Heat shock effects and population survival in the polar dinoflagellate Polarella glacialis

Journal of Experimental Marine Biology and Ecology 438 (2012) 100–108

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Heat shock effects and population survival in the polar dinoflagellate Polarella glacialis☆ Shuxian Zheng a, b, Guizhong Wang a, Senjie Lin b,⁎ a b

College of Oceanography and Environmental Science, Xiamen University, Xiamen 361005, China Department of Marine Sciences, University of Connecticut, Groton 06340, USA

a r t i c l e

i n f o

Article history: Received 14 May 2012 Received in revised form 4 September 2012 Accepted 5 September 2012 Available online 1 November 2012 Keywords: Cell cycle Growth PCNA Global warming Rubisco

a b s t r a c t The rare bipolar dinoflagellate Polarella glacialis forms blooms in the Antarctic sea ice and the Arctic water column every year. Recently, P. glacialis-like genotypes were found in temperate waters. Here, we investigated how P. glacialis would respond if it were transported from polar to temperate waters by shifting cultures of P. glacialis (strain CCMP2088) from 4 °C to 10° and 15 °C. After a 4-day lag phase, the cultures remaining at 4 °C grew exponentially at 0.10 ± 0.17 d−1 for 22 days before entering early stationary phase. Consistent to this growth pattern, flow cytometric analysis on samples collected at the same time showed higher percentages of S-cells on day 9 and day 12 than day 33 and day 37. Western blot analysis of the CO2-fixing enzyme Rubisco 1 (typically 55 kDa) and the cell cycle-related protein PCNA2 (typically 36 kDa) revealed active expression of both proteins, and higher expression in exponential growth than in early stationary phase. In comparison, cultures shifted to 10° and 15 °C first experienced a 4-day lag phase plus a 13–15 day declining phase (growth rates were − 0.21 ± 0.10 d −1 and − 0.23 ± 0.08 d −1 respectively), then a steady cell density period (growth rates were 0.05 ± 0.12 d −1 and 0.01 ± 0.15 d −1 respectively). Strikingly, the percentages of the G2/M phase (%G2M) in the 10 °C-cultures remained at high levels and G1 population at a low level, suggesting that a subpopulation of the cells might still be actively dividing. %G2M of the 15 °C-culture remained to be the lowest among the three temperature treatments (P b 0.05), indicative of least active cell division. Molecular apparatus of photosynthesis and cell division cycle appeared to be substantially damaged as the culture stayed longer at the elevated temperatures, as evidenced by the progressive degradation of Rubisco and aggregation of PCNA. Our results show that although extreme heat shock as exerted by the temperature rise from 4 °C to 10° or 15 °C caused impairment of molecular engines of photosynthesis and cell division cycle and extensive population decline in P. glacialis, this polar dinoflagellate seems to be able to survive the extreme temperature insults and potentially can be spread by humans to temperate regions. © 2012 Elsevier B.V. All rights reserved.

1. Introduction Dinoflagellates are generally believed to prefer warm temperatures and presumably may do better in face of temperature increases. For example, Prorocentrum donghaiense was able to grow at temperatures ranging from 10 to 27 °C, and achieved its maximum specific growth rate of 0.77 d−1 at 27 °C (Xu et al., 2010). For P. minimum, growth rates increased from 0.25 d−1 at 4 °C to 0.98 d−1 at 20 °C, with a Q10 of 2.3 (Lomas and Glibert, 1999a, 1999b). Recently, a study showed that phytoplankton exposed to temperature rise at nuclear power plant thermal effluent tipped toward dinoflagellates both in terms of species number and

