Aquatic Botany 101 (2012) 28–33
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Meristematic activity of Mediterranean seagrass (Posidonia oceanica) shoots Neus Garcias-Bonet a,∗ , Carlos M. Duarte a,b , Núria Marbà a a b
Department of Global Change Research, IMEDEA (CSIC-UIB), Institut Mediterrani d’Estudis Avanc¸ats, Miquel Marquès 21, 07190 Esporles, Spain The UWA Oceans Institute, University of Western Australia, 35 Stirling Highway, Crawley 6009, Australia
a r t i c l e
i n f o
Article history: Received 29 March 2011 Received in revised form 23 March 2012 Accepted 30 March 2012 Available online 6 April 2012 Keywords: Seagrass Posidonia oceanica Meristems Meristematic activity Flow cytometry
a b s t r a c t Shoot meristematic activity of Mediterranean seagrass Posidonia oceanica has been assessed in eleven different meadows located around Balearic Islands (Spanish Mediterranean). Moreover, in six of them, the meristematic activity has been determined hourly (or every 2 or 3 h, depending on the meadow) for at least 24 h, with the aim of detecting a possible circadian rhythm in the % of dividing nuclei. Meristematic activity was inferred by applying flow cytometry techniques combined with DNA labeling to determine the percentage of nuclei in each phase of the cell cycle (i.e. G0 + G1, S, G2). The percentage of nuclei in G2 phase of the cell cycle reflects the percentage of nuclei that are dividing in a specific moment. In the meristems of vertical shoots of P. oceanica the percentage of nuclei in G2 phase was on average 7 ± 0.11%, and it ranged from 2% to 12% across the meadows studied. The average percentage of nuclei in the G2 phase in P. oceanica meristems is lower than reported for other plants. No circadian rhythms were detected in meristems of P. oceanica. The variability observed for meristematic activity across meadows suggests that it could be used as indicator of seagrass stress and, thus, to assess impacts to meristems before population declines could be observed. © 2012 Elsevier B.V. All rights reserved.
1. Introduction Meristems are multicellular plant tissues formed by undifferentiated cells in continuous division to produce new cells that will develop and differentiate into new tissues and organs. Therefore, meristems are the major sites of cell divisions responsible for plant growth. Meristems are also responsible for forming primordia that will develop into new leaves and flowers, and other plant tissues such as rhizomes or stolons and, hence, they are responsible of the vegetative spread of clonal plants (Tomlinson, 1974). In a meristem, different cell layers and zones can be distinguished with cells being different in size and shape. Also, there is heterogeneity of growth rates within the apex and heterogeneity in cell cycle duration can be detected in the cells of a single meristem (Lyndon, 1998). Meristematic activity, assessed as temporal changes in the number of cells per apex, the rate of cell division (Lyndon, 1998) or/and the cell cycle duration, has been widely studied in terrestrial angiosperms (Lyndon, 1998). On the contrary, the dynamics of meristems of seagrasses, i.e. marine angiosperms is largely unknown (but see
∗ Corresponding author. Tel.: +34 971610896; fax: +34 971611761. E-mail addresses:
[email protected] (N. Garcias-Bonet),
[email protected] (C.M. Duarte),
[email protected] (N. Marbà). 0304-3770/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.aquabot.2012.03.013
Garcias-Bonet et al., 2008) despite its central role in clonal seagrass growth (Tomlinson, 1974). Posidonia oceanica is a Mediterranean endemic seagrass and the dominant species, covering an extension of 50 000 km2 (Bethoux and Copin-Montegut, 1986), along the Mediterranean Sea. Like all seagrasses, P. oceanica is a clonal plant that occupies the space largely by growing vegetatively repeating the structural unit called ramet. In P. oceanica, shoots, which have basal meristems, emerge at the apexes of the rhizomes. Hence, in this species, shoot meristems are responsible for both shoot and rhizome elongation (Tomlinson, 1974). P. oceanica ranks among the largest and the slowest-growing seagrass species (Duarte, 1991). Despite the slow growth rate of P. oceanica rhizomes (1–6 cm yr−1 , Marbà and Duarte, 1998), the resulting clones may spread across several kilometers (Diaz-Almela et al., 2007; Arnaud-Haond et al., 2012) and, thus, achieve millenary ages, while the ramets may live for several decades (e.g. Marbà et al., 1996). P. oceanica plays an important role in ecological processes, such as shoreline protection, sediment retention and carbon burial, and enhancement of coastal biodiversity (Hemminga and Duarte, 2001). P. oceanica is very sensitive to coastal deterioration and is suffering a widespread decline in the Mediterranean (Marbà et al., 2005). Detection of P. oceanica decline from thinning of the meadows identifies the problem at a time when it may be difficult to revert, and because the dynamics of P. oceanica shoot populations is largely the outcome of the dynamics of its meristems, P. oceanica meristematic activity may allow a rapid assessment of the health
