Protoplasma DOI 10.1007/s00709-015-0809-2

ORIGINAL ARTICLE

Effects of high temperature on the ultrastructure and microtubule organization of interphase and dividing cells of the seagrass Cymodocea nodosa M. Koutalianou 1 & S. Orfanidis 2 & C. Katsaros 1

Received: 23 October 2014 / Accepted: 20 March 2015 # Springer-Verlag Wien 2015

Abstract Short-time temperature effects (34–40 °C) on microtubule (MT) organization and on cell structure of young epidermal leaf cells of the seagrass Cymodocea nodosa were investigated under laboratory conditions using transmission electron microscopy (TEM) and tubulin immunofluorescence. The interphase MT network was affected by the increased temperature, the effect being time dependent and expressed in both the form and the orientation of the MT bundles. After 1 h at 38 °C, there was also a severe disturbance in dividing cells with thick and short MTs in the mitotic spindle and atypically organized phragmoplasts, while after 2 h at 38 °C the mitotic index was tenfold reduced compared with the control material. After 6 h at 38 °C, a large number of telophase cells were observed, meaning that cytokinesis was blocked. TEM observation revealed cells with uncompleted cell plates consisting of swollen vesicles and branched cisternae, with no phragmoplast MTs. These cells bear a nucleolus with segregated fibrillar and granular zones, an increased number of swollen mitochondria, and numerous parallel arrays of endoplasmic reticulum cisternae in the cortical cytoplasm. The possible relationship of these changes in C. nodosa with a response mechanism in order to face elevated temperature effects of climate change is discussed.

Handling Editor: Anne-Catherine Schmit * C. Katsaros [email protected] 1

Faculty of Biology, University of Athens, Athens 157 84, Greece

2

Hellenic Agricultural Organization-Demeter, Fisheries Research Institute, 640 07 Nea Peramos, Kavala, Greece

Keywords Cymodocea nodosa . Heat stress . Microtubules . Seagrass . Ultrastructure

Introduction Seagrasses constitute a unique group of fully submerged flowering plants which provide various important ecological services to the marine environment (Costanza et al. 1997; Beal and Schmit 2000; Orth et al. 2006). Unfortunately, currently they are in serious decline with their loss accelerating from 1 % year−1 before 1940 to 7 % year−1 presently (Waycott et al. 2009). This is probably due to the higher rates of changes in coastal waters today than previously because of recent climatic changes and severe anthropogenic stress to the coastal zone (Orth et al. 2006; Koch et al. 2013). Global climate change is characterized by both the change in mean variables and the increase in extreme events such as heat waves (Easterling et al. 2000). Since these changes may be too fast to allow seagrasses to adapt, species thriving in shallow or in relatively enclosed waters (lagoons, bays) are expected to suffer most (Pergent et al. 2014) and therefore are good candidates to study their adaptation potential and foreseen impacts (Ondiviela et al. 2014). Cymodocea nodosa (Ucria) Ascherson is a common seagrass forming patchy meadows along Mediterranean and adjacent seas (den Hartog 1970; Reyes et al. 1995; Barbera et al. 2005; Mascaró et al. 2009) and in Mediterranean coastal lagoons (Agostini et al. 2003; Nicolaidou et al. 2005; Pasqualini et al. 2006). Since undergoing or future increase of the mean water temperature may have a rather positive effect on the distribution of this warm-temperate species, the declines reported, in agreement with field data, are mainly attributed to intense human disturbances (Duarte et al. 2008; Hughes et al. 2009; Waycott et al. 2009; Orfanidis et al. 2010;

