Caryologia. International Journal of Cytology, Cytosystematics and Cytogenetics 73(4): 55-63, 2020 Firenze University Press www.fupress.com/caryologia ISSN 0008-7114 (print) | ISSN 2165-5391 (online) | DOI: 10.13128/caryologia-788 Caryologia International Journal of Cytology, Cytosystematics and Cytogenetics Citation: V. Neves, W. Viegas, A. D. Caperta (2020) Effects of high temperature on mitotic index, microtubule and chro- matin organization in rye (Secale cere- ale L.) root-tip cells. Caryologia 73(4): 55-63. doi: 10.13128/caryologia-788 Received: December 20, 2019 Accepted: July 27, 2020 Published: May 19, 2021 Copyright: © 2020 V. Neves, W. Viegas, A. D. Caperta. This is an open access, peer-reviewed article published by Firenze University Press (http://www. fupress.com/caryologia) and distributed under the terms of the Creative Com- mons Attribution License, which per- mits unrestricted use, distribution, and reproduction in any medium, 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. Funding Statement: This work was funded by Portuguese national funds through Fundação para a Ciência e a Tecnologia (www.fct.pt/) pro- ject PTDC/AGRPRO/4285/2014, UI/ AGR/04129/2013, and grant LEAF- AGR/04129/BPD/2015 to ADC. Effects of high temperature on mitotic index, microtubule and chromatin organization in rye (Secale cereale L.) root-tip cells Vânia Neves, Wanda Viegas, Ana D. Caperta* Linking Landscape, Environment, Agriculture and Food (LEAF), Instituto Superior de Agronomia (ISA), Universidade de Lisboa, Tapada da Ajuda, 1349-017 Lisboa, Portugal *Corresponding author. E-mail: anadelaunay@isa.ulisboa.pt Abstract. Stressful high temperatures on plants can limit whole-plant function and decrease crop productivity. However, little is known regarding heat stress effects on microtubule cytoskeleton and chromatin in roots from intact plants. Here we studied high temperature effects on cell division, microtubule and chromatin organization pat- terns in rye root tips from intact plants subjected to 40ºC for 4 h and after different recovery periods (0RT, 7RT, 24 RT). We showed that heat stress induced changes in nuclear morphology as detected by the unusual presence of interphase cells with irreg- ularly shaped nuclei, probably associated with changes in chromosome segregation at anaphase, leading to micronuclei formation as well as changes in the mitotic index. These alterations were associated to differential effects in microtubules organization in both heat-stressed interphase and mitotic cells at 0RT and 7RT. Although no changes in the distribution of H3 phosphorylation of Ser 10 residues on chromatin were found in cells from heat-stressed plants, marked alterations in chromatin DNA methylation patterns were detected. These effects included higher agglutination of 5-methylcytosine domains in both interphase and metaphase cells compared to controls. Taken together these results seem to suggest that alterations in microtubule conformation upon heat stress influences nuclear chromatin organization and cell cycle progression. However, when seedlings recovered from stress (24RT), root tip cells presented microtubule con- figurations and chromatin organization patterns similar to controls. We conclude that in spite of heat stress markedly altered cell cycle progression and distribution of epi- genetic marks, these responses are transient to cope with such stress conditions in the roots. Keywords: DNA methylation, heat stress, histone H3 Ser 10 phosphorylation, micro- tubules, root. INTRODUCTION High temperature is one of major environmental factors limiting crop growth and yield worldwide causing many physiological changes that affect crop yield and quality (Suzuki et al. 2014). Most studies on these effects focus on the above-ground tissues such as shoot and reproductive organs, 56 Vânia Neves, Wanda Viegas, Ana D. Caperta although roots can also be subjected to high tempera- ture stress which can limit whole plant function and decrease productivity (Heckathorn