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Table 1 Summary of Posidonia oceanica meadows sampled in this survey, indicating decimal coordinates. The length and the frequency of sampling are shown for each station. Station
Coordinates
Cabrera Island Es Port C Es Port Fe Es Castell St Maria
39.14◦ N; 2.93◦ E 39.14◦ N; 2.93◦ E 39.15◦ N; 2.93◦ E 39.15◦ N; 2.94◦ E
Mallorca Island Magalluf Illetes Portals Vells Pollenc¸a Cala Millor Porto Colom Sa Colonia Cap Salines
39.50◦ N; 2.54◦ E 39.53◦ N; 2.59◦ E 39.48◦ N; 2.52◦ E 39.89◦ N; 3.09◦ E 39.60◦ N; 3.39◦ E 39.41◦ N; 3.27◦ E 39.31◦ N; 2.99◦ E 39.28◦ N; 3.04◦ E
Depth (m) 17 17 5 7 6 9 6 4 7 6.5 4 6
status of the plants. Indeed, there is evidence of a marked decrease in the percentage of dividing cells in shoot meristems of P. oceanica when sediments conditions become adverse to plant survival, such as accumulation of sulfides (Garcias-Bonet et al., 2008). Eukaryotic cell division cycles comprise the interphase and the mitosis phase. The interphase consists of a G1 phase (post-mitotic phase), where the cell grows; an S phase (DNA synthetic phase), where the DNA is replicated; and a G2 phase, where the cell has doubled the DNA content and the nuclear proteins and is preparing to enter the mitotic phase. There are many regulation points across the cell division cycle. In this regulation, protein-kinases are involved regulating the transition from the G1 phase to the S phase and from the G2 phase to the mitosis (Huntley and Murray, 1999; Dewitte and Murray, 2003). The percentage of nuclei that have doubled their DNA content provides information on the percentage of meristematic cells that are dividing at a specific time (Chen and Setter, 2003). However, this measure does not provide information on cell division rates (Erickson and Sax, 1956). A reduction in the percentage of dividing cells would be expected to result in decreased shoot growth (Wang et al., 2000), which may propagate to the population level to affect net population growth. Cell division parameters have been estimated based on: (1) the observation of individual cells and determination of the time between two successive division events; (2) using cell cycle blocking methods, arresting the progression of the cycle at any point of the cell cycle and determining the rate at which this fraction of cells increases; and (3) using cell suspension cultures and determining the rate at which the cell number increases over time, in most of the cases using synchronized cell cultures (Fiorani and Beemster, 2006). These methods combine labeling techniques with microscopy or flow cytometry. Another metric informing of the cell division parameters is the mitotic index, which is the percentage of the total number of cells in a sample that are in mitosis (Baskin, 2000). The mitotic activity of plant meristems can also be assessed by measuring the percentage of cells that are dividing, as demonstrated for intact tissues of Nicotiana tabacum (Galbraith et al., 1983). Studies on macroalgae Ulva pseudocurvata (Chlorophyceae) and Porphyra yezoensis (Rhodophyceae) detected a circadian rhythm in cell division rates (Oohusa, 1980; Titlyanov et al., 1996). Flow cytometry has been extensively used in research with animal cells and bacteria but not so much with plant cells, because of the difficulties they present. However, flow cytometry techniques have been applied in plants to analyze the DNA content of cells or isolated nuclei suspensions (Galbraith et al., 1983; Le Gall et al., 1993; Koce et al., 2003), to elucidate the duration of the cell cycle in cell cultures of Solanum aviculare (Yanpaisan et al., 1998), to determine the rate of cell division in phytoplankton (Mann and Chisholm, 2000; Sosik et al., 2003; Agawin and Agusti, 2005) and to examine the response
Sampling date
Sampling length
Sampling frequency
July 2004 July 2004 July 2004 July 2004
30 h 30 h 1 event 1 event
1–2 h 1–2 h – –
July 2004 July 2004 June 2004 October 2005 August 2004 June 2004 July 2004 May 2006
24 h 24 h 48 h 22 h 1 event 1 event 1 event 1 event
1h 1h 3h 1h – – – –