☆ Most of this work was conducted in the Department of Marine Sciences of the University of Connecticut when Shuxian Zheng visited as a visiting graduate student. ⁎ Corresponding author. Tel.: +1 860 405 9168; fax: +1 860 405 9153. E-mail address: [email protected] (S. Lin). 1 Rubisco, the abbreviation for ribulose-1,5-bisphosphate carboxylase oxygenase. 2 PCNA, the abbreviation for proliferating cell nuclear antigen. 0022-0981/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.jembe.2012.09.003

cell abundance (Li et al., 2011). Some dinoflagellates were found to produce heat-shock proteins to stabilize protein secondary structure in response to thermal stress (Alexandrov, 1994). Heat shock protein 70 was induced in Alexandrium tamarense when subjected to a 10 °C jump from its acclimated temperature 20 °C (Kobiyama et al., 2010). Yet temperatures higher than the organisms' limit can cause denaturation of proteins and damage to enzymes and membranes (Redeke, 1933), resulting in decreased photosynthesis, making photosynthesis a sensitive indicator of thermal stress in plants (Berry and Björkman, 1980). Elevated temperature, even by as little as 2–3 °C, can cause increased metabolic activity and growth of some species and create stress to others, thus changing species composition and competition (Beardall and Raven, 2004). Dinoflagellate Polarella glacialis is a small, nonthecate species representing a rarely documented bipolar organism. Along with Symbiodinium, the reef coral endosymionts in tropical and subtropical seas, P. glacialis is affiliated in the order of Suessiales (Montresor et al., 2003a). It was first recorded from the upper land-fast sea ice in

S. Zheng et al. / Journal of Experimental Marine Biology and Ecology 438 (2012) 100–108

Antarctic McMurdo Sound (Stoecker et al., 1997) and in the water column in the Canadian Arctic (Montresor et al., 2003b). This extreme halotolerant species was found to bloom, with no obvious harmful effect to the ecosystem, in sea-ice brine channels during the spring, when grazing pressure is relatively low, and subsequently encysts at the beginning of the austral summer, contributing a substantial fraction of the phytoflagellate biomass and primary production (Stoecker et al., 1998, 2000). More recently, a diversity of 18S rRNA and cytochrome b gene sequences (18S rDNA and cob) closely related to P. glacialis was found in temperate aquatic environments such as Long Island Sound (salinity 32) and Mirror Lake (freshwater) (Lin et al., 2009, 2010), raising a question if this species can tolerate and grow at temperate temperatures. Frequent human activities in the polar region can introduce the Antarctic and Arctic algae to warmer regions, for instance through ballast-tank waters and sediments of bulk cargo vessels (Hallegraeff and Bolch, 1992). Little physioecological and even less molecular genetic information is available for P. glacialis. In the current study, we studied the effects of temperature shock on the growth of the dinoflagellate P. glacialis by monitoring its physiological and biochemical responses to temperature rises from 4 °C to 10 and 15 °C. We examined growth rate and expression of two important genes for this alga. Rubisco is the primary CO2-fixing enzyme whereas PCNA is a key protein required for DNA replication, the hallmark of the S phase of the cell division cycle. Among typical dinoflagellates (containing peridinin as the major carotenoid accessory pigment), Rubisco has been studied only for Symbiodinium sp. (Rowan et al., 1996), Lingulodinium polyedrum (Morse et al., 1995), P. minimum (Zhang and Lin, 2003), and Heterocapsa triquetra (Patron et al., 2005). These lineages contain form II Rubisco, instead of the form I Rubisco typical of all other algae, presumably originated from bacteria through horizontal gene transfer (Morse et al., 1995). The abundance of PCNA is generally growth stage dependent and shows a cell cycle specific pattern with the highest expression in the early S phase and low in G1 and G2/M phases (Cachon, 1989; Lin, 1997; Lin and Corstjens, 2002). In dinoflagellates, PCNA abundance has rarely been studied but in one study was measured in the heterotrophic species Pfiesteria piscicida, in which it was modestly higher in the exponential growth phase than in the stationary growth phase (Zhang et al., 2006). 2. Materials and methods 2.1. Algal culture Before the experiments, the stock culture of P. glacialis strain CCMP2088 was maintained in autoclaved 0.45-μm filtered seawater (32–34‰) amended with L1 medium nutrients (Guillard and Hargraves, 1993). The vitamins were added after autoclaving of the medium. Temperature was kept at 4 °C and illumination at 70 μmol photons·m −2 s −1with cool white light bulbs under 14/10 L/D photocycle. Growth was monitored by cell count of triplicate samples every other day with a Sedgwick-Rafter counting chamber. 2.2. Temperature treatment and sample collection Experimental cultures were inoculated from the stock culture grown at 4 °C during the mid-exponential growth phase. Three groups of 500-mL cultures, with initial cell density of 5.1 × 104cells·ml−1, were put into 4°, 10° and 15 °C culture rooms at the same time, each in triplicate. The starting cell density was chosen to ensure that enough protein could be sampled for protein analysis. In the preliminary experiment, the culture with this cell density was in the middle of exponential growth. Illumination conditions and media were the same as the stock culture. A 1-mL sample was taken every other day for cell count to follow growth pattern. Samples were collected on days 9 and 12 during exponential growth phase and days 33 and 37