et al. 2013). A high degree of complexity in plant responses at the molecu- lar, physiological and biochemical levels were described, largely controlled by different, and sometimes opposing, signaling pathways that may interact and inhibit each other (Suzuki et al. 2014). However, knowledge concern- ing combined microtubule (MT) cytoskeleton organiza- tion and chromatin nuclear topology upon heat stress in intact plants is scarce, particularly in roots. In the plant cell cycle, the MT cytoskeleton com- posed of heteropolymers of α- and β-tubulin undergo dynamic conformational changes in a process known as dynamic instability in response to the needs of the cell (Horio and Murata 2014). Particularly, during cell divi- sion in somatic cells, MTs are arranged into character- istic structures like the interphase cortical MTs (CMT), pre-prophase band, mitotic spindle and phragmoplast (Baluska et al. 1998). Moreover, MTs can undergo a number of posttranslational modifications that act to control specific MTs-based functions in plants, including tyrosination, detyrosination, acetylation (Smertenko et al. 1997a), phosphorylation (Blume et al. 2008), polyglu- tamylation (Wang et al. 2004), and transamidation (Del Duca et al. 2009). In plant cells disruption of MTs occurs in response to various environmental factors namely to extreme temperatures such as heat stress (Nicotiana tabacum, Smertenko et al. 1997a; Smertenko et al. 1997b; Arabi- dopsis thaliana, Müller et al. 2007), low temperature and abscisic acid treatments (Triticum aestivum, Khokhlova et al. 2003), hyperosmostic stress (Triticum turgidum, Komis et al. 2002), and affects post-translational modi- fications of tubulin as in cadmium stress (Glycine max, Gzyl et al. 2015). Furthermore, plant MTs in addition to their role in cell division and axial cell expansion, also have a thermosensory function that is of agronomical relevance in osmotic or cold stress conditions (Triticum aestivum, Abdrakhamanova et al. 2003; Nick 2012). Moreover, plants response to drought, cold and high salinity stress involve several epigenetic regula- tory mechanisms like both DNA and histone meth- ylation and generation of small RNAs implicated in genome regulation and structure (Mirouze and Pasz- kowski 2011; Asensi-Fabado et al. 2017). Recent research has shown that environmental cues and abiotic stresses activate a stress memory that is mediated by epigenetic and chromatin-based mechanisms including chroma- tin modifications, such as cytosine methylation of DNA, histone methylation and nucleosome occupancy (Lämke and Bäurle 2017). For example, exposure of Arabidop- sis plants to stresses, including salt, UVC, cold, heat and flood, resulted in a higher homologous recombina- tion frequency, increased global genome methylation, and higher tolerance to stress in the untreated progeny. However, this transgenerational effect did not persist in successive generations (Boyko et al. 2010)..By contrast, prolonged heat stress induces transcriptional activation of several repetitive elements of Arabidopsis thaliana that requires minor changes in histone modifications but does not involve DNA demethylation (Pecinka et al. 2010). Patterns of DNA and histone modification can be altered in root tip cells of soybean seedlings grown at different temperatures (Stępiński 2012). During male meiosis in Secale cereale plants with B and without B chromosomes, heat exposure causes differential anoma- lies in chromatin structure in pachytene cells (Pereira et al. 2017). Heat-damaged pachytene cells displayed eas- ily recognizable paired chromosome fibres and a single amorphous and heterochromatic mass closely associ- ated with the nuclear periphery as well as disruption of the organization of sub-telomeric chromosome regions. However, no changes in DNA methylation patterns were detected in untreated and treated plants (Pereira et al. 2017). In this work we investigated the effects of heat stress (40ºC 4 h) on the organization of MT arrays