of cell division of P. oceanica meristems to sediment deterioration due to macroalgae invasions (Garcias-Bonet et al., 2008). The goal of this study is to determine the variability of P. oceanica meristematic activity by quantifying the percentage of nuclei in each phase of the cell cycle in meristems of vertical shoots collected in 11 different meadows along the Balearic Islands. Moreover, we examine the diel fluctuations of the percentage of nuclei in each phase of the cell cycle in vertical shoot meristems in 6 (out of the total 11) meadows sampled to elucidate if the cellular divisions are synchronized and/or adjusted to a circadian rhythm. 2. Methodology 2.1. Sampling approach We collected P. oceanica vertical shoots from 11 meadows located along the Balearic Sea (8 off the coasts of Mallorca Island and 3 off those of Cabrera Island) growing at depths between 4 and 17 m (Table 1). In one of these meadows, Es Port (Cabrera Island), we collected shoots to examine the meristematic activity from plots with sediments that were fertilized with iron for two years (station Es Port Fe, Marbà et al., 2007) and unfertilized plots (station Es Port C). The sediments of all the meadows studied, as those in most of the Balearic Sea, are biogenic, carbonate rich and iron deficient (Holmer et al., 2003). The biogeochemical condition of these Balearic sediments renders seagrass meadows iron limited and highly vulnerable to organic inputs (Marbà et al., 2008). Cabrera Island is the largest island of Cabrera Archipelago, declared a Terrestrial-Maritime National Park since 1991. In Cabrera Island, the access to the sites Santa Maria and Es Castell is restricted to visitors whereas Es Port is open to visitors, with 50 permanent moorings. In Mallorca Island, the meadow at Ses Salines is free of anthropogenic pressures, while the meadows at Magalluf, Illetes, Portals Vells, Pollenc¸a, Cala Millor, Porto Colom, Sa Colònia de Sant Jordi (referred to as Sa Colonia) are adjacent to tourist destinations. The meadows were sampled during the growing season of P. oceanica (Marbà et al., 1996; Barrón and Duarte, 2009; Table 1). One hundred healthy shoots were collected randomly by SCUBA divers in each station and maintained in aerated aquaria with seawater from the same location. The shoots harvested were 3–4 years old, close to the half-life of shoot population in the meadows studied (Marbà et al., 2005). The samples were immediately transported to the laboratory and the meristematic activity was measured one day after sample collection. 2.2. Meristematic activity In five of these eleven sampled P. oceanica meadows, the activity of vertical shoot meristems was estimated hourly (or every 2–3 h,
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Table 2 Average and standard error (SE) of the percentages of nuclei in each phase of the cell cycle (i.e. G1, S, G2) for all the Posidonia oceanica meadows tested in this study. The time of the day when the analysis was performed is detailed. n: number of shoot meristems analyzed. Station
Time of day Number of shoot meristems analyzed
Average % of nuclei SE of % of nuclei Average % of nuclei SE of % of nuclei in in G1 phase in G1 phase in S phase S phase
Average % of nuclei SE of % of nuclei in G2 phase in G2 phase
Cabrera Island 12:00 Es port C 12:00 Es Port Fe 11:00 Es Castell 11:00 St Maria
3 3 5 5
85.96 87.09 78.87 83.39
1.06 1.06 1.99 1.36
5.55 5.72 14.81 9.35
0.69 1.47 2.12 1.80
8.50 7.19 6.32 7.25
1.18 0.46 0.74 0.73
Mallorca Island Magalluf 11:00 Illetes 11:00 Portals Vells 11:00 11:00 Pollenc¸a Cala Millor 11:00 Porto Colom 11:00 Sa Colonia 11:00 Cap Salines 12:00
3 3 5 6 4 4 5 6
77.68 76.61 82.53 86.57 89.73 91.16 88.25 73.67
2.75 2.95 3.51 1.09 1.82 1.12 1.15 2.69
16.85 17.47 11.81 5.66 4.86 4.82 6.78 21.03
3.24 4.65 4.68 0.90 0.93 0.64 1.87 3.16
5.46 5.92 5.75 7.78 5.41 4.03 4.97 5.30
0.52 1.84 1.19 0.51 0.89 0.71 1.09 1.52
depending on the meadow) in experiments of duration ranging from 22 to 48 h (Table 1). In the other six P. oceanica meadows, the activity of rhizome meristems was estimated only once, always between 11:00 and 12:00 am (Table 2). The meristematic activity was calculated by quantifying the percentage of nuclei in each phase of the cell cycle (i.e. G0 + G1, S, G2). The base of the leaves of vertical shoots, containing the meristematic zone, was dissected and the nuclei were isolated using a Partec® extraction kit and stained with propidium iodide (PI) for 1 h in darkness at 4 ◦ C. The number of nuclei in each phase of the cell cycle (i.e. G0 + G1, S, G2) was quantified using a Beckton–Dickinson flow cytometer equipped with an argon-ion laser measuring the red fluorescence emitted by the PI. For each meristem, ten thousand nuclei were analyzed (Fig. 1). The histograms of nuclei fluorescence obtained from the flow cytometer were analyzed using cell cycle analysis software (ModFit, Verity Software House), which provided the percentage of nuclei in G1, S, G2 phases of each meristem sampled (e.g. Sandoval et al., 2003) (Fig. 1).