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Short et al. 2011; Tuya et al. 2014). However, negative effects for this species are expected in very shallow waters and coastal lagoons due to the maximal warming in the summer (up to 5 °C at the end of this century under the A2 IPCC scenario for Venice lagoon) (IPCC 2000), very close to species temperature survival. In order to predict future shifts, it is essential to understand species temperature thresholds and mechanisms for adaptation and interaction (Koch et al. 2007). Until now, there are publications reporting the effects of increased temperatures on the presence and distribution, external morphology, growth, photosynthesis, and metabolic activities (like electron transport system) of different seagrass species (Barber and Behrens 1985; Lee and Dunton 1997; Ralph 1998; York et al. 2013). In addition, there are few publications dealing with the effects of abiotic factors on the morphology and development of different seagrass species. These factors are mainly different salinity, light, and temperature conditions (Jagels and Barnabas 1989; Iyer and Barnabas 1993; Benjamina et al. 1999; York et al. 2013; Serra et al. 2013). However, there is no information on the effects of high temperature on the cell fine structure and cytoskeleton organization. Contrary to seagrasses, in other, non-marine angiosperms, there are studies examining the effect of heat shock on the fine structure of the cells and the cell organelles (Ciamporova and Mistrik 1993; Collins et al. 1995; Zhang et al. 2005; Zhang et al. 2009a, b; Huang et al. 2013). Among others, it has been reported that cold, heat, and osmosis stress in plants modify the physical properties of biological membranes (Falcone et al. 2004; Matos et al. 2007) which may enclose sensory devices capable of detecting and transducting signals (Los and Murata 2004). The cytoskeleton may play an essential role in regulating intracellular signaling and cell architecture (Saidi et al. 2011) as a response to environmental stress (Simon 1978; Bush 1995; Webb et al. 1996). Cold-induced physiological and biochemical changes have been repeatedly investigated, in contrast with heat stress response, but both resulted in protein unfolding, affect membrane fluidity and metabolism, and cause cytoskeleton rearrangement (Ruelland and Zachowski 2010; Nick 2013). In terrestrial angiosperms, MTs are additional components of cold sensing (Nick 2013). In cell suspension cultures of Nicotiana tabacum and root cells of Arabidopsis thaliana, it has been found that MT organization is susceptible to heat stress (Smertenko et al. 1997; Muller et al. 2007). The existing fine structural studies on marine angiosperms deal with the morphology and anatomy in leaf cells of Cymodocea rotundata, Cymodocea serrulata, Thalassia hemprichii (Doohan and Newcomb 1976), Zostera capensis (Barnabas et al. 1977), Posidonia australis (Kuo 1978), Zostera muelleri (Kuo et al. 1990), and Syringodium isoetifolium (Cymodoceaceae) (Kuo 1993). A general

overview of the morphology and ultrastructure of seagrasses is also given by Kuo and den Hartog (2006). Regarding the cytoskeleton, the only existing publication is that of Malea et al. (2013), in which the effect of heavy metals (Ni, Cu, Cr) on the organization of MTs in cells of C. nodosa is examined. In this paper, it was reported that treatment with Ni (5–40 mg/L) for 3 days causes a depolymerization of MTs, while high concentrations of Cu for 7 days cause also a disarrangement of MTs, and treatment with Cr for 9 or 11 days caused an extensive MT bundling. The present study is the first report on the effect of short-term elevated temperatures on the fine structure and MT cytoskeleton organization in interphase and dividing epidermal cells from young leaves of the seagrass C. nodosa.

Materials and methods Seagrass sampling and stock culture conditions C. nodosa (Ucria) Ascherson plants were collected from 0.8 m depth in the western coast of the South Evoikos Gulf (38°24′ N, 23°40′ E), Greece. At this site, C. nodosa grows in depths from 0.3 to around 3 m, forming a rather continuous patchy meadow. Intact shoots were transported to the laboratory in plastic containers containing seawater from the collection site. After being transported to the laboratory, the shoots were cleaned of debris and placed in artificial seawater (dH2O with 3.1 % Red Sea Salt—Red Sea). The cultures were grown under 88 μmol m−2 s−1 of cool white fluorescent lighting on a 14:l0 light/dark cycle, at room temperature conditions at 21 °C. The material used in all the experiments, for both tubulin immunofluorescence and TEM examination, was taken from the meristematic region of either the smallest juvenile leaf or the second smallest juvenile leaf. In both cases, small pieces of 0.5 cm height×0.3–0.7 cm length were cut. For comparison, the present study is focused on the epidermal cells of the above regions. Experimental culture conditions The above-described light regime was used for all experiments. For the experiments performed in high temperature conditions (34, 38, and 40 °C), the plants were cultivated in a thermal incubator (OilBath ONE; Memmert) as follows: at 34 °C for 6 h; at 38 °C for 30 min, 1 h, 2 h, 4 h, and 6 h; and at 40 °C for 1 h. TEM examination was applied only in plants cultivated for 6 h at 38 °C. For the recovery experiments, plants

