based on 300 cells per sample taken from three independent experiments.

Single-cell microgel electrophoresis: alkaline versus neutral comet assay

The first step of the comet assay procedure consisted in the preparation of agarose-embedded V. faba protoplasts according to the method described by Tegeder et al. [25] with minor modifications. Briefly, 5-mm sections of root tips (n = 30 for each series) were incubated for 4 h in 10 mM citrate buffer (pH 4.8, 37°C) supplemented with 5% (w/v) cellulase R10, 1% (w/v) pectinase, 1% (w/v) macerozyme R-10 from Rhizopus sp. (Serva), 1% (w/v) hemicellulase from Aspergillus niger (Sigma) and 2.5% (w/v) pectolyase. The macerat was filtered through a ø 48 μm nylon mesh. The protoplast suspension was centrifuged at 100 G for 5 min at 4°C. The pellet was washed 2 times with the mannitol/MES buffer (0.5 M mannitol and 20 mM MES, pH 5.5). The sediment was suspended in a small amount of 0.5 M mannitol in the same buffer.

SSBs and DSBs were assessed with alkaline and neutral single-cell microgel electrophoresis (comet assay), respectively according to the method described by Potocki et al. [26]. Briefly, protoplasts originating from V. faba root tips were mixed with 0.7% low melting (LM) agarose, added to agarose-LM slides, lyzed overnight with proteinase K (0.5 mg ml^-1) and reduced glutathione (2 mg ml^-1) in a lysis solution (1.25 M NaCl, 50 mM EDTA, 100 mM Tris-HCl, 0.01% N-lauroylsarcosine sodium salt, pH 10) for 2 h at 37°C. The slides were then placed in a horizontal gel electrophoresis unit (Bio-Rad). The neutral comet assay buffer was as follows: 100 mM Tris-HCl, 0.5 M NaCl, 1 mM EDTA, 0.2% DMSO, pH 10; and the alkaline comet assay buffer was as follows: 1 mM EDTA, 0.2% DMSO, 300 mM NaOH, pH >12. Next, the slides were stained with 0.25 μM YOYO-1 (Invitrogen Corporation, Grand Island, NY, USA) in 2.5% DMSO and 0.5% sucrose, mounted with a coverslip and stained with DAPI (0.4 μg ml^-1; Sigma-Aldrich). Observations were made immediately using an AxioImagerA1 fluorescence microscope (Zeiss, Jena, Germany) equipped with a blue light filter (excitation 470/40 nm; emission 525/50 nm) for YOYO-1, and a UV filter (UV light; excitation 365 nm; emission 445/50 nm) for DAPI. All images were recorded at exactly the same time of integration using an AxioCam ERc5s CCD camera (Zeiss, Jena, Germany) and AxioVision 4.8 software (Zeiss, Jena, Germany). Comet analyses were carried out automatically using OpenComet ImageJ software. Histograms were transferred manually to a diagram using Photoshop CS5 (Adobe Systems). The tail moment (TM) was calculated for every image of nuclei as the product of tail DNA (i.e. amount of DNA in the comet tail) times tail length (i.e. length of the comet tail measured from right border of head area to end of tail, according to the method described by Olive et al. [27] and Kołodziejek et al. [28]). We analyzed 30 images of nuclei from each experimental series. All experiments were repeated five times. Statistical analysis was carried out using Excel 2003.

Terminal deoxynucleotidyl transferase-mediated dUTP (2'-deoxyuridine, 5'-triphosphate) nick end-labeling (TUNEL) assay in Click-chemistry technology

For detection of apoptosis TUNEL-assay (Click-iT^® TUNEL Alexa Fluor^® 594 Imaging Assay for microscopy and HCS, Invitrogen-Life Technologies, #C10246) was performed according to the protocol provided by the manufacturer (using slightly modified methods described by Jones et al. [29] and Gladish et al. [30]). In this assay, free 3'-OH ends of fragmented DNA were enzymatically labeled with fluorescein-modified nucleotide (i.e. fluorescein-dUTP) using terminal deoxynucleotidyl transferase (TdT). The Click-iT^® TUNEL Alexa Fluor^® 594 Imaging Assay was able to detect apoptotic cells of V. faba under the investigated conditions. Additionally for each batch, a negative control without the addition of TdT enzyme and a positive control with DNase I treatment, to generate strand breaks, were always included to ensure the reproducibility of the assay. The percentage of positive-labeled cells (red fluorescence typical of Alexa Fluor^® 594 azide: 590/615 nm) represents averages from three repetitions.

For each sample, 5-mm sections of root tips (n = 30 for each series) were fixed using 4% paraformaldehyde (Polysciences, #18814) in PBS for 45 min at room temperature. The fixation step was followed by a permeabilization step with 0.25% Triton X-100 in PBS for 20 min at room temperature. Next, terminal deoxynucleotidyl transferase-mediated dUTP (2'-deoxyuridine, 5'-triphosphate) nick end-labeling (TUNEL) assay was performed following the manufacturer's instructions strictly (Click-iT^® TUNEL Alexa Fluor^® Imaging Assay Protocol) and the nuclei were stained for 3 min with 0.3 mg ml^-1 4',6-diamidino-2-phenylindole (DAPI). Finally, the cells were mounted in Vectashield embedding medium (Vector Laboratories, CA, USA). Images were collected with an AxioImagerA1 fluorescence microscope (Zeiss, Jena, Germany) equipped with a green light filter (excitation 545/25 nm; emission 605/70 nm) for AlexaFluor^®594, and a UV filter (UV light; excitation 365 nm; emission 445/50 nm) for DAPI. All images were recorded at exactly the same time of integration using an AxioCam ERc5s CCD camera (Zeiss, Jena, Germany) and AxioVision 4.8 software (Zeiss, Jena, Germany). Image processing was done in Adobe Photoshop 7.0 (Adobe Systems).