and chro- matin in rye root tips from intact seedlings, immediately after stress (0RT) and at different recovery periods (7RT, 24RT). In-depth cytological analyses of chromatin and microtubular organization were performed in root tip cells with DAPI, and immuno-labelling with antibodies against tubulin, 5-methylcytosine (5-mC) and histone H3 phosphorylated at serine 10 residue (H3S10ph). MATERIALS AND METHODS Plant material and heat stress conditions Rye (Secale cereale L., 2n = 14 chromosomes) seeds were kindly supplied by Neil Jones (Aberystwyth, UK). Seeds were placed in Petri dishes with moistened fil- ter paper in the dark at 4°C for 3 days. Seedlings were transferred to a growth chamber with controlled light- temperature (Rumed), with a photoperiod of 18 h light and 6 h dark, at 25°C ± 2ºC for 2 days. Then, seedlings were subdivided in two sets of experiments: (i) kept at 25°C ± 2ºC with a photoperiod of 18 h light and 6 h dark (controls); and (ii) exposed to a ramp of increasing temperature from 25ºC to 40ºC (temperature increases 2°C / h), remaining 4 h at 40°C, after which the tem- perature fell gradually to 25°C (temperature decreases 2°C / h). The heat stress temperature was chosen based 57Effects of high temperature on mitotic index, microtubule and chromatin organization in rye root-tip cells on agronomical relevant temperatures shown to have a significant effect in cool season grasses such as rye (Xu, Zhan et al. 2011). All seedlings were kept moist dur- ing heat stress. Root tips were collected from plants in control conditions and from treated plants immediately after exposure to heat stress 40°C (0RT), and 7 h (7RT) and 24 h (24RT) during stress recovery. Immunolabelling For immunostaining of α-tubulin root tips were prepared as described in Caperta et al. (2006). Briefly, roots were fixed in freshly prepared 4% paraformalde- hyde solution (PFA) containing MT stabilizing buffer (1xMTSB) for 45 min at room temperature, and then rinsed twice in 1xMTSB for 5 min. For H3S10ph immu- nodetection, root tips were fixed in freshly prepared 4% PFA solution containing phosphate-buffered saline (1xPBS, pH 7.3) for 30 min, and then washed three times for 5 min in 1x PBS according to Caperta et al. (2008). Immunostaining of 5-mC was performed in root tips fixed in ethanol:acetic acid (3:1) as described in Carvalho et al. (2010). For both H3S10ph and 5-mC immunolabelling, root tips were digested by treating with a pectolytic enzyme mixture [2% cellulase (Sigma), 2% cellulase ‘‘Onozuka R-10’’ (Serva), and 2% pectinase enzyme (Sigma) solution in 1xEB at 37º] until the mate- rial became soft. The macerated material was squashed in 1xPBS or 1xMTSB on a slide. Slides were incubated for 1 h at 37ºC in a moisture chamber with a blocking solution (3% bovine serum albumin (BSA) in 1x PBS/1xMTSB, 0.1% Tween 20), followed by an incubation at 10ºC overnight with the primary antibody diluted in 1xPBS/MTSB sup- plemented with 1% BSA. After three washes in 1xPBS/ MTSB for 5 min, the secondary antibodies diluted in 1xPBS/MTSB supplemented with 1% BSA were applied for 45 min at 37ºC. After final washes with 1xPBS, slides were counterstained with 4’6-diamino-2-phenylindole (DAPI) and mounted in 1mg/ml Citifluor antifade medi- um (AF1, Agar Scientific). For MTs immunolocalization a mouse monoclonal antibody to α-tubulin (clone DM1A, Sigma, 1:100) was used, and the tyrosinated form of α-tubulin was detected with a rat antibody (YOL 1/2, Serotec, 1:100). An anti- mouse Alexa 488 antibody (Molecular Probes) and an anti-rat antibody conjugated with biotin (Serotec, 1:200) were used as secondary antibodies. This latter antibody was further detected with a streptavidin-Cy3 conjugate antibody (Sigma, 1:700). For detection of H3S10ph, a rab- bit antibody (Upstate, 1:200) was utilized and revealed using anti-rabbit rhoda mine-conjugated antibody (Dianova, 1: 100). For revealing 5-mC a primary mouse antibody (Abcam, 1:200) and a secondary antibody anti- mouse-Cy3 (Sigma, 1:100) were