Effects of high temperature on Cymodocea nodosa

were cultivated at 38 °C for 6 h or at 40 °C then transferred at normal temperatures for 72 h. Tubulin immunofluorescence Tubulin immunofluorescence was applied on epidermal cells, following the protocol of Katsaros and Galatis (1992) with a modified enzyme solution for the softening of the cell walls which contained 2.5 % (w/v) cellulase Onozuka (Yakult Honsha Co., Tokyo, Japan), 2 % (w/v) macerozyme Onozuka (Yakult), 1 % (w/v) driselase from Basidiomycetes sp. (Sigma, St. Louis, MO, USA), and 1 % (v/v) β-glucuronidase type HP-2 from Helix pomatia (Sigma) in PEM buffer (0.1 mM pipes, 2 mM EGTA, 1 mM magnesium sulfate), pH 5.6, for 80 min. Following rinsing with PEM, the leaves were squashed on coverslips coated with 1 mg/

Fig. 1 a–f Young epidermal cells of C. nodosa control material appearing after tubulin immunofluorescence (1 in all figures and c2), Hoechst staining of DNA (2 except c2), and DIC optics (3). a Interphase cell with cortical MTs perpendicular to the long leaf axis (arrow). b Preprophase cell showing preprophase MT band. c Prophase cell showing both remnants of the preprophase MT band and prophase spindle. d Metaphase cell. e Telophase-early cytokinetic cell with phragmoplast MTs. f Advanced cytokinetic cell with phragmoplast MTs. Scale bar= 10 μm

mL poly-L-lysine (Sigma) and left to allow the separated cells to dry. For the tubulin labeling, both anti-atubulin (YOL1/34; AbD Serotec, Kidlington, UK) and FITC-anti-rat secondary antibody (Sigma) diluted at 1:40 were used. DNA was counterstained with 10 μg/ mL Hoechst 33258 (Sigma), and the cells or small leaf pieces were finally mounted in an anti-fade solution of 1.6 mg/mL p-phenylenediamine (Sigma) diluted in a solution containing 2:1 glycerol/PBS. The samples were examined with a Zeiss Axioplan microscope equipped with an ultraviolet source, proper filters, and a Zeiss Axiocam MRc5 digital camera. Measurements of the number of MT bundles diverging more than 30° from the typical parallel distribution (Fig. 5) were made by examining young epidermal cells under the Zeiss Axioplan microscope. Only cells with a normal shape in DIC optics were used for measurement. Frequency of

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Fig. 2 a–d TEM micrographs of epidermal cells of C. nodosa control material. a Cortical microtubules (MTs) (arrows) at the periphery of an interphase cell. b Longitudinal sections of MTs (arrows) in surface paradermal section. c Nucleus with nucleolus and electron-dense masses