DNA extraction and separation

1.5-mm-long sections of roots (n = 30 roots for each series; repeated twice) were homogenized in a mortar according to the method described by Byczkowska et al. [8] using 600 μl extraction buffer (2% SDS; 0.5 M NaCl; 100 mM Tris-HCl, pH 8.0; and 50 mM EDTA, pH 8.0) for 60 s. The homogenates were incubated at 65°C for 40 min, vortexed, chilled on ice for 10 min and centrifuged (12,000 G, 10 min, 4°C). Then, chloroform/isoamyl alcohol (24:1) was added (1.0 volume per each sample). Next, the samples were vigorously mixed (by inversion) for 2 min and centrifuged at 12,000 G for 1 min at 4°C. The supernatant was transferred to a fresh Eppendorf tube and extracted with 0.8 volume of cold isopropanol for 2 min. 500 μl of 70% ethanol was added to the pellet, microcentrifuged for 2 min (minispin, Eppendorf), dried and re-suspended with 40 μl of TE buffer (10 mM Tris HCl, 1 mM EDTA, pH 8.0; BioShop^® Canada Inc., Burlington) containing RNase A (20 μg ml^-1). Isolated samples of DNA were dissolved in distilled nuclease-free water, and separated on 1.5% agarose gel with 0.5 μg ml^-1 ethidium bromide. As a DNA marker, 1 kb DNA ladder was used, 250–10,000 bp (Fermentas, Thermo Fisher Scientific). The separated DNA samples were visualized under UV light.

Total protein extraction and Western blotting

Proteins were extracted from 1.5-mm-long sections of roots (n = 30 roots for each series; repeated twice) using TriPure Isolation Reagent (Roche Diagnostics Corporation, Indianapolis, IN, USA) according to the instructions of the manufacturer. Total protein concentrations in the cell lysates were determined using an Ultrospec 110pro (Amersham Biosciences, Austria). Total protein extracts were fractionated on 4–12% polyacrylamide-SDS gel and blotted onto nitrocellulose membrane (Ø 0.45 μm, Schleicher & Schüell, Germany). Signals were visualized with NBT/BCIP (Nitro blue tetrazolium chloride/5-bromo-4-chloro-3-indolyl phosphate, toluidine salt, Sigma-Aldrich, Saint Quentin, France) as substrates. Actin protein was used as an internal control (according to Rybaczek and Kowalewicz-Kulbat [14]).

Tissue printing

1.5-cm-long apical fragments of roots (n = 15 roots for each series; repeated twice) were dissected longitudinally and transversely (cross-section at the level of the meristematic zone) and blotted onto a nitrocellulose membrane according to the method described by Cassab [31]. The following primary antibodies were used: (1) anti-H2AXS139Ph; (2) anti-PARP-2, as well as secondary antibodies conjugated to alkaline phosphatase. The color reaction was induced (for 10 min) using substrates for alkaline phosphatase (nitroblue tetrazolium; NBT and 5-bromo-4-chloro-3-indolyl phosphate; BCIP) in a buffer containing: 100 mM Tris, pH 9.5; 100 mM NaCl; 5 mM MgCl₂.

Root tissue prints were made using a Stemi 2000C stereoscopic microscope (Zeiss, Jena, Germany) and images were recorded on an AxioCam ERc5s CCD camera (Zeiss, Jena, Germany). Image processing was done in Adobe Photoshop 7.0 (Adobe Systems).

Immunocytochemistry

Immunocytochemical assays were performed according to the method described earlier (Rybaczek and Maszewski [32] and Rybaczek et al. [33]). Excised apical parts of V. faba roots (1.5-mm-long sections; n = 30 for each series) were fixed for 45 min (18°C) in PBS-buffered 3.7% paraformaldehyde, washed several times with PBS and placed in a citric acid buffered digestion solution (pH 5.0; 37°C for 45 min) containing 2.5% pectinase (Fluka), 2.5% cellulase (Onozuka R-10; Serva) and 2.5% pectolyase (ICN). After removing the digestion solution, the root tips were washed 3 times in PBS, rinsed with distilled water and squashed onto Super Frost Plus glass slides (Menzel-Gläser). The air-dried slides were pretreated with PBS-buffered 5% BSA at 20°C for 50 min and incubated overnight in a humidified atmosphere (4°C) with a primary antibody (raised against proteins indicated in the subsection Chemicals and antibodies) dissolved in PBS containing 1% BSA (at a dilution of 1:100). Following incubation, the slides were washed 3 times in PBS and incubated for 1 h (18°C) with Agrisera secondary goat anti-rabbit IgG DyLight^®488 antibody (AS09 633; 1:1000). Nuclear DNA was stained with 4’,6-diamidino-2-phenyl-indole (DAPI, 0.4 μg ml^-1; Sigma-Aldrich). Following washing with PBS, the slides were air dried and embedded in Vectashield mounting media for fluorescence assessment (Vector Laboratories). The labeling index (LI) was calculated as the ratio of immunofluorescence-positive cells to all the cells in a meristematic population. Observations were made using an AxioImagerA1 fluorescence microscope (Zeiss, Jena, Germany) equipped with a blue light filter (excitation 470/40 nm; emission 525/50 nm) for AlexaFluor^®488-conjugated antibodies, a green light filter (excitation 545/25 nm; emission 605/70 nm) for AlexaFluor^®555-conjugated antibodies, and a UV filter (UV light; excitation 365 nm; emission 445/50 nm) for DAPI. All images were recorded at exactly the same time of integration using an AxioCam ERc5s CCD camera (Zeiss, Jena, Germany) and AxioVision 4.8 software (Zeiss, Jena, Germany). Fluorescence signals were computed using ImageJ 1.37c software (Public Domain by Wayne Rasband) according to Abràmoff et al. [24]. Analysis was performed based on 500 cells (100 cells per each preparation) for each experimental series. The experiments were done in triplicate.

Transmission electron microscopy (TEM)

TEM was used to examine the morphology of nuclei from untreated V. faba root meristem cells and the cells treated with HU or the mixture of HU/CF. The apical parts of roots (1.5-mm-long sections, n = 30 for each series) were fixed in 2% glutaraldehyde in 1% cacodylate buffer (pH 7.3) for 3 h at 4°C, post-fixed in 1% osmium tetraoxide in the same buffer for 3 h, and dehydrated in an ascending ethanol series. After infiltration with a medium consisting of Epon 812 and Spurr’s resin, ultrathin sections were double stained with uranyl acetate and lead citrate according to Reynolds [34]. The sections were examined and photographed in a JEM-1010 transmission electron microscope (JEOL, Ltd.). Observations were based on ultramicroscopic photographs taken of at least 50 cell nuclei. The experiments were done in triplicate (i.e. the whole TEM-related procedure was performed three times).