2.3. Statistical analysis We assessed the variability of percentages of nuclei in each phase of the cell cycle among stations and among sampling times using ANOVA. Differences among means were evaluated using Tukey’s post hoc test.
3. Results Most of the nuclei observed were in the G1 phase whereas those in the G2 phase accounted for the smallest fraction of the nuclei populations (Table 3 and Fig. 2). A distinct temporal variability in the percentage of nuclei in the different phases was apparent (Fig. 2). Over diel or longer time scales, the percentage of nuclei in the G2 phase of the cell cycle varied between 1.5 fold (Es Port C) and 4.6 fold (Portals Vells, Table 3), the percentage of nuclei in G1 phase between 1.1 fold (Es Port Fe) and 1.2 fold (Magalluf) and that of nuclei in S phase between 2.8 fold (Portals Vells) and 4.2 fold (Es Port Fe, Table 3). However, minimum and maximum diel values of % of nuclei in G1 and G2 phases were only significantly different (ANOVA test, p < 0.0001) in shoots growing at Pollenc¸a, and those of the percentage of nuclei in S phase were only significantly different (ANOVA test, p < 0.0001) in the shoots growing at Es Port Fe and Pollenc¸a. The diel fluctuations in meristematic activity observed in the shoots of P. oceanica did not exhibit a circadian rhythm (Fig. 2). Moreover, temporal trends in cell division of P. oceanica meristems differed across meadows (Table 3 and Fig. 2).
The meristematic activity in P. oceanica shoots (i.e. percentage of nuclei in each phase of the cell cycle) at noon varied substantially across the 11 meadows studied (Table 2). At this time of the day, most meristematic nuclei in the shoots of all stations were in the phase G1 of the cell cycle accounting for 74–91% of the total nuclei population (Table 2). The percentage of nuclei in the phase S ranged between 5% and 21%, and between 4% and 9% of nuclei were in the phase G2 of the cell cycle (Table 2). 4. Discussion The percentage of dividing cells in P. oceanica meristems, quantified as the percentage of cells in the G2 phase of the cell cycle, is 7 ± 0.11% on average for all the meadows studied here. This value suggests that cell division rates in P. oceanica are low when compared with those reported for other plant species, encompassing terrestrial and aquatic plants and macroalgae (Table 4). In most studies assessing cell division of vegetation tissues, the rate of cell division is reported as mitotic index (sum of cells in prophase, metaphase, anaphase and telophase as a percentage of the total), and only few studies provide data on the percentage of cells in each phase of the cell cycle. The mitotic indexes reported in the literature indicate that they vary between 1 and 30% in other plant tissues (Table 4). Less data on the percentage of cells in each phase of the cell cycle, specifically for the G2 phase, are available, and range from 22 to 56% (Table 4). The low percentage of dividing cells observed in P. oceanica meristems is consistent with the very slow growth of this species, which ranks among the slowest-growing plants among the seagrass flora (Duarte, 1991). The low fraction of dividing cells observed in this study could also reflect an underestimation of the amount of dividing cells due to the methodology used. During nuclei extraction process we are loosing all the nuclei in the mitosis phase, because the nuclear membrane disappears to allow the separation of the chromosomes, which makes it impossible to recover those nuclei for flow cytometry analysis. However, this underestimation should be small since during the temporal experiments no clear peaks in the G2 phase, i.e. nuclear phase prior to the mitosis phase, were detected (Fig. 2). This study quantifies meristematic activity during the growing season of P. oceanica, and thus when cell division may be the fastest in the year. This, however, should be tested by seasonal studies. Similarly, it would be interesting to analyze whether the percentage of dividing cells in the meristems of vertical shoots varies with shoot age, as rhizome growth appears to decline with shoot age (Tomasello et al., 2007) and there is evidence of shoot age coupling to flowering events (Balestri and Vallerini, 2003).