Fig. 3 a–f TEM micrographs of mitotic cells of C. nodosa control material. a1 Prometaphase cell. a2 Higher magnification of an area (marked by the white rectangle in a1) showing MTs (arrow) connected with the chromosomes. b1 Metaphase cell. b2 Higher magnification of an area (marked by the white rectangle in b1) showing spindle MTs (arrow). c1 Anaphase cell. c2 Higher magnification of an area (marked by the white rectangle in c1) showing kinetochore-directed and interzonal MTs (arrows). d Cell plate with phragmoplast MTs (arrows). e Advanced cytokinetic cell showing almost completed cell plate. Scale bars=0.2 μm (a2, b2, c2, d) and 1 μm (a1, b1, c1, e)

mainly arranged at the nuclear periphery. d Preprophase MT band (white bracket) located along the equatorial site of the cortical cytoplasm, close to the plasmalemma of the longitudinal walls (marked by the white rectangle in c). Scale bars=1 μm (c) and 0.2 μm (a, b, d)

Effects of high temperature on Cymodocea nodosa

Fig. 4 a–h Interphase epidermal cells of young leaves of C. nodosa under heat stress. In all figures: 1 tubulin immunofluorescence, 2 Hoechst staining of DNA, 3 DIC optics. a Incubation at 34 °C for 6 h: thick MT bundles oriented more or less perpendicularly to the long leaf axis (arrow in a3). b Incubation at 38 °C for 30 min: MT bundles showing a slightly aberrant orientation. c Incubation at 38 °C for 1 h: fragmented MT bundles with slightly aberrant orientations. d Incubation at 38 °C for 2 h: thick, short MT bundles with aberrant orientations. e

Longer incubation for 4 h at 38 °C: depolymerization and disassembly of interphase MTs with loss of their orientation. f Incubation for 6 h at 38 °C: MT network atypically organized with thick fragmented MTs and loss of the transverse orientation. g Group of cells after incubation in 38 °C for 6 h. MT organization appears almost completely disorganized. h Incubation at 40 °C for 1 h: severe disruption of the cytoskeleton with short and thick fragmented MTs with aberrant orientations. Scale bar= 10 μm

average number of MTs diverging more than 30° was plotted as a histogram using MS Excel software (Microsoft Corp.)

Standard error bars of repeated experiments were used. At least 100 cells for each experiment were examined.

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Electron microscopy For the transmission electron microscopy (TEM) study, the protocol reported by Katsaros et al. (1983) was followed. Thin sections of epidermal cells were examined with a Philips 300 electron microscope and a JEOL 100S TEM. The number and size of the mitochondria was measured on digitalized TEM micrographs captured as Btiff^ files. The average number and size of the mitochondria was plotted as a histogram (Fig. 9) using MS Excel software (Microsoft Corp.). Standard error bars of repeated experiments were used. At least 100 interphase young epidermal cells from each experiment were used for the measurements.

Results Microtubule organization Control Tubulin immunofluorescence and TEM examination of control material revealed that the MT cytoskeleton of interphase epidermal cells was organized in parallel bundles oriented more or less perpendicularly to the long leaf axis (Figs. 1a and 2a, b). As the cells start entering mitosis, preprophase MT bands were observed (Fig. 1b). TEM examination revealed that each preprophase MT band consists of 15–20 MTs surrounding the nucleus in a median plane and located along the cortical cytoplasm, close to the plasmalemma of the longitudinal walls (Fig. 2c, d). When the cell was entering prophase, prophase spindles were formed at opposite Bpoles^ of the nucleus (Fig. 1c). During prometaphase, the chromosomes are directed towards the cell equator and kinetochoredirected MTs are also visible (Fig. 3a1, a2). In metaphase, a normal spindle was developed and the chromosomes were aligned at the metaphase plate (Figs. 1d and 3b), while during both metaphase and anaphase, kinetochore-directed and interdigitating spindle MTs were observed (Fig. 3b, c). During telophase-cytokinesis, a normal phragmoplast-cell plate system was gradually formed (Figs. 1e, f and 3d, e). High temperature effect—interphase epidermal cells For the examination of the effect of high temperatures on MT organization, three parameters were used to distinguish the normal from the abnormal epidermal cells: the orientation, the length, and the thickness of the MT bundles. Using these criteria, the examined epidermal cells after high-temperature treatment showed abnormalities in a variable degree. In immunofluorescence preparations of epidermal cells of plants incubated at 34 °C, a slight disturbance of MTs was observed only after 6 h. Thick MT bundles were formed in the cortical