Statistical analysis

Statistical analyses were performed with STATISTICA 8.0 PL software (StatSoft INC, Tulsa, Oklahoma). All of the experiments were done at least in triplicate. All data were expressed as mean ± SD. Differences between groups were assessed by the non-parametric Mann–Whitney U test (for impaired data). Student's t-test was used for data normally distributed. P ≤ 0.05 was considered significant according to Iglesias-Guimarais et al. [35]. Means values of the number of micronucleus (MN) per 1,000 cells were calculated for significance among all experimental series tested and the DMSO control. Comet assay-related data were assessed by ANOVA and Tukey's a posteriori test. All statistical calculations were performed with the sequence of actions typical for statistical analyses: (1) verification of data distribution, (2) verification of homogeneity of variance, (3) evaluation of differences between the examined objects, followed by (4) analysis of differences using t Student, Tukey or Mann-Whitney U tests. The incidence of an association was investigated: (1) between the control and the HU; (2) between the control and the HU→HU/CF (i.e. PCC); as well as (3) between the HU and the HU→HU/CF (i.e. PCC).

Co-treatment with HU/CF triggers either premature chromosome condensation (PCC) or apoptosis like-programmed cell death (AL-PCD) in V. faba root meristem cells

First, V. faba root meristems were used as a model system to study whether CF had a role in the induction of AL-PCD in HU-synchronized and next HU/CF co-treated cells. The final concentrations of the agents (2.5 mM HU and 5 mM CF) were chosen in a series of preliminary tests using different chemicals, various doses, different protocols and various durations of treatment [21,33,36]. Cytological symptoms of PCC comprise aberrant mitoses with (i) irregular condensation of chromatin in prophase, (ii) chromosome fragmentation, (iii) disturbances in the chromosome's metaphase system; (iv) lost and lagged chromatids and chromosomes during anaphase, and (v) segregation defects e.g. chromosomal bridges (S1 Fig). Differentiation between particular phenotypes (S-PCC versus G2-PCC) was based on the fragmentation degree of chromosomes forced to undergo premature mitosis. Numerous fragmentations without chromatid-like pair elements indicated S-PCC phenotype (S1 Fig), while a relatively small number of breakpoints i.e. <20, leading to the loss of relatively large fragments of chromosomes during anaphase were characteristic of the cells showing G2-PCC phenotype (S1 Fig).

In the negative control series (32-h incubation in water) we observed 11.7% ± 1.5 of mitotic cells (all of them with a correct morphology; S1A and S1A'' Fig). In the cells treated with HU for 32 h (S1B and S1B' Fig), the mitotic index decreased to 2.1% ± 0.8, and chromosomes showing some aberrations appeared (6.1% ± 0.4 population of mitotic cells within the HU-treated series; S1B'' Fig). The cells subjected to 24-h blocking in 2.5 mM HU and then transferred into HU/CF mixture showed PCC symptoms in 60.9% ± 2.4 cells derived from the population of dividing cells (estimated as 10.8% ± 1.3; S1C'' Fig). The value 10.8% is the sum of cells showing a set of abnormalities shared by both phenotypes typical of PCC-type aberrant mitoses [i.e. S-PCC (8.0% ± 0.9) and G2-PCC (1.9% ± 0.4)] as well as small fraction of cells showing normal succession of chromosomal events despite of HU/CF co-treatment (i.e. 0.9% ± 0.3). Differences in the percentage of V. faba cells during consecutive stages of mitosis or PCC were significant (p ≤ 0.01). An association was found between the control and HU, as well as between the control and PCC (i.e. HU/CF co-treatment in HU-synchronized cells).

Quantitative analysis revealed that AL-PCD cells (i.e. cells containing a nucleus with extremely strongly condensed chromatin) were only observed in the HU/CF co-treated series (5.3% ± 1.1; S1E Fig) and not detected in either the control (32-h water-incubated; negative control) or the HU-treated cells (32-h; positive control). Additionally, in all the experimental series tested (S1A' Fig, S1B' Fig and S1C' Fig), the MN-test was used to determine the frequencies of micronuclei in interphase V. faba cells. The number of micronuclei per 1,000 cells were 0.6 ± 0.4, 2.1 ± 0.9, 6.9 ± 1.7, for the control, HU and HU/CF series respectively (S1D Fig). All the correlations related to the MN-test were significant. An association was found between the control and HU (p ≤ 0.05), as well as between the control and PCC (i.e. HU/CF co-treatment in HU-synchronized cells, p < 0.001).

The results obtained seemed to support our preliminary idea that in some populations of meristematic cells in which PCC was forced by CF, the induction of AL-PCD resulted from the aberrant course of premature mitoses, while the appearance of MN additionally indicated disturbances in the division of the genetic material into two opposite poles in the cell.

The induction of PCC is crucial for the generation of DNA damage. HU mainly induced double-stranded breaks (DSBs) and HU/CF co-treatment induced single-stranded breaks (SSBs)

Previously we described that prolonged HU treatment led to rapid phosphorylation of histone H2A variant H2AX on S139 resulting in the formation of phospho-H2AXS139 foci along megabase chromatin domains near the sites of DSBs [37]. We also showed that the breakage of restrictive interactions of intra-S-phase checkpoints during PCC induction resulted in the accumulation of SSBs (co-locatization experiments using anti-ssDNA and anti-H2AXS139ph antibodies; [38]). Here, through quantitative immunocytochemical, tissue printing-related and biochemical analyses, we finally proved that both replication stress and PCC induction resulted in DNA damage (Fig 1A and 1B) and HU induced DSBs while HU/CF SSBs (Fig 1 and Fig 2).

10.1371/journal.pone.0142307.g001Fig 1

Histone H2AX phosphorylated at S139 (H2AXS139Ph) is a marker of double-stranded breaks (DSBs), while poly(ADP-ribose) polymerase-2 (PARP-2) is a marker of single-stranded breaks (SSBs).