101

during early stationary phase (when growth became almost linear). For more in-depth cell cycle analysis, diel sampling was conducted on days 39, 37 and 35, respectively for the cultures at 4°, 10° and 15 °C. The cultures were evenly divided into 250-mL subcultures a day before the diel sampling. After over 24-hour acclimation, the cell density was monitored every 6 h for 24 h by cell counting, with 5 time points in total. At each time point, three of the 250-mL cultures were harvested for flow cytometric and western blot analyses. Division of the cultures to subcultures was to avoid disturbing the growth of cultures repetitively throughout the diel sampling period (Estrada and Berdalet, 1998). 2.3. Flow cytometry analysis For flow cytometry, each sample must contain more than 106 cells to assure enough events in each run. Cells in 250-mL samples were harvested by centrifugation at 3000g at 4 °C for 15 min; the cell pellets were washed twice with phosphate buffer saline (PBS, pH 7.40) and resuspended in 1 mL PBS (pH = 7.40). One mL 4% paraformaldehyde (PFA) was added to fix the cells at 4 °C overnight. Cells were centrifuged at 3000g for 5 min to remove PFA next day. After washing by PBS twice, samples were permeabilized by the addition of 2 mL of −20 °C 100% EtOH overnight to extract cellular pigments and stored at 4 °C. Before flow cytometry analysis, the fixed samples were washed twice with PBS (pH = 7.40) and spun down to 1 mL at 3000g for 10 min to remove the ethanol and the soluble pigments. Then, they were incubated with DAPI (diluted to 0.4 μg mL −1 in PBS) for 10 min at room temperature. Samples were kept in darkness until analysis to avoid fluorescence decay. Prior to loading the samples to the flow cytometer, samples were filtrated through 20-μm filters to prevent clogging. Analysis was performed on a BD FACS Aria™II flow cytometer with a 405 nm argon laser. Histograms of relative DNA content were analyzed using Modfit LT (Verity Software House) in order to quantify the percentage of cells at each stage (G1, S, G2/M). Each sample was measured three times (N = 3). 2.4. Western blot analysis At least 16,000 cells were collected by centrifugation at 3000g for 15 min at 4 °C. Cell pellets were resuspended in Laemmli buffer (Laemmli, 1970), and stored at −80 °C until protein extraction. The cell pellet in Laemmli buffer later was homogenized using micropestle as described in Feinstein et al. (2002). The cell slurry was boiled at 100 °C for 5 min to denature the proteins, then centrifuged at 12,000g for 5 min to obtain supernatant. Fifteen microliters of the resulting supernatant (equivalent to 5000 cells) were loaded to a gradient (4%– 20%) sodium dodecyl sulfate-polyacylamide gel (SDS–PAGE, Bio-Rad). Electrophoresis was carried out at 150 V for 130 min and the resolved proteins were transferred onto a nitrocellulose membrane (Amersham). For immunodetection, the nitrocellulose membrane was first blocked with TTBS containing 3% gelatin at room temperature for 1 h. All antibodies were diluted in TTBS (TBS with 0.1% Tween 20). After two washes in TTBS (10 min each), the protein blot was incubated with a dinoflagellate Rubisco antibody (PmiRBC1-2, an antiserum raised against the Prorocentrum minimum form II Rubisco as reported in Zhang and Lin (2003)) at a dilution of 1:1000 for 1.5 h at room temperature followed by one 15-min and three 5-min washes before incubation in horseradish peroxidase (Amersham)-linked goat anti-rabbit Ig using a dilution of 1:1000 for 1 h at room temperature (Zhang and Lin, 2003). After rinsing in TTBS one time for 15 min and 3 times for 5 min, results of Western blots were visualized on X-ray film (Fuji) with ECL Plus western blotting detection reagents (Amersham or Beyotime). The protein bands on the Western blots were scanned through GelDocTM XR170-8170 (Bio-Rad). The optical density for each band was determined using Quantity one (Bio-Rad) and expressed in arbitrary units. After stripping with the stripping buffer (Beyotime Company), the western membranes were reprobed with antibody