used. After three final washes, the slides were counterstained with DAPI and mounted in 1mg/ml Citifluor antifade medium (AF1, Agar Scientific). All samples were examined using a Zeiss Axioskop 2 epifluorescence microscope, images were obtained using a Zeiss AxioCam digital camera, and the digital images were processed with Photoshop (Adobe Systems). Quantitative analysis of cell cycle progression and MT organization The nuclear morphology of well-preserved cells from control and in distinct recovery periods (0, 7 and 24 RT) after treatment was determined and the percent- age of DAPI stained cells with either regular or irregular shaped nuclei, and cells with micronuclei were calcu- lated. Cell cycle was evaluated by calculating the mitotic index as the percentage of mitotic cells identified in at least n = 200 cells for each treatment. The number of mitotic cells at different phases was moreover evaluated through tubulin immunolocalization of particular MTs configurations (e.g. preprophase band, mitotic spindle or phragmoplast) in both control and heat-stressed cells after distinct periods of stress recovery. Effects of heat stress on CMTs organization were also evaluated in 200 cells from each treatment through the quantification of cells with normal or new MTs arrangements in distinct recovery periods. The Chi-square test (χ2, P< 0. 05) was utilized for statistical analysis. RESULTS AND DISCUSSION Changes in nuclear morphology and in mitotic index after heat stress are associated with new, transient MT arrange- ments Interphase cells were classified as normal, when nuclei present regular shape and well-defined con- tour; abnormal, those showing nuclei with irregular shape; and cells with micronuclei. Most control cells (n = 200) showed normal interphase nuclei (94%, Table 1; Fig. 1a,b) and the mitotic index was 6%, but decreased immediately after heat stress (0RT – 3%, n=302). In 0RT cells a significant difference in nuclei types was detected in comparison with controls (χ2 = 35.31, P< 0. 05), with an increase in the frequency of cells with abnormal nuclei (21%) and cells with micronuclei (3%). At 7RT cells (n = 300) significant differences between 58 Vânia Neves, Wanda Viegas, Ana D. Caperta heat-stressed cells and controls were found (χ2 = 92.36, P< 0.05) with a frequency of abnormal nuclei more than doubled (41%), and a small increase in cells with micro- nuclei (6%). The observed increase of micronuclei fre- quency attributable to root tips heat exposure is moreo- ver associated with heat stress effects detected on mitotic cell cycle progression. The mitotic index was three times higher at 7RT (18%, Table 1) than in controls. Contrast- ingly, 24RT cells (n = 275) presented a high frequency of normal nuclei (90%) and a decrease in mitotic index (9%). The frequency of abnormal nuclei (7%) and micro- nuclei markedly decreased (3%). These findings support the hypothesis of mitotic arrest after 7RT of exposure to heat stress. Root tips exposure to diverse chemical substances including fertilizers, heav y metals, herbi- cides, pesticides and radioactivity also affect the mitotic index in varying frequencies in Allium cepa (Bonciu et al. 2018). Nonetheless, contrasting effects in the mitotic index were also observed in Secale cereale plants exposed to chemical stresses like the MTs-depolymerizing agent colchicine where mitotic arrest occurred in the low- concentration treatment, whereas c-metaphase cells were able to progress into the cell cycle in the high-concentra- tion treatment (Caperta et al. 2006). Heat stress effects in nuclear morphology can also result from perturbations of CMTs organization as previously described for colchi- cine treatments (Caperta et al. 2006). MTs configurations were analyzed in untreated and heat-treated root tips through tubulin immunolocaliza- tion using antibodies that recognize α-tubulin (DM1A) and tyrosinated tubulin (YOL 1/2). Our results show that both antibodies presented coincident and similar immuno-signal