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Fig. 1. Flow cytometry plots: red fluorescence (FL3-H) vs side scatter (SSC-H) showing the nuclei detected in each sample (in red), and the corresponding histogram of nuclei fluorescence (FL3-A) with the cell cycle phases analysis (percentage of nuclei in each phase of the cell cycle: G1, S, G2) by ModFit software for three different sampling hours at Illetes station. Three different situations are shown: high (Illetes 00:00 h), intermediate (Illetes 10:00 h) and low (Illetes 07:00 h) percentages of nuclei in G2 phase of the cell cycle.
The variability of the percentage of nuclei in the G2 phase observed across P. oceanica meadows may reflect different local ambient conditions for seagrass growth and the extent of synchronization of cell cycles among populations. The largest percentages
of nuclei in the G2 phase were observed in P. oceanica shoot meristems growing at the relatively pristine meadows from Cabrera Island (Table 2). The percentage of nuclei in the G2 phase observed in seagrasses from Cabrera rank within the range reported for
Table 3 Average (AVG), standard error (SE), coefficient of variation (CV), maximum and minimum values and the time of the day when they were observed (MAX, MIN, Sampling hour) of the percentages of nuclei in each phase of the cell cycle (i.e. G1, S, G2) for the Posidonia oceanica meadows that have been analyzed for 24 h or 48 h. Stations with average % of nuclei in G1, S or G2 phases statistically (Tukey post hoc test) similar are indicated with the same letter (i.e. A, B, C, D). Cabrera Island
Mallorca Island
Es port C (n = 72)
Es Port Fe (n = 72)
Magalluf (n = 71)
Illetes (n = 72)
Portals Vells (n = 84)
Pollenc¸a (n = 126)
% of nuclei in G1 phase AVG SE CV MAX Sampling hour MIN Sampling hour
86.09A 0.36 2826.35 88.29 14:00 78.09 15:00
85.8A 0.27 3945.05 87.96 08:00 80.16 15:00
81.02B 0.59 1636.44 86.05 03:00 71.18 12:00
80.51B,C 0.63 1526.95 85.91 07:00 71.19 16:00
78.67C 0.8 1083.08 83.38 23:00 70.39 08:00
81.75B 0.43 1696.68 86.56 11:00 74.91 03:00
% of nuclei in S phase AVG SE CV MAX Sampling hour MIN Sampling hour
7.02B 0.39 212.39 16.43 15:00 4.93 02:00
6.9B 0.29 287.99 13.26 15:00 3.19 08:00
13.78A 0.71 232.86 22.65 12:00 7.86 00:00
13.45A 0.74 216.44 24.91 16:00 8.12 01:00
15.56A 0.97 176.42 27.77 08:00 9.85 14:00
8.50B 0.42 179.09 14.66 01:00 4.45 15:00
% of nuclei in G2 phase AVG SE CV MAX Sampling hour MIN Sampling hour
6.89B,C 0.17 490.13 8.49 12:00 5.47 18:00
7.29B 0.15 567.26 8.85 08:00 5.59 16:00
5.2D 0.25 250.79 6.84 10:00 2.37 07:00
6.04C,D 0.22 325.41 7.71 21:00 3.89 16:00
5.67D 0.31 199.65 8.44 14:00 1.83 08:00
9.76A 0.2 425.15 12.16 04:00 7.71 19:00
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Fig. 2. Percentage of nuclei in each phase of the cell cycle: G1 (white bars), S (gray bars) and G2 (black bars) in meristems of Posidonia oceanica vertical shoots measured at different frequencies for at least 24 h. Shaded areas correspond to night times.