(A) Results of the immunocytochemical analysis and the method of tissue printing. (a-a', b-c) the presentation of superimposed fluorescence images (DAPI-related in blue and H2AXS139Ph-related in green) after the immunocytochemical detection of H2AXS139Ph in (a) control, (b) after 2.5 mM hydroxyurea-treatment (HU) for 32 h, (c) after 24-h synchronization under the influence of 2.5 mM HU and 8-h co-treatment with 2.5 mM HU and 5 mM caffeine (CF). (a') negative control; incubation exclusively with secondary antibodies. The values of marking indices (expressed in percents) are presented in the top left corner on the following images: (a)–for control series; (b)–after 32-h treatment with HU; (c)–after the induction of premature chromosome condensation (PCC) under the influence of HU/CF. Scale bars in a-a', b-c are 20 μm. (d-f) identification of H2AXS139Ph in the top sections of Vicia faba roots by the method of tissue printing, negative images. In the top left corner of each negative image, there is a miniature of the same fragment of nitrocellulose membrane in color, i.e. stained in the reaction of NBT/BCIP (d'-f'). (d-d') control, (e-e') HU, 32 h, (f-f') HU for 24 h and co-incubation HU/CF for 8 h (total incubation time: 32 h). Scale bars in d'-f' and d-f are 10 mm. (g-g', h-i) presentation of superimposed fluorescence images (DAPI-related in blue and PARP-2-related in green) after the immunocytochemical detection of PARP-2: (g) control, (h) after HU-treatment for 32 h, (i) after 24-h synchronization under the influence HU and 8-h co-treatment with HU/CF. (g') negative control; incubation exclusively with secondary antibodies. The values of marking (expressed in percents) are presented in the top left corner of the following images (g) control series; (h) after 32-h treatment with HU; (i) after the induction of PCC under the influence of HU/CF. Scale bars in g-g', h-i are 20 μm. (j-l) identification of PARP-2 in the top section of V. faba roots by the method of tissue printing, negative images. In the top left corner of each negative image, there is a miniature of the same fragment of nitrocellulose membrane in color, i.e. stained in the reaction of NBT/BCIP (j'-l'). (j-j') control, (k-k') HU, 32 h, (l-l') HU for 24 h and co-incubation HU/CF. Scale bars in j'-l' and j-l are 10 mm. (B) Identification of proteins H2AXS139Ph and PARP-2 by the method of Western blot. (a-a') expression levels of the H2AXS139Ph by Western blot analysis. Data shown are the representatives of three independent experiments. The relative levels of H2AXS139Ph after normalization for actin, as determined by densitometry analysis of the bands, are shown in the histogram (a'; the pixel values [pv; 1–255] categorized according to densitometry analysis of the band intensities and expressed in arbitrary units [a.u.]). Columns, mean from three independent experiments; bars, SD. * p ≤ 0.001 (Control/HU, Mann-Whitney U test); ▲ p ≤ 0.01 (Control/PCC, Mann-Whitney U test). (b-b') expression levels of the PARP-2 by Western blot analysis. Data shown are representative of three independent experiments. The relative levels of PARP-2 after normalization for actin, as determined by densitometry analysis of the bands, are shown in the histogram (b'; the pixel values [pv; 1–255] categorized according to densitometry analysis of the band intensities and expressed in arbitrary units [a.u.]). Columns, mean from three independent experiments; bars, SD. ▲ p ≤ 0.01 (Control/HU, Mann-Whitney U test); * p ≤ 0.001 (Control/PCC, Mann-Whitney U test).

10.1371/journal.pone.0142307.g002Fig 2

Single strand breaks (SSBs) and double strand breaks (DSBs) were assessed with alkaline (pH ≥ 13) and neutral (pH = 10) variants of comet assay, respectively.

SSBs were generated during replication stress (i.e. after 2.5 mM hydroxyurea [HU] treatment) and DSBs were connected with the induction of premature chromosome condensation (PCC) due to co-treatment with HU and caffeine (CF). (A-F) fluorescence images of nuclei from individual protoplast of Vicia faba, stained with YOYO-1 after electrophoresis (comet assay); h, comet head; t, comet tail. (A-C) alkaline variant of comet assay dedicated to detection of SSBs; (D-F) neutral variant of comet assay assigned to DSBs. Scale bar in 'A' = 5 μm is applied to all figures presented. (A'-F') the intensity of DNA fluorescence after staining with YOYO-1 of the nuclei shown in (A-F). (A, D) nuclei of a protoplasts isolated from the untreated (control) V. faba roots. (B, E) nuclei of protoplasts isolated from the roots of V. faba treated with HU for 32 h. (C, F) nuclei of protoplasts isolated from the roots of V. faba treated with HU for 24 h and next co-treated with HU/CF for 8 h.

Immunocytochemical identification of the phosphorylated form of H2AX histone was done with rabbit polyclonal antibodies (α-H2AXS139ph) directed against a synthetic peptide (CKATQA[pS]QEY) corresponding with a fragment of human H2AX histone (amino acids 134–142). According to our previous results [32–33] a small number of untreated cells emitted weak phospho-H2AXS139 fluorescence (2.1% ± 0.5) from areas distributed randomly within the cell nucleus (Fig 1Aa). H2AXS139 labeling index was significantly elevated in the HU-treated cells (61.9 ± 2.4; Fig 1Ab). In this series, apart from foci dispersed over the whole nucleus area, a new category of foci appeared, localized at the border of the nucleolus and nucleoplasm, probably connected with labeling of the perinucleolar heterochromatin fraction (Fig 1Ab). The addition of caffeine (HU→HU/CF), an inhibitor of superior kinases (of ATR/ATM type) that phosphorylates H2AX histones at serine 139, resulted in more than a 5-fold decrease in the quantity of specifically labeled nuclei, and additionally led to a decrease in the number of foci per nucleus from 16.9 ± 1.5 after HU to 7.5 ± 0.5 after HU+CF (Fig 1Ab and 1Ac). In the control series, the average number of foci per nucleus was 4 ± 1.0, while in the negative control (not incubated with primary antibodies) no cells were labeled (Fig 1Aa and 1Aa'). Immunocytochemical observations were confirmed by biochemical analysis (Fig 1Ad, Fig 1Ad', Fig 1Ae, Fig 1Ae', Fig 1Af, Fig 1Af', Fig 1Ba and 1Ba'). The tissue printing technique revealed strong phosphorylation of S139 in meristematic zones and a slightly weaker signal in supra-meristematic zones of roots treated with HU (Fig 1Ae and 1Ae'). In the roots co-treated with HU/CF, the positive reaction was limited to the meristem zone, while in the higher zones of the roots, strong signals were observed in the form of streaks, probably corresponding to the order of cells in the boundary zone between the primary cortex and the central cylinder (Fig 1Af and 1Af'). The results of the SDS-NuPAGE/Western blot analysis of the total extract from V. faba root tip cells revealed one band close to 16 kDa (Fig 1Ba), as well as a strong increase (over 10-fold) in the amount of H2AXS139ph after HU-treatment, and an increase (over 4-fold) in the quantity of H2AXS139ph after co-treatment with HU/CF, in comparison with the control band on the same blot (Fig 1Ba and 1Ba'). The indicators point to the statistical significance of the results obtained (Mann-Whitney U test, p ≤ 0.001: * Control/HU; Mann-Whitney U test, p ≤ 0.01: ▲ Control/PCC i.e. HU/CF co-treatment in HU-synchronized cells).