Fig. 5 Histogram showing the effect of high temperatures on MT orientation. Every MT bundle diverging more than 30° from the typical parallel distribution of MTs was counted as MT with aberrant orientation

cytoplasm of interphase cells oriented more or less perpendicularly to the long leaf axis (Figs. 4a and 5). Slight changes in the organization of MTs of interphase cells were also observed after incubation for 30 min at 38 °C, where short, wavy in shape MTs were formed with aberrant orientations (Figs. 4b and 5). These alterations were more obvious after 1 (Fig. 4c) and 2 h (Fig. 4d) of incubation at 38 °C, with the formation of a reduced number of short, fragmented MT bundles with loss of their orientation (Fig. 5). Longer incubation for 4 h (Fig. 4e) and 6 h (Fig. 4f, g) at 38 °C caused further disturbance in both shape, density, and orientation of MTs. Interphase MT network was atypically organized with loss of the transverse orientation (Fig. 5) and presence of thick, fragmented MT bundles. However, the cells were still alive after this treatment and fully recovered after 72 h of cultivation in normal temperature (data not shown). On the other hand, incubation for 60 min at 40 °C resulted in a severe disruption of cytoskeletal MTs which did not recover after 72 h of incubation in normal temperature (data not shown). Short and thick MT bundles were observed, together with MTs with aberrant orientations (Fig. 4h). Quantitative data on the effect of high temperatures to MT orientation are shown in Fig. 5. High temperature effect—dividing cells In immunofluorescence preparations of epidermal cells of plants incubated at 34 °C for 6 h, there was no disturbance of mitotic MTs. Incubation at 38 °C showed a time-dependent Table 1 Comparison of the frequency (%) of dividing cells at different incubation times at 38 °C with control conditions (21 °C) Temperature (°C) Incubation Interphase cells (%) Dividing cells (%) 21 38

30 min 1h 2h 6h

58.30 89.30 89.80 95.30 96.50

41.70 10.70 10.20 4.70 3.50

Effects of high temperature on Cymodocea nodosa Table 2

Comparison of the frequency (%) of mitotic phases of the epidermal cells at different incubation times at 38 °C with control conditions (21 °C)

Temperature (°C)

21 (control) 38

Incubation

30 min 1h 6h

Dividing cells Prophase (%)

Metaphase (%)

Anaphase (%)

Telophase-cytokinesis (%)

34.23 7.13 2.36 –

2.37 1.78 – –

1.96 0.46 – –

3.14 1.33 7.84 3.5

effect in the mitotic index. In the examined meristematic region of the control (21 °C), usually 41.70 % of the epidermal cells were found at a mitotic stage. This percentage was drastically reduced to 10.70, 10.20, and 4.70 % in plants incubated at 38 °C for 30 min, 1 h, and 2 h correspondingly. Longer incubation at 38 °C for 6 h resulted in further decrease of the mitotic index to 3.5 % (Table 1). Mitotic spindle was normal after 30 min of incubation, but after 1 h, cells with preprophase band were not found and mitotic spindles appeared consisting of thick and short MT bundles (Fig. 6a). The most susceptible cells were those which at the time of treatment were at telophase-cytokinesis. The phragmoplast appeared to lose its normal shape and showed disordered, not parallel, MTs and loss of its polarity (Fig. 6b, c). The mitotic index was measured in more than 500 cells, and as shown in Table 2, preprophase-prophase cells were decreased from 7.13 % after 30 min of incubation to 2.36 % after 1 h. The inhibition of cytokinesis resulted in an accumulation of