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raised against the PCNA from Pfiesteria piscicida (Zhang et al., 2006) by a dilution of 1:5000, following the same procedure as described above. After another stripping, the monoclonal anti-α-tubulin antibody (Sigma, St. Louis, MO) was used at a dilution of 1:1000 in TTBS as a reference for total protein loaded to each lane. 2.5. Statistical analysis One-way and Two-way ANOVA were applied to evaluate the difference among treatments. And calculation of specific growth rate (μ, d −1) was performed using the formula: μ¼ ðlnN 2 –lnN1 Þ=ðt 2 −t 1 Þ N2 and N1 were cell concentrations at times t2 and t1 (Guillard, 1973). 3. Results 3.1. Effects of temperature rises on growth patterns of P. glacialis At the beginning of the temperature treatment, all the cultures showed a 4-day lag phase (Fig. 1). For the cultures remaining at 4 °C, after the lag phase with a growth rate of −0.10 d −1, the population grew slowly from day 5 to day 26 with a mean growth rate (μ) of 0.10 ± 0.17 d −1 (mean doubling time = 9.8 days). The exponential growth lasted 22 days, then population growth slowed down. At the end of the experiment (day 49), the culture still showed a small positive growth rate (0.03 d −1) resulting the final cell density of 6.3 × 10 5 cells mL −1. Dead cells, cell agglomerate or cells sticking to the bottle surface were rarely seen under microscope by then, indicating a good condition of the culture. At 10 °C, after the lag phase the cell concentration maintained stable until day 8 with a mean growth rate of 0.01±0.00 d−1, then quickly decreased at a mean rate of −0.21±0.10 d−1 from 1.1×104 cells mL−1 on day 9 to 5.4×102 cells mL−1 on day 24 (Fig. 1). After 15-day population decline, the cultures recovered to a steady state (mean rate=0.05± 0.12 d−1). The cell concentration slightly increased from 6.5×102 cells mL−1 on day 25 to 9.3×102 cells ml−1 on day 36. For the cultures grown at 15 °C, following the lag phase the cell concentration decreased at a mean rate of −0.23 ± 0.08 d−1 from 7.4× 10 3 cells mL−1 on day 5 to 5.4× 10 2 cells mL−1 on day 18, a 13-day decrease period that occurred earlier than the 10 °C culture (Fig. 1). Subsequently, the culture recovered to a relatively stable cell concentration by a mean rate of 0.01±0.15 d−1 from day 19 to day 45. The cell density ranged from 3.2×102 cells mL−1 to 7.2×102 cell mL−1.