P. oceanica growing in sediments with low concentration of sulfides (Garcias-Bonet et al., 2008), a toxic compound for plant cells (Raven and Scrimgeour, 1997). P. oceanica has been demonstrated to be highly sensitive to sediment sulfide concentrations as reflected by a more rapid decline in P. oceanica shoot density when sediment sulfide concentration exceeds 10 M (Calleja et al., 2007). At high sediment sulfide concentrations there is evidence that sulfides intrude into seagrass tissues (e.g. Frederiksen et al., 2007) and may damage plant meristems (Greve et al., 2003). Indeed, a sharp decrease in meristematic activity of P. oceanica shoots when sediment sulfide concentration increases, due to enhanced sediment sulfate reduction rates of invasive Caulerpa species, has been reported (Garcias-Bonet et al., 2008), and suggests that meristematic activity below 5% of nuclei in the G2 phase could reflect sulfide intrusion. According to Garcias-Bonet et al. (2008), the percentage of nuclei in G2 phase observed in our study in P. oceanica shoots growing at Porto Colom and Sa Colonia (Table 2) could reflect sulfide stress and, hence, sediment conditions compromising seagrass
growth and survival. These are indeed ecosystems that receive substantial organic inputs from dense tourist developments on shore. Cell division in the meristems of P. oceanica shoots does not exhibit a circadian rhythm nor does it display consistent temporal fluctuations across meadows. The absence of consistent rhythms in the percentage of meristematic cell division can be explained because meristems of vascular plants present many areas of division that have different rates and different lengths in the cell cycle (Lyndon, 1998), which prevents detection of synchronization of cell division. The lack of temporal rhythms in cell division allows quantification of the average of the percentage of cells that are dividing, assessed as the % of nuclei in the G2 phase, without requiring following cell divisions across time. Further studies are needed to understand how seagrass meristems are regulated and how these percentages of dividing cells are changing across the year, as leaf growth and rhizome elongation rates in temperate seagrass, as P. oceanica and others marine angiosperms, exhibit marked seasonality.
Table 4 Values of dividing cells in plant tissues by applying different approaches found in literature. Plant species
Tissue analyzed
Approach
Value (%)
Reference
Lemna minor Arabidopsis sp. Dactylis glomerata Dactylis glomerata Arabidopsis thaliana Vicia faba Stenocereus gummosus Ferocactus peninsulae Pachycereus pringlei Nicotiana tabacum Nicotiana tabacum Nicotiana tabacum Nicotiana tabacum Solanum aviculare Vicia faba Posidonia oceanica
Root tips Seedling cotyledons Shoot meristems Roots Shoot apical meristem Root tips Root meristem Root meristem Root meristem Leaf primordia Leaf Root Leaf Cell suspension Root tips Shoot meristems
Mitotic index Mitotic index Mitotic index Mitotic index Mitotic index Mitotic index Mitotic index Mitotic index Mitotic index Metaphase index Nuclei in G2 phase by flow cytometry Nuclei in G2 phase by flow cytometry Nuclei in G2 phase by flow cytometry Cells in G2 phase by flow cytometry Cells in G2 phase by flow cytometry Nuclei in G2 phase by flow cytometry
7.1–7.7 20–30 7.3–7.8 5.2–6.9 1 9.1 4.8 8.8 6.9 5 22.3 55.9 27 40 26.5 6.99
Samardakiewicz and Wozny (2005) Stoynova-Bakalova et al. (2004) Kinsman et al. (1997) Kinsman et al. (1997) Jacqmard et al. (2003) Dolezel et al. (1992) Dubrovsky et al. (1998) Dubrovsky et al. (1998) Dubrovsky et al. (1998) Cockcroft et al. (2000) Galbraith et al. (1983) Galbraith et al. (1983) Chen et al. (2001) Yanpaisan et al. (1998) Dolezel et al. (1992) This study