telophase-cytokinetic cells which increased from 1.33 % (30 min) to 7.84 % (1 h). After incubation at 38 °C for 6 h, actually all the dividing cells were blocked at telophasecytokinesis (Tables 1 and 2). Further examination with transmission electron microscopy confirmed the absence of phragmoplast MTs and the presence of incomplete cell plates in cytokinetic cells (see below, Fig. 8d, e). Incubation at 40 °C, even for short times, severely affected the dividing cells, with the formation of thick and short MTs in the mitotic spindle (Fig. 6d) and atypically organized phragmoplasts. Cell structure Control In median paradermal sections of young leaves of C. nodosa, the epidermal cells appeared rectangular in shape and characterized by a large nucleus occupying most of the cell space (Fig. 7a, b, e). TEM examination revealed dense masses of condensed chromatin usually distributed along the nuclear periphery while a prominent nucleolus was present in a central nuclear position (Fig. 7b, e). A variable number of undifferentiated chloroplasts were observed at the perinuclear cytoplasm (Fig. 7b, c). Mitochondria were spherical to ovoid with typical tubular cristae and evenly distributed in the cytoplasm (Fig 7d, e). High temperature effect

Fig. 6 a–d Dividing epidermal cells of young leaves of C. nodosa under heat stress, after tubulin immunofluorescence (1), Hoechst staining of DNA (2), and DIC optics (3). a Prophase cell after incubation at 38 °C for 6 h with abnormal mitotic spindle consisting of mainly perinuclear thick and short MT bundles. b, c Telophase-cytokinetic cells with abnormal phragmoplast with disordered, not parallel, MTs and loss of polarity. d Prophase cell after incubation at 40 °C for 1 h. Prophase spindle is disassembled, showing thick and short MTs. Scale bar=10 μm

TEM examination of material incubated for 6 h at 38 °C revealed many epidermal cells in which the nucleus appeared to have masses of condensed chromatin around the nucleolus (Fig. 8a, b). Fibrillar and granular zones of the nucleoli were segregated, while numerous electron-dense granules were present around them (Fig. 8b). In surface paradermal sections, the MT network was found atypically organized with loss of the transverse orientation and fragmented MTs (Fig. 8c; compare with Fig. 2b). Cytokinetic cells bear uncompleted cell plates, with no phragmoplast MTs and accumulation of swollen, independent aggregates of vesicles and branched cisternae (Fig. 8d, e). A very obvious effect was the formation of

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parallel bundles of endoplasmic reticulum (ER) cisternae arranged in the cortical cytoplasm (Fig. 8f). In most cells, there were three to four rows of parallel, sometimes swollen ER cisternae under the plasma membrane (Fig. 8f; compare with Fig. 7b, e). In addition, mitochondria were also affected after 6 h at 38 °C. Their number was increased, and their shape appeared swollen, containing stroma with very few cristae (Fig. 8g, h; compare with Fig. 7d, e). Figure 9 shows the effect of heat treatment on mitochondria. It is obvious that both the number and size of mitochondria after incubation at 38 °C for 6 h have almost doubled compared with the control.

Discussion Control The MT organization in interphase cells of C. nodosa was found similar to that found in other, terrestrial angiosperms, i.e., cortical MT bundles traversing the cells in a direction perpendicular to the leaf axis (see also Malea et al. 2013). Moreover, ultrastructural analysis and immunofluorescence examination of MT organization revealed that mitosis and cytokinesis are more or less similar to those described for terrestrial angiosperms (Smirnova 2012). The fine structure of C. nodosa young epidermal cells examined in the present study is also similar to that reported for other seagrass species by Doohan and Newcomb (1976), Barnabas et al. (1977), Kuo (1978, 1990, 1993), and Kuo and den Hartog (2006). Fig. 7 a–e TEM micrographs of young leaf cells of C. nodosa control material. a Epidermal cells appearing in paradermal leaf section. b Higher magnification of a young epidermal cell. Note its rectangular shape and large nucleus. ER is shown by a black arrowhead. c Typical undifferentiated chloroplast with few thylakoid bands. d Mitochondrion showing typical cristae. e Young epidermal cell. Mitochondria are marked by arrows and ER by arrowhead. Scale bars=2 μm (a), 1 μm (b, e), 0.5 μm (c), and 0.2 μm (d)