Our previous results showed that labeling cell nuclei using antibodies recognizing PARP2 gene product, i.e. poly(ADP-ribose) polymerase 2 (PARP-2), was an equally sensitive test detecting SSBs within DNA molecules [38]. Immunocytochemical analysis showed a low constitutive level of PARP-2 protein in the control cells (1.5% ± 0.3), an over 14-fold increase in PARP-2 protein after treatment with HU (21.3% ± 1.9) and specific labeling of almost half of the cells forming the root meristem (46.2% ± 2.8) in the series in which PCC was induced with CF (Fig 1Ag, Fig 1Ag', Fig 1Ah and Fig 1Ai). A 24-h incubation in 2.5 mM HU contributed to the formation of numerous fine specific PARP-2 foci, localized first of all in the perinucleolar region, as well as on the area of the entire nucleus and–in a characteristic way–on the periphery of cell nuclei in the region connected with the nuclear envelope (Fig 1Ah). The incubation in HU/CF, apart from an increase in the number of labeled cells, resulted in a considerable increase in the size of PARP-2 positive foci, as well as in disappearance of labeling in the perinucleolar heterochromatin and strong labeling of the boundary area of the nucleoplasm (i.e. those areas of the nucleus that adhered to the nuclear envelope; Fig 1A). In turn, the results obtained by the tissue printing method were not unequivocal, since strong labeling was observed in the case of root imprints derived from all experimental series (including the control); at the same time, a slightly more intensive labeling exactly of the meristematic zone was observed after the treatment with HU and after the PCC induction (Fig 1Aj, Fig 1Aj', Fig 1Ak, Fig 1Ak', Fig 1Al and Fig 1Al'). The results of the Western blot analysis of the total extract from V. faba root tip cells revealed only one band close to 66 kDa (Fig 1Bb) as well as showed a 5-fold increase in the amount of PARP-2 after HU-treatment and an over 11-fold increase in the quantity of PARP-2 after the HU/CF co-treatment, compared with the control band on the same blot (Fig 1Bb and 1Bb'). The indicators point to the following statistical significance: Mann-Whitney U test, p ≤ 0.01: ▲ Control/HU; Mann-Whitney U test, p ≤ 0.001: * Control/PCC i.e. HU/CF co-treatment in HU-synchronized cells. The performed analyses clearly show that both the formation of H2AXS139ph and PARP-2 foci are sensitive tests revealing the presence of structural damage to the genome (Fig 1A and 1B).

The type of DNA fragmentation can be also distinguished in comet assay [28,39]. Regardless of the method (alkaline or neutral variant), intact DNA remains in the head of the comet and DNA from regions with strand breaks appears in the tail. The amount of DNA in the tail is proportional to the number of DNA breaks. Fig 2A, Fig 2A', Fig 2B, Fig 2B', Fig 2C and 2C' present the alkaline variant of comet assay whereas Fig 2D, Fig 2D', Fig 2E, Fig 2E', Fig 2F and 2F' the neutral one. In the V. faba cells exposed for 24 h to HU, the percentage of DNA in the comet tails showed a higher level of DNA migration in both alkaline and neutral pH conditions. In the neutral variant, changes observed were as follow: 62% ± 1.1 of DNA in the comet tail for HU-treated cells, (Fig 2E and 2E'), control (2.9% ± 0.6; Fig 2D and 2D'); whereas in the alkaline variant: 34% ± 0.8 for HU-treated cells, Fig 2B and 2B', and for control 0.9% ± 0.3, Figs 2A and 1A'. During the PCC induction the results we observed were reversed, i.e. a higher level of DNA in the tail of comets in the alkaline version of the comet assay, compared to the natural version: 41% ± 2.3 and 20.6% ± 0.9, respectively. The results presented in Fig 2B', Fig 2C' and Fig 2E' are statistically significant (p < 0.001 compared to the control [presented in Fig 2A' and Fig 2D', respectively]; ANOVA and Tukey's a posteriori test).

AL-PCD is a secondary result of CF-induced PCC in HU-synchronized V. faba roots

DNA cleavage is a PCD marker. Nucleases cleave DNA between nucleosomes which results in 180 bp fragments [3]. The effect of this process can be visualized on electrophoresis gel [3,8,28] indicating apoptotic cells (ladder pattern) and showing immediate DNA degradation during necrosis (smear), while DNA in living cells is not fragmented [3,28].

To determine whether the PCC induction is connected with PCD-type DNA degradation, we separated the isolated samples of DNA on agarose gel and visualized under UV light. Electrophoresis showed a large scale DNA fragmentation only in the HU/CF co-treated series (S2 Fig, lane 3, arrows). Laddering was not detected in the HU-incubated series nor in the control (S2 Fig, lane 2 and lane 1, respectively). Typical internucleosomal DNA fragmentation was undetectable.

Terminal deoxynucloetidyl transferase-mediated dUTP nick and labeling (TUNEL) assay, which positively stains apoptotic nuclei, can be also used to determinate the type of DNA fragmentation. TUNEL assay allows determination of the presence of free 3’-OH ends in chromatin [40]. The TUNEL assay was used here to detect 3'-OH termini in nuclear DNA (Fig 3). For DNase I-treated series (positive control), all cells were TUNEL-positive (Fig 3A, Fig 3A' and Fig 3A''). In the control series (32-h incubation in water) no TUNEL reaction was evident as no AlexaFluor 594-related red fluorescence was observed (Fig 3, the top panel). In the HU-treated series, we observed a TUNEL-positive sign in a small part of meristematic cells only (< 11% ± 1.3; Fig 3). In contrast, the cell population treated with the mixture of HU/CF exhibited up to 48% ± 2.4 of TUNEL-positive nuclei (Fig 3B, Fig 3B' and Fig 3B'', the bottom panel, TUNEL-positive indicated by arrows). Statistical results analyzed by Student t-test indicate that differences in the percentage of TUNEL-positive cells between the control and HU-treated cells, as well as between the control and PCC-induced cells (i.e. HU/CF co-treated) are significant, p ≤ 0.01.