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Acknowledgements This work was funded by the Fundación BBVA (project “Praderas”), by CSIC (project “Desarrollo de nuevas técnicas de detección temprana de esters en angiospermas marinas: actividad meristemática”) and by the Spanish Ministry of Science and Innovation (project “MEDEICG” (CTM2009-07013)). We thank L. Navarro, O. Olivares and Pepe for initial discussions on plant meristematic activity, S. Agustí for advise on Flow Cytometry techniques, and Regino Martínez, Rocío Santiago and María Sánchez for field assistance. N. Garcias-Bonet was supported by a PhD grant from the Government of Balearic Islands (reference FPI04 43126005Q). References Agawin, N.S.R., Agusti, S., 2005. Prochlorococcus and Synechococcus cells in the central Atlantic Ocean: distribution, growth and mortality (grazing) rates. Vie et Milieu 55, 165–175. Arnaud-Haond, S., Duarte, C.M., Diaz-Almela, E., Marbà, N., Sintes, T., Serrão, E.A., 2012. Implication of extreme life span in clonal organisms: millenary clones in meadows of the threatened seagrass Posidonia oceanica. PLoS One 7 (2), e30454. Balestri, E., Vallerini, F., 2003. Interannual variability in flowering of Posidonia oceanica in the North-Western Mediterranean Sea, and relationships among shoot age and flowering. Botanica Marina 46, 525–530. Barrón, C., Duarte, C.M., 2009. Dissolved organic matter release in Posidonia oceanica meadow. Marine Ecology Progress Series 374, 75–84. Baskin, T.I., 2000. On the constancy of cell division rate in the root meristem. Plant Molecular Biology 43, 545–554. Bethoux, J.P., Copin-Montegut, G., 1986. Biological fixation of atmospheric nitrogen in the Mediterranean-Sea. Limnology and Oceanography 31, 1353–1358. Calleja, M., Marbà, N., Duarte, C.M., 2007. The relationship between seagrass (Posidonia oceanica) decline and porewater sulfide pools in carbonate sediments. Estuarine, Coastal and Shelf Science 73, 583–588. Chen, J.G., Shimomura, S., Sitbon, F., Sandberg, G., Jones, A.M., 2001. The role of auxin-binding protein 1 in the expansion of tobacco leaf cells. Plant Journal 28, 607–617. Chen, C.T., Setter, T.L., 2003. Response of potato tuber cell division and growth to shade and elevated CO2 . Annals of Botany 91, 373–381. Cockcroft, C.E., den Boer, B.G.W., Healy, J.M.S., Murray, J.A.H., 2000. Cyclin D control of growth rate in plants. Nature 405, 575–579. Dewitte, W., Murray, J.A.H., 2003. The plant cell cycle. Annual Review of Plant Biology 54, 235–264. Diaz-Almela, E., Arnaud-Haond, S., Vliet, M.S., Álvarez, E., Marbà, N., Duarte, C.M., Serrao, E.A., 2007. Feed-backs between genetic structure and perturbationdriven decline in seagrass (Posidonia oceanica) meadows. Conservation Genetics 8, 1377–1391. Dolezel, J., Cihalikova, J., Lucretti, S., 1992. A high-yield procedure for isolation of metaphase chromosomes from root-tips of Vicia faba L. Planta 188, 93–98. Duarte, C.M., 1991. Allometric scaling of seagrass form and productivity. Marine Ecology Progress Series 77, 289–300. Dubrovsky, J.G., Contreras-Burciaga, L., Ivanov, V.B., 1998. Cell cycle duration in the root meristem of Sonoran Desert Cactaceae as estimated by cell-flow and rateof-cell-production methods. Annals of Botany 81, 619–624. Erickson, R.O., Sax, K.B., 1956. Rates of cell division and cell elongation in the growth of the primary root of Zea mays. Proceedings of the American Philosophical Society 100, 499–514. Fiorani, F., Beemster, G.T.S., 2006. Quantitative analyses of cell division in plants. Plant Molecular Biology 60, 963–979. Frederiksen, M.S., Holmer, M., Diaz-Almela, E., Marbà, N., Duarte, C.M., 2007. Sulfide invasion in the seagrass Posidonia oceanica at Mediterranean fish farms: assessment using stable sulfur isotopes. Marine Ecology Progress Series 345, 93–104. Galbraith, D.W., Harkins, K.R., Maddox, J.M., Ayres, N.M., Sharma, D.P., Firoozabady, E., 1983. Rapid flow cytometric analysis of the cell-cycle in intact plant-tissues. Science 220, 1049–1051. Garcias-Bonet, N., Marbà, N., Holmer, M., Duarte, C.M., 2008. Effects of sediment sulfides on seagrass Posidonia oceanica meristematic activity. Marine Ecology Progress Series 372, 1–6.
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