3.2. Cell cycle patterns in different growth stages after the temperature rises At 4 °C, the percentages of cells in G1 (%G1), S (%S) and G2+ M (%G2M) phases did not differ much from day 9 to day 12, day 33 and to day 37 (Table 1), although %G1 increased slightly and %S decreased slightly from day 9 and day 12 (the late exponential stage) to day 33 and day 37 (the early stationary stage). This pattern was consistent with the growth rate shift on day 26 described above. And the flow cytometric DNA histograms further proved the shift (Fig. 2). At 10 °C, %G1 remained the lowest among the three temperature treatments (P b 0.05) and %G2M the highest among these treatments (P b 0.05) (Table 1, Fig. 2). Both %G2M and %S increased substantially from day 9 to day 12 (Table 1) when the population was still decreasing (growth rate − 0.30 d −1 on day 12) (Fig. 1). The %G2M increased to 40.5% on day 37 (Table 1), the highest among 4 days. At 15 °C, the overall %G1 was also lower than at 4 °C (P b 0.05) and %S increased from day 12 (Table 1). %G2M on days 33 and 37 were lower than that on days 9 and 12. The shift was more evident from the DNA histograms (Fig. 2). These observations were indication of less active cell cycle progression at 15 °C. 3.3. Protein expression patterns in different growth stages after the temperature rises Western blot with Rubisco antibody showed that the culture grown at 4 °C was expressing Rubisco normally, i.e. protein of ~ 55 kDa, with high abundances (thick and dark bands in the gel) in late exponential growth and early stationery growth phases (Fig. 3). Western blot with PCNA antibody showed a protein of ~ 55 kDa in the two growth stages (Fig. 4). The abundances of PCNA normalized to α-tubulin were higher in the late exponential stage (day 9 and day 12) than in the early stationery stage (day 33 and day 37) (P b 0.05). And the maximum abundance of Rubisco when normalized to α-tubulin occurred on day 12 among the four sampling days. At 10 °C, the ~55 kDa Rubisco band was less abundant than at 4 °C (Fig. 3), and it decreased from day 9 to day 12 when the culture was declining (Fig. 1). Multiple bands appeared on day 33 when the culture was in the steady-cell-density stage, with the densest band at ~14.8 kDa (Fig. 3). Similarly, single bands of ~55 kDa PCNA protein were observed on days 9 and 12 at 10 °C (Fig. 4), with its abundance decreasing over this time period. Multiple bands were detected on day 33 with two major bands of ~115.5 kDa and ~55 kDa. The ~115.5 kDa protein was more abundant than the ~55 kDa protein (the ratio of the two >1; Table 2).

Cell density (cells.ml-1)

1000000

100000

4°°C

10000

10°°C 15°°C 1000

100 0

2

4

6

8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 44 46 48 50 52 54

Day Fig. 1. Growth of P. glacialis at 4°, 10° and 15 °C. The arrows indicate days when daily sampling was conducted and the circles indicate the days when diel sampling was carried out.

S. Zheng et al. / Journal of Experimental Marine Biology and Ecology 438 (2012) 100–108 Table 1 Percentages of cells in the cell cycle phases of three temperature cultures on days 9, 12, 33 and 37. Culturing temperature (°C)

Phases Average percentages of cells in each cell cycle phase (%)

4

G1 S G2+M G1 S G2+M G1 S G2+M

10

15

Day 9

Day 12

Day 33

Day 37

86.87 ± 0.66 7.33 ± 1.32 5.80 ± 0.75 71.23 ± 6.44 9.41 ± 3.22 19.35 ± 3.30 83.79 ± 4.95 9.78 ± 2.37 6.43 ± 2.80

85.62 ± 2.89 6.82 ± 1.60 7.55 ± 1.37 44.37 ± 7.18 30.79 ± 14.26 24.84 ± 12.96 80.43 ± 3.52 12.97 ± 1.66 6.61 ± 1.84

87.71 ± 0.54 5.06 ± 0.80 7.23 ± 0.45 58.94 ± 1.35 19.96 ± 6.44 21.10 ± 7.53 76.74 ± 2.06 22.00 ± 1.93 1.26 ± 0.70

87.60 ± 0.60 5.84 ± 0.59 6.56 ± 0.17 50.90 ± 1.53 8.61 ± 1.95 40.50 ± 2.15 79.41 ± 2.55 15.61 ± 4.01 4.98 ± 1.53

Day 9

103

At 15 °C, no band was detected by western blot with Rubisco antibody on days 9 and 12, but a single band of ~ 14.8 kDa appeared on days 33 and 37 (Fig. 3) when the culture was in steady population (Fig. 1). For PCNA, two weak bands of ~ 115.5 kDa and ~ 55 kDa were detected on day 9 (Fig. 4). Three bands of ~ 181.8 kDa, ~ 115.5 kDa and ~ 55 kDa were observed on day 12, with the latter two bands being denser than on day 9. Then four bands (~ 181.8 kDa, ~ 115.5 kDa, ~ 55 kDa and ~ 25.9 kDa) appeared on days 33 and 37 (Fig. 4), of which the bands of ~ 115.5 kDa and ~ 55 kDa were the most abundant (P b 0.05). The ~ 115.5 kDa protein grew more abundant than the latter from day 12 (Table 2). As shown in Fig. 5, barely any bands of α-tubulin were detected in 10 and 15 °C samples. On the same western blot, samples from the 4 °Cculture had the regular band of α-tubulin. This indicated the degradation