10.1371/journal.pone.0142307.g003Fig 3

Terminal deoxynucleotidyl-dUTP nick end labeling (TUNEL) assay in Click-iT technology in the untreated control (negative), DNase-treated control (positive), HU-treated and HU/CF-co-treated (i.e. PCC-induced) Vicia faba root meristem cells.

Left panel—DNA fragmentation in V. faba cells detected by TUNEL reaction and visualized by AlexaFluor 594. Central panel—DAPI stained nuclei. Right panel—Merged images (AlexaFluor 594 + DAPI). Positively stained nuclei appear in the DNase-treated cells (e.g. A-A'') and in the HU/CF co-treated cells (e.g. B-B''). Positively stained nuclei in the HU-treated series are indicated by arrowheads. Non-reacting nuclei can be seen in the negative control sections (as indicated in the highest panel described as 'CTRL'). Scale bar = 20 μm.

Double staining with acridine orange (AO) and ethidium bromide (EB) is another method used to specify the type of cell death, as well as being a useful tool in detecting and quantifying the states living, dying and dead (Fig 4, Fig 5 and S3 Fig). Fluorescence microscopy revealed images indicating the occurrence of living cells (fluoresce green), dying cells (green-yellow, yellow, yellow-orange and bright orange) and dead cells (dark orange and bright red) in the control (Fig 4A), after both HU treatment (Fig 4B) and after PCC induction (Fig 4C). The diagram presenting the color spectrum resulting from the quantitative measurements of fluorescence intensity of nuclear chromatin stained with AO/EB was made in order to determine the degree of DNA damage in the control nuclei (Fig 4A') as well as in the nuclei derived from stressed roots of V. faba (Fig 4B and 4C'). The highest number of dead cells (13.4%) was observed in HU/CF treated material (Fig 4C''). Thus, it was shown that the number of dead cells after PCC induction was over 6-fold higher compared to the control and 4.5-fold higher, compared to the HU series (Fig 4A'', 4B'' and 4C''). The fractions of living, dying and dead cells in the control, compared with the HU series were similar; we only observed an increase in the number of dead cells in the latter (1.5-fold; Fig 4A'' and 4B''). Further analysis consisted in comparing the numbers of living cells or being at various cell death stages (early-to-late) in particular zones of V. faba roots [from 'quiescent center' through the zones of cell division, i.e. root meristem (zone marked as II in Fig 5A) up to the zone of elongation (marked as III in Fig 5A)]. Our results suggest that the increase in the number of dead cells in the meristematic zone of V. faba roots after HU/CF treatment was statistically significant (p < 0.01) in relation to both the control and HU-treated series (Fig 5C). In the meristematic zone (II) the relatively high percentage (Fig 5C) populations of dying (Fig 5C, yellow-colored columns) and dead cells (i.e. 19.9%, 13.4%, and 28.8% in the control, HU-treated, and PCC-induced series, respectively) resulted from some imperfection in the method of intravital AO/EB staining, and from the fact it is not possible to omit from the calculation the population of rhizodermis cells in which the occurrence of the PCD phenomenon is typical (comp. Fig 5Aa, Fig 5Ab, Fig 5Ac, Fig 5Ad and Fig 5C; this could be the reason that no significant differences were observed herein). In the other zones of V. faba roots induced to PCC, the number of dead cells was about 1.5-fold higher than in the control and almost 2-fold higher than in the HU-treated material (Fig 5A and 5B). In turn, in the elongation zone (III, over-meristematic, Fig 5A), the highest index of dead cells was observed under the influence of HU/CF (7.7%, i.e. 1.5-fold and 3.4-fold higher than in the control and HU-series, respectively; Fig 5D). However, it seems more interesting to compare the numbers of dead cells in the meristematic zone than in the elongation zone; in the latter it was lower, where in the control, the HU and the HU/CF series, decreases of 4, 6 and 3.7 times were observed, respectively (comp. Fig 5C and 5D). S3 Fig shows longitudinal intersections of the three most representative roots stained with the intravital AO/EB method, as well as shows the proportions of living (green), dying (range: yellow-to-orange) and dead (red) cells, and their distribution in the meristem, supra-meristematic zone and in the rhizodermis. In all zones (particularly in the area of a root cap) and in all experimental series we observed red fluorescence indicating PCD processes eliminating cells, particularly from the rhizodermis, present in the external layers of the root (S3 Fig). The arrows on S3b, S3b' and S3b'' Figs show a distinct widening of the HU-treated roots forming an easily visible protuberance in which the accumulation of dead cells can be observed. These protruberances may result from the appearance of aerenchymatic-like spaces in the root cortex cells of V. faba (comp. [8]).

10.1371/journal.pone.0142307.g004Fig 4

Double in vivo staining with acridine orange (AO) and ethidium bromide (EB) as a useful tool for detecting and quantifying the state of dead, dying and living cells in root meristems of Vicia faba.

(A-A'') control. (B-B'') hydroxyurea-induced replication stress. (C-C'') caffeine-induced premature chromosome condensation (PCC). (A,B,C) fluorescence micrographs of nuclei in living (green), dying (range: yellow-to-orange), and dead (red) cells. (A',B',C') diagrams presenting the color spectrum resulting from measurements of the fluorescence intensity of nuclear chromatin stained with AO/EB, in order to determine the degree of damage in the nuclei of stressed roots of V. faba. (A'',B'',C'') circle diagrams presenting the percentage of living (green), dying (range: yellow-to-orange) and dead cells (red). The data shown in the pie charts in A'',B'',C'' indicate that the correlations were significant with reference to the number of dead cells for all experimental series reported herein: an association was found between the control and HU (p ≤ 0.05, Mann-Whitney U test), between the control and PCC (p ≤ 0.01, Mann-Whitney U test), and between the HU-treated and PCC-induced cells (i.e. HU/CF co-treated; p ≤ 0.01, Mann-Whitney U test). Scale bar in (A) = 20 μm is also applied to the figures presented in the pictures (B) and (C).