High temperature effect Cultivation of C. nodosa plants for 6 h at 38 °C caused visible alterations in the structure of cell organelles. The electrondense granules observed within nuclei are similar to the Bheat-shock^ granules described by Fransolet et al. (1979), where heat shock selectively affected the nucleolar structure and the pre-rRNA synthesis. In parallel, in the present study, the fibrillar and granular zones of the nucleoli appear segregated, similar with the results observed in root cells of Allium cepa at 45 °C, Zea mays at 46 °C, and Glycine max at 40 °C by Ciamporova and Mistrik (1993). High temperature, also, in a thermosensitive line in rice caused severe damage on the nucleus as most of the nuclear membrane ruptured and became blurred and a lot of fibrillar-granular materials appeared (Zhang et al. 2009a, b). Furthermore, heat stress seriously affects the mitochondria. They become swollen and increased significantly in number. These findings are in agreement with those reported by Grigorova et al (2012) in Triticum aestivum L. The swollen shape of mitochondria may also be associated with membrane alterations (Ristic and Cass 1992) and/or degradation of membrane phospholipids (Utrillas and Alegre 1997). Stress promotes directly or indirectly mitochondria inner membrane permeabilization and leads to mitochondria swelling (Li and Xing 2011). The increased number of mitochondria is related to the increasing demand of ATP production under stress conditions, when photosynthesis is damaged (Silva et al. 2010), and to the synthesis of mitochondrial stress proteins (Rizhsky et al. 2002; Rizhsky et al. 2004). C. nodosa increased numbers of mitochondria after high temperature treatment could be also

Effects of high temperature on Cymodocea nodosa Fig. 8 a–h TEM micrographs of young leaf epidermal cells of C. nodosa after incubation at 38 °C for 6 h. a Group of epidermal cells. Compare with Fig. 7a. b Nucleus with nucleolus showing segregated fibrillar and granular zones and the presence of electron-dense granules around it. c Surface section of an epidermal cell showing short MTs with aberrant orientations (arrows). d Median area of a cytokinetic cell with uncompleted cell plate consisting of accumulating swollen vesicles and branched cisternae. e Higher magnification of the one end of an uncompleted cell plate. No phragmoplast MTs can be observed. f Epidermal cell with parallel bundles of endoplasmic reticulum (ER) cisternae in the cortical cytoplasm (arrowheads). g Group of swollen mitochondria showing disorganized matrix. h Epidermal cell with increased number of swollen mitochondria (arrows). Scale bars=2 μm (a), 1 μm (b, d, e, f, h), 0.5 μm (g), and 0.2 μm (c)

Fig. 9 Histogram showing the number and size of the mitochondria in control material (light gray) and under heat stress at 38 °C for 6 h (dark gray)

compared with the results of Kaniewska et al (2012) on reef coral (Acropora millepora) where there was an increase in mitochondrial transcripts for ATPase due to warming ocean. Another finding after the incubation at 38 °C for 6 h was the formation of parallel cortical bundles of ER along the walls. These changes in ER morphology may be related with the increased synthesis of stress proteins from the lamellae which remain active (Pareek et al. 1997). In an investigation of different stress factors in plants (Ciamporova and Mistrik 1993), accumulation of ER cisternae into whorls was observed that could be a reason for the failure to supply new cisternae to the Golgi apparatus or a way to protect the other parts of the cytoplasm (Ishikawa 1996). Apart from the above cellular changes, heat stress makes cortical MTs vulnerable almost at every high temperature examined in the present study and the disturbance was time dependent. The severe effects of high temperature on the