distributions (Fig. 2). Control cell MTs exhibited both α-tubulin and tyrosinated tubulin arrays Table 1. Percentage (%) of interphase cells with nuclear normal morphology (DAPI), tubulin immunolabeled cells in interphase (CMTs) and mitosis, and mitotic index. Control and heat-stressed cells analyzed after 0 (0 RT), 7 (7 RT), and 24 (24 RT) h of recovery from the stress. The presence of cortical microtubules (CMT), preprophase band (PPB), mitotic spindle (SP) and phragmoplast (P) was scored. Seedlings treatments Frequencies (%) of DAPI stained interphase cells with normal nuclear topology Mitotic Index Frequencies (%) of interphase cells with typical organized CMTs arrays Frequencies of mitotic cells (%) at distinct phases Number of mitotic cells analysedPPB SP P Control 94a 6a 98a 64a 24a 12 33 0 RT 76b 3b 16b 78b 11b 11 37 7 RT 53c 18c 37c 48c 27a 25 48 24 RT 90a 9d 95a 33d 48c 19 60 Figure 1. DAPI-stained Secale cereale meristematic interphase root cells. 1a – Control cells with a well-defined contour; and 1b. heat-stressed cells with irregular nuclei and/or with micronuclei (arrowed). Bar: 5 μm. Figure 2. Indirect immunodetection of α-tubulin (MT) in con- trol cells. Nuclei, chromatin and chromosomes are stained with DAPI (blue). Tubulin containing arrays are detected with DM1A α-tubulin (green) and YOL1/2 tyrosinated α-tubulin (red) antibod- ies. a- a’’ Interphase cell with cortical microtubules; b- -b’’ prophase cell with the preprophase band; c -c’’ meta/anaphase cell with spin- dle; d- d’’ ana/telophase cell with the phragmoplast. Bar: 5 μm. 59Effects of high temperature on mitotic index, microtubule and chromatin organization in rye root-tip cells configurations characteristic of higher plant cells: CMTs at interphase, pre-prophase band, mitotic spindle and phragmoplast at the end of telophase (Fig. 1). Control cells presented organized CMTs (98%, Table 1), whereas at 0RT significant differences occurred (χ2 = 687. 57, P< 0.05), in which the majority of cells exhibited branched and wavy CMTs with a disorganized orientation (84%) (Fig. 3a-a’). Previous studies showed that heat stress caused dissem- blance of MTs in mitotic tobacco suspension cultured cells after 30 min at 42ºC (Smertenko et al. 1997b). In the present study we showed higher resilience of rye MTs to heat stress since exposure of 2 days seedlings to 4 h at 40ºC only induced CMTs disorganization at 0RT with- out total disruption. However, knowledge is inexistent with regard to the heat response of the rye variety used in this study (heat-resistant or heat-sensitive). Compared to controls, at 7RT there was still a significant difference in the frequency of interphase cells with a disorganized and branched wavy-like MTs configuration (63%, Table 1, Fig. 3d-d’) (χ2 = 424. 97, P< 0.05), although the frequency of interphase cells with normal MTs arrangements doubled. At 24RT 95% of interphase cells present typical organized CMT arrays (Table 1). Disorganized wavy MTs arrange- ments were also observed in root cells exposed to high colchicine concentration conditions (Lazareva et al. 2003; Caperta et al. 2006). Therefore, it is tempting to suggest that new MTs re-orientations result from perturbations induced by heat stress on CMTs associations with plasma membrane, although the nature of MT attachments to the membrane is not yet totally clear both in animal (Wolff 2009) and in plant cells despite all efforts to understand it (Liu et al. 2015). It was demonstrated that CMTs can change their orientation in response to a broad range of abiotic signals (Nick 2013) by controlling the direction of cellulose deposition and reinforcing axial cell expansion (Geitmann and Ortega 2009). In the current work, the high frequency of interphase cells with abnormal nuclear morphology observed at 7RT probably reflects changes in CMTs organization by inducing differential cytosol com- partmentalization. Earlier studies moreover reported that different MTs arrays presented distinct sensitivities to