10.1371/journal.pone.0142307.g005Fig 5

Acridine orange (AO) and ethidium bromide (EB) staining of living, dying and dead cells in Vicia faba root meristem cells in relation to (A) the localization in particular zones, and (B-D) the surface area occupied by the cells in particular zones.

(A) Fluorescence picture of AO/EB stained V. faba roots in planta. (a) schematic figure presenting the outline of the roots of the control series, with marked (I) root cap, (II) zone of cell division i.e. root meristem, (III) zone of elongation, and the quiescent center. (b) the control roots, (c) the roots treated with 2.5 mM hydroxyurea (HU) for 32 h, (d) the roots treated with 2.5 mM HU for 24 h and then co-treated with 2.5 mM HU and 5 mM caffeine (CF). Scale bar = 1 mm. The schematic outline of the root from scheme (a) was placed over a root from the control series (b) on which the root outline from figure 'a' and figure 'b' are precisely overlapped, (c) a root from the series, in which seedlings were subjected to replication stress and (c) a root that was induced to premature chromosome condensation (PCC). (c-d) The continuous line marks those root fragments that in terms of size and shape were the same as the analogous areas in the roots of the control series (a-b versus c-d), while the broken line (in figures [c] and [d]) marks the root areas that indicated the appearance of aerenchymatic-like spaces that had formed in the roots that had been subjected to treatment with HU (c) or co-treated with the mixture of HU/CF (d). In places indicated by broken lines, roots of the series (c) and (d) were distinctly wider than the control (b). (B-D) quantitative presentation of surface area (%) occupied by the green, yellow-orange and red colors (that correspond to the populations of living, dying and dead cells, respectively) in the particular zones of V. faba roots. (B) quiescent center, (C) zone of cell division, i.e. root meristem, marked also as zone II, and (D) zone of elongation, marked as zone III.

Vacuolar/autolytic (V/A) AL-PCD, following CF-induced PCC in HU-synchronized V. faba roots

The aberrant course of prematurely induced mitotic division as a rule leads to cell apoptosis, PCD (or AL-PCD in plant cells). In order to establish a possible cause-and-effect relationship between the induction of PCC and of AL-PCD, and to determine the type of AL-PCD, precise ultrastructural investigations were performed of the meristematic zone in V. faba roots. The results presented in Figs 6 and 7 and S4–S7 Figs show that PCC induction increased the number of root cells with AL-PCD symptoms, and that the electron microscope images distinctly indicate the occurrence of a vacuolar/autolytic type of AL-PCD [(V/A) AL-PCD].

10.1371/journal.pone.0142307.g006Fig 6

Electron micrographs of Vicia faba root meristem cells.

(A) control (32-h incubation in water); (B) hydroxyurea-treated (32-h); (C-C', D-D', E-F) hydroxyurea (HU) synchronized for 24 h and then HU/CF co-treated (for a successive 8 h; total incubation time: 32 h). The arrows in picture (C') point out vesicles of the Golgi apparatus. The arrows in picture (D') indicate lytic vacuoles localized in the vicinity of plasmodesmata. The square in the bottom right corner of picture (E) contains an enlarged image of the cell from picture (F). Asterisk (*), the visible light in the vacuoles presented in pictures (C and D) indicates the places of accumulation of deposits in the vacuoles, showing that these vacuoles function as lytic vacuoles. All the photos presented in figures (C-F) are derived from the series in which PCC was induced. However figure (C) shows the morphology of the root cuticle cells, from which the plastids seen in the picture (precisely amyloplasts, marked as 'p') are filled with statolith starch grains (marked as 's'). Successive figure (D) presents the appearance of a typical V. faba meristematic cell, whose morphology (apart from the deposits seen in the lytic vacuoles and indicated by the asterisk) does not significantly differ from the morphology of the control cells (comp. A and D). Two further pictures (E and F) illustrate the morphology of meristematic cells that entered the path of apoptosis-like programmed cell death (AL-PCD), while picture (E) shows premature vacuolization stadium, and picture (F) demonstrates: (1) extensive vacuolization within the whole meristematic cell space, (2) the presence of swollen ER compartments (indicated by arrows), and (3) the existence of autophagosome-like structures, created from ER (the structures inside the squares). a-l autophasome-like structure, c cytoplasm, cw cell wall, dch dense chromatin, ER endoplasmic reticulum, G Golgi structure, lv lytic vacuole, m mitochondrion, n nucleus, ne nuclear envelope, no nucleolus, nov nucleolus vacuole, p plastid, pd plasmodesmata, s starch, v vacuole. Scale bar = 5 μm.

10.1371/journal.pone.0142307.g007Fig 7

Spreading of (presumably) lytic enzymes from the interior of almost totally degenerated dead cells to adjacent cells through plasmodesmata.

(A-B) The direction of enzyme propagation is indicated with arrows. Double arrow-heads indicate cytoplasm residues pushed towards plasmalemma with suspended organelles already partially digested. The asterisk in picture (A) indicates electron lucent areas, newly formed inside a regular meristem cell (i.e. a cell that does not show changes in other regions, other than those marked with an asterisk where the wave of lytic enzymes arrived). Picture (B) Gradual, sequential propagation of a cleared up region appearing, presumably, due to the action of lytic enzymes in a cell neighboring a dead cell due to AL-PCD, towards further areas of the adjacent cell. c cytoplasm, cw cell wall, dch dense chromatin, lv lytic vacuole, m mitochondrion, mls multilamellar structure; n nucleus, p plastid, pd plasmodesmata, s starch; p(s) swollen plastids; m(s) swollen mitochondium. Scale bar = 5 μm.

The AL-PCD process is connected with several metabolic-biochemical changes in the cell. They can concern either the nuclear compartment or the extranuclear regions (in the latter case these can be connected with changes observed in the cytoplasm or organelles present in it). Early AL-PCD events were found to be connected with changes occurring in the cytoplasm [e.g. progressing vacuolization and formation of greater and greater lytic vacuoles (see Fig 6B, Fig 6C, Fig 6D, Fig 6E and 6F in comparison with Fig 6A)]. It has also been shown that the beginning of changes concerning the nuclear compartment took place only when the changes observed in the cytoplasm were considerably advanced (comp. Fig 6F with Fig 6D). Unexpectedly, no changes were observed in the structure of mitochondria (S4C Fig).