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structure and organization of MTs observed are in contrast to the results of Smertenko et al (1997). These authors found that in tobacco suspension culture cells, cortical microtubules showed normal organization even after 24 h at 38 °C whereas in the present study C. nodosa cells cultivated at 38 °C for 6 h exhibited disturbance in both shape and orientation of MTs. As mentioned in the BIntroduction^ section, the only study dealing with MT organization in C. nodosa is that by Malea et al. (2013) where after treatment with low concentrations of heavy metals (Cu, Ni, and Cr) there was MT disturbance in leaf meristematic cells. The above authors suggested that MT cytoskeleton is a sensitive indicator of stress conditions (Malea et al. 2013). Similar results have been reported for the reef coral A. millepora in which experiments with high CO2 concentrations and high temperature for 28 days revealed a differential expression of the genes involved in membranecytoskeleton interactions and cytoskeletal remodeling that indicates that elevated temperature triggers changes on cytoskeleton (Kaniewska et al. 2012). An interesting question which has not been answered until now is Bwhat happens with the dividing cells and how the cell cycle is influenced by elevated temperatures.^ According to our results, the organization of mitotic MTs is more susceptible than interphase MT network. We observed thick and short MTs in the mitotic spindle and atypically organized phragmoplasts which agreed with the results of Smertenko for N. tabacum cells. It is interesting that incubation at 38 °C showed a time-dependent effect in the mitotic index and resulted in a tenfold reduction after 2 h (from 41. 70 % at control material to 4.70 % at 38 °C—2 h). After 1 h, cells with preprophase bands were not found, and after 2 h, the phragmoplast appeared to lose its normal shape. The reduction of the mitotic index suggests that cell division procedure is blocked (see Table 2), starting at a preprophase stage by suppression of the mitotic entry, while cytokinesis is also arrested, resulting in the accumulation of telophase-cytokinetic cells. Similar observations were made by Smertenko et al. (1997). The possible cytoskeletal target of heat shock, according to Chan et al. (1999), might be MT subunits and/or MAPs (microtubule-associated proteins). According to Nick (2013), Bmicrotubules are proposed as elements of sensory hub that decodes stress-related signal signatures^. At low temperatures, the plant thermometer seems to be involved in membrane fluidity and MT disassembly (Nick 2013). It is proposed that disassembly of MTs represents one of the mechanisms to maintain a more fluid plasma membrane which may be important for its functioning (Ciamporova and Mistrik 1993). Although it is not clear how cytoskeletal changes are related to signaling events, MT disassembly is likely due to a chemical reaction of tubulin subunits in response to high temperatures (Muller et al. 2007). Considering the above, according to our observations, we could assume that MT cytoskeleton in cells of the seagrass

exhibits variable sensitivity to heat stress, and in this way, it seems to participate to a heat stress response. Abiotic changes are likely to elicit cellular stress response (CSR), a universally conserved mechanism to protect cells from the potential damage that biological stressors may cause. The CSR can temporarily increase the tolerance to the stressor and remove already damaged cells through apoptosis (Kultz 2005). Although responses to elevated temperature have been studied in detail in terrestrial plants, it is difficult to predict from these models how seagrasses will respond to it. In the present study, we observed potential cellular damage and cytoskeleton disturbances in C. nodosa that are an indication of CSR. The ability of marine macro-autotrophs and angiosperms to rapidly upregulate CSR in elevated temperatures is critical for species subjected to stress on a daily basis, either in the intertidal zone or sub-tidally (Murthy and Sharma 1989; Collen and Davison 1999; Dummermuth et al. 2003; Dring 2005; Lesser 2006; Ross and Van Alstyne 2007). In order to understand the exact effects of this mechanism at a cellular level, further studies have to be done. Acknowledgments The authors wish to express their thanks to Prof. Konstantinos Fasseas (Agricultural University of Athens) for the access to TEM facilities. This research has been co-financed by the European Union (European Social Fund—ESF) and National funds through the Operational Program BEducation and Lifelong Learning^ of the National Strategic Reference Framework (NSRF)—Research Funding Program: THALES (MIS: 375425) and by the University of Athens (program BKapodistrias^). Conflict of interest The authors declare that they have no conflict of interest.

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