temperature stress (Smertenko et al. 1997b; Abdrakhamanova et al. 2003; Müller et al. 2007). The most heat-sensitive MT arrays are those of the mitotic spindle and the phragmo- plast in tobacco cultured cells (Smertenko et al. 1997b). In Triticum chilling sensitive species, CMTs are extreme- ly cold-sensitive, whereas they persist at low tempera- tures in chilling-tolerant species (Abdrakhamanova et al. 2003). In this study, MTs from the pre-prophase band (PPB) appeared to be very sensitive to heat stress since most prophase cells at 0RT (94%) presented slightly dis- organized pre-prophase bands (Fig. 3b-b’). Such marked changes in frequencies of prophase cells with abnormal PPB at 0RT were however not associated with perturba- tions in other mitotic phases since all metaphase (Fig. 3c-3c’) and anaphase cells exhibited well-formed spin- dles, and telophase cells showed phragmoplast MTs orthogonally disposed to the division plane. After 7RT, most prophase cells presented a reduction of abnormal pre-prophase bands (65%), although in some cells altered spindle and phragmoplast configurations were revealed (Fig. 3f-f ’), which were absent at 24RT. Compared to controls, the frequency of cells with PPBs presented the highest value at 0RT (78%) and the Figure 3. Indirect immunodetection of α-tubulin (MT) in heat- stressed cells after 0 (0 RT), 7 (7 RT), and 24 (24 RT) h of recovery from the stress. Nuclei, chromatin and chromosomes are stained with DAPI (blue). Tubulin containing arrays are detected with YOL1/2 tyrosinated α-tubulin (red) antibody. a-a’ and d-d’ Inter- phase cells showing branched, stringy CMTs with disorganized orientation; b-b’ prophase cell with a slightly disorganized pre-pro- phase band with MTs without the usual parallel orientation; meta- phase cell (c-c’) and anaphase cell (e-e’) with normal spindles; f-f ’ telophase cell with thick CMTs arrays and with remnants of phrag- moplast. Bar: 5 μm. 60 Vânia Neves, Wanda Viegas, Ana D. Caperta lowest one at 24RT (33%). These findings contrasted with frequencies of cells with spindles and phragmoplasts, which have low frequencies at 0RT (22%) and high fre- quencies at 7RT (52%), with a maximum of at 24RT (67%). Taken together, the drastic dropping of mitotic index values at 0RT seems to be associated with pertur- bations on CMTs organization as well as disturbances on PPB allowing however the progression of subsequent mitotic phases as only cells with normal spindles and phragmoplasts were detected. On the other hand, at 7RT the observed beginning of normal CMT organization reestablishment seemed to allow cells transition to mitosis associated with the marked increase in the mitotic index. The high frequencies of cells in metaphase, anaphase and cytokinesis at 7RT, associated with some perturbations in spindle and phragmoplast organizations appear to indi- cate why heat-stressed cells stay longer in mitosis. After 24 RT the MT cytoskeleton configurations in interphase and prophase cells were like the ones observed in controls, although the high frequencies of cells at metaphase, ana- phase and cytokinesis revealed a delayed reestablishment reflected in the high mitotic index yet observed. Nonethe- less, it is not yet clear which microtubule structures, corti- cal MTs or mitotic MTs, are susceptible to tubulin modifi- cation induced by heat stress. Heat stress induces changes in DNA methylation patterns but no alterations in H3S10ph distribution patterns In both control and heat-stressed cells H3S10ph marks were absent during interphase. However, they were present in prophase cells in the pericentromeric heterochromatin and restricted to one nuclear hemi- sphere revealing Rabl configuration (Fig. 4). The Rabl configuration, characteristic of rye genome (Caperta et al. 2002) is maintained after heat stress as centromeres are all aligned in one nuclear pole. In interphase cells no labelling was found (Fig. 4a-a’), which contrasted with prophase (Fig. 4b-b’), metaphase (Fig. 4a-a’ and c-c’) and