One of the first symptoms of AL-PCD concerning the nucleus was a considerable increase in the condensation degree of chromatin fibrils (the first stadium of this process is shown in Fig 6F, while an advanced stadium is presented in S5A and S5C Fig). The occurrence of the V/A-type of AL-PCD induced during the co-treatment with HU/CF was indicated on the basis of the following symptoms: (i) extensive vacuolization within the whole cell space (early stages of vacuolization are presented in Fig 6E, and late stages in Fig 6F), (ii) the presence of deposits within the lytic vacuoles (Fig 6C and 6D), and (iii) the existence of autophagosome-like structures created from, among other things, the swollen ER (Fig 6F). AL-PCD symptoms were not observed in either the control series or the HU series (S4A and S4B Fig). The majority of cells induced to enter PCC also showed no AL-PCD symptoms (apart from insignificant changes in their morphology and e.g. the formation of vacuoles with a lytic character; S4C Fig). However 5.3% ± 1.1 of cells subjected to PCC entered the (V/A) AL-PCD pathway. The qualification of particular cells to those in which (V/A) AL-PCD symptoms were detected took place when changes indicating the occurrence of AL-PCD concerned the nuclear compartment (S5A, S5B and S5C Fig). We used this classification in this paper, which is consistent with Dominguez and Cejudo [41], who considered the degradation of cellular nucleus to be the symptom of the final and irreversible stage of PCD (although the final degradation of nucleus was caused by metabolic changes, for example those occurring in the cytoplasm in cells undergoing PCD).

The electron microscopy observations of cells induced to PCC and then entering the AL-PCD pathway showed that the most visible changes took place in the nucleus. In the V. faba nuclei the increasing transparency of decondensed nucleoplasm was the basic morphological indicator of the successive stages of AL-PCD. In addition, it served as a convenient background against which it was easy to distinguish the extremely condensed fibers of condensed chromatin. These strongly condensed areas of chromatin were often adjacent to the nuclear envelope (S5A and S5C Fig). The other characteristic features indicating the occurrence of AL-PCD include, among others: (1) shrinkage of the protoplast (S5B Fig); (2) formation of sections of a multi-layer nuclear envelope (S5A and S5C Fig); (3) formation of multi-membrane structures either in the region of plasmalemma or nuclear envelope (S5A, S5B, S6B and S5C Figs); (4) degradation of organelles inside lytic vacuoles (S5A, S6B, S6C, S7A and S7B Figs); and (5) formation of autophagosome-like structures (S5C Fig). Additionally, the triggering of (V/A) AL-PCD was accompanied by the appearance of specific structures inside the cell: (1) either showing indistinct/unclear morphology (cloudy morphology; S6A, S6B and S6C Fig); or (2) having a clear myelin character (S6B Fig). It was also shown in this work that the 'signal transmission' (from one cell to another cell) proceeded, among other things, through plasmodesmata (Fig 7, comp. Fig 6D and 6D'), i.e. cytoplasmic channels selectively displacing metabolites and signal molecules present inside lytic vacuoles (Fig 6D and 6D'). The cytoplasm of the cells showing symptoms of (V/A) AL-PCD was relatively bright, as caused by the reduction in the number of ribosomes (S6B, S7A and S7B Figs). Plastids, mitochondria and other organelles were gradually pushed towards the cell walls (S5B, S7A and S7B Figs). Compact Golgi structures accompanied by quite large vesicles filled with an electron-transparent material (Fig 6C') were easily distinguishable (Fig 6B and 6E).

Finally, fragmentation of the nuclei and their progressing marginalization were among the final stages of (V/A) AL-PCD proceeding in the meristematic cells of V. faba root (however, this stage was observed only when almost all the organelles in a given cell were subjected to degradation by -presumably—lytic enzymes). The description of the final stage of cell degradation should be as follows: when the cell interior is almost totally filled with a huge lytic vacuole and most organelles have been degraded (and those that have not been completely digested are pushed towards border cell areas, towards plasmalemma), organelles show strong changes in their morphology; changes that resemble swelling from the long-lasting influence of (presumably) lytic enzymes on the intercellular structures and preceding the moment of their final digestion (Fig 7A and 7B). Fig 7 also showed that a cell that had died as a result of (V/A) AL-PCD was still able to transmit a stream of lytic enzymes derived from its own lytic vacuole through the system of plasmodesmata into an adjacent cell (even when the morphology of the adjacent cell was normal).

The results of the investigation performed (summarized in Fig 8) allow us to put forward the thesis that the induction of (V/A) AL-PCD in the V. faba cells may, and even should, be perceived as a consequence of previously initiated PCC process and the DNA damage occurring during its course.

10.1371/journal.pone.0142307.g008Fig 8

Scheme depicting experimental procedures used to reveal and to support the fact that the activation of vacuolar/autolytic type of plant-specific, apoptosis-like programmed cell death [(V/A) AL-PCD] is a secondary result of the caffeine-induced premature chromosome condensation (PCC) in hydroxyurea (HU) synchronized Vicia faba root meristem cells.

The scheme shows that PCC leads to the formation of cells with different phenotypes (phenotype A–typical of cells not diverging from the morphology of regular cells; phenotype B–characteristic of cells with the morphology typical of PCC induction from G2 phase; and phenotype C–cells with strongly fragmented chromosomes, induced to PCC, probably in early or middle sub-periods of S phase). The scheme shows that the induction of PCC is associated with the generation of DNA damage (mainly single-strain breaks [SSB] but also to a lesser extent, double-strain breaks [DSBs]). The occurrence of various types of DNA damage during PCC induction has been proven following the results of many experimental procedures (immunocytochemistry, with the use of antibodies against specific marker proteins, tissue printing methods, Western blot and comet tests–both alkaline and neutral). In turn, double staining with acridine orange and ethidium bromide allowed us to distinguish in planta, the sub-fractions of living, dying and dead cells. Preliminary histochemical analyses (classic staining with Schiff’s reagent in Feulgen’s method) showed the presence of such changes in the structure of cellular nuclei that could indicate the occurrence of apoptosis-like programmed cell death (AL-PCD). On the other hand, more detailed analyses, i.e. DNA electrophoresis and TUNEL reaction, confirmed by ultrastructural tests (performed at a level of transmission electron microscopy) proved the existence of vacuolar/autolytic type of AL-PCD (V/A-type AL-PCD) in stressed V. faba roots.