anaphase (Fig. 4a-a’) cells. In mitotic cells, a marked labelling was also found in chromosomes pericentro- meric regions but no detectable changes were observed in signal dimensions or intensities in both control and heat-stressed cells (Figs. 4d-d’, 4e-e’, 4f-f ’). Instead, cold treatment of plant root meristems resulted in additional chromosomal sites of H3S10ph, besides the usual ones in pericentromeric regions (Manzanero et al. 2000). Also, up-regulation of the stress-inducible genes in Arabidop- sis T87 and tobacco BY-2 cell lines is associated with increased phosphorylation of histone H3 at serine 10 residue at high salinity, cold and abscisic acid treatments (Sokol et al. 2007). Control cells showed interphase nuclei with dis- perse, dotted and intense 5-mC immunosignal all over the nucleus both in highly condensed (heterochromatin) and decondensed (euchromatin) chromatin (Fig. 5a-a’’). Instead, in heat-stressed cells at 0RT and 7RT a distinct, heterogeneous DNA methylation distribution pattern with aggregated immunosignal was preferentially found more concentrated in the nuclear periphery (Fig. 5c-c’’ and 5d-d’’). In metaphase cells, at both 0RT and 7RT a heterogeneous and discontinuous 5-mC labeling was also detected along chromosome arms (Fig. 5e-e’’). After 24 RT, the distribution patterns of 5-mC were like those observed in control interphase and metaphase cells (Fig. 5f-f ’’’). The results presented here in rye somatic cells from control plants are in accordance with earlier stud- ies in rye metaphase chromosome spreads, which dis- played a punctuated and uniform pattern of methylated DNA residues along both the As and Bs chromosomes, without any particular sites of accumulation (Carchilan et al. 2007). However, no differences were found in the nuclear distribution of methylated cytosines between meiocytes of heat-stressed and control rye plants with Figure 4. Indirect immunodetection of histone H3 phosphoryl- ated at serine 10 residue in Secale cereale meristematic root cells. Nuclei, chromatin and chromosomes are stained with DAPI (blue). Chromatin and chromosomes are detected with histone H3 phos- phorylated at serine 10 residue (H3S10ph, green) antibody in control (a-a’- c-c’) and heat-stressed cells (d-d’- f-f ’). a-a’ Interphase cell without H3S10ph labelling and an anaphase cell with H3S10ph marks; b-b’ prophase cells showing H3S10ph marks in the pericentromeric heterochromatin and restricted to one nuclear hemisphere reveal- ing Rabl configuration; c-c’ metaphase cell presenting H3S10ph dots only in a discrete region in pericentromeric chromatin; d-d’ cell at interphase showing no H3S10ph labelling and a metaphase cell with pericentromeric H3S10ph immnosignals; e-e’ cell at prophase with H3S10ph marks in the pericentromeric heterochromatin; and f-f ’ early anaphase cell revealing H3S10ph dots only in pericentromeric chromatin. Bar: 5 μm. 61Effects of high temperature on mitotic index, microtubule and chromatin organization in rye root-tip cells 0B or 2B chromosomes (Pereira et al. 2017). A study on the effects of heat stress in the repetitive sequence genome fraction (coding and non-coding sequences) using leaves of intact rye plants suggests marked differ- ences between these sequences that most likely reflect their distinct roles in the plant pathways involved in the stress response (Tomás et al. 2013). Hence, it appears that there are different tempera- ture chromatin sensitivities accordingly with chromatin fractions, cell types and tissue origins. For instance, in soybean at normal temperature, root hairs were more hypermethylated than were stripped roots whereas in response to heat stress, both root hairs and stripped roots showed hypomethylation (Hossain et al. 2017). These changes in DNA methylation were directly or indirectly associated with expression of genes and trans- posons within the context of either specific tissues/ cells or heat stress (Hossain et al. 2017). 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