Day 37

G1 peak 4oC

G2+M peak

The fitted S peak

10oC

15oC

Fig. 2. DNA histograms of the cultures grown at 4°, 10° and 15 °C on days 9 and 37.

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kDa Day 9

kDa

Day 9

181.8 115.5 82.2

181.8 115.5 64.2 48.8 37.1

64.2

4oC

10oC

15oC

Day 12

37.1

4oC

25.9 19.4 14.8

15oC

10oC

25.9 19.4 14.8

Day 12 181.8 115.5 64.2 48.8 37.1 25.9 19.4 14.8

181.8 115.5 82.2 64.2 37.1

4oC Day 33

10oC 10oC

15oC 15oC

25.9 19.4 14.8

4oC

Day 33

181.8 115.5 82.2 64.2

10oC

15oC

10oC

15oC 181.8 115.5 64.2 48.8 37.1

37.1 25.9 19.4 14.8

4oC Day 37

4oC

10oC

15oC

25.9 19.4 14.8

4oC

181.8 115.5 82.2 64.2 37.1

Day 37

25.9 19.4 14.8

15oC

10oC

181.8 115.5 64.2 48.8 37.1 25.9 19.4 14.8

4oC

Fig. 3. Western blots of Rubisco in the 4°, 10° and 15 °C cultures on days 9, 12, 33 and 37. Each lane was loaded with the total protein from 5,000 cells.

of this cytoskeleton protein at the elevated temperatures. In this situation, the role of α-tubulin in the cytoskeleton might have been complemented by other forms of tubulin. 3.4. Diel cell cycle progression and expression of Rubisco and PCNA At steady cell concentration, the culture could be arrested at a cell cycle stage or cell division could be balanced by cell death. To determine what happened during the temperature treatments, diel samples were collected and cell cycle analyzed. The 4 °C-culture was in the early stationary phase on day 39. Consistent to this, the cell cycle profile did not show clear cell division during the diel cycle (Fig. 6A). The %G2M increased mildly at hour 12 (in the dark period) and hour 24 (in the light period), and %S slightly increased at hour 24 (in the light period). The cell cycle seemed to complete in 24 h but only a very small fraction of the population progressed through. Rubisco and PCNA expressed and their abundances changed through the diel cycle without an evident rhythm (Fig. 6B, C). And their varying trends differed. The maximum expression of Rubisco was observed at the beginning of the diel experiment (hour 0, which was 4 h before light turn-off). The next expression peak appeared 12 h later (in the dark period). The minimum expression was observed at hour 6 (in the dark period). For PCNA, the maximum expression was observed at hour 18 (in the light period), and the minimum was at hour 0, coincident with the minimum %S (Fig. 6A).

Fig. 4. Expression of PCNA-like proteins in the 4°, 10°and 15 °C cultures on days 9, 12, 33 and 37; the same western blots as in Fig. 3 were stripped off the Rubisco antibodies and reprobed with the PCNA antibodies.

Compared with the other cultures, %G1 in the 10 °C-culture on day 37 was sustained as the lowest (P b 0.01) and %G2M remained the highest (P b 0.01) (Fig. 7). In the diel cycle, %S had its peak at hour

Table 2 The damaged to normal protein ratios observed over time under three different temperatures. Culturing temperature (°C)

Rubisco

PCNA

4 10 15 4 10 15

Damaged to normal protein ratioa Day 9

Day 12

Day 33

Day 37

0 0 0 0 0 0.8

0 0 0 0 0 1.37

0 2574.65 2991.33 0 2.10 1.28

0 – 1295.49 0 – 1.48

a Protein abundances were measured from the optical density of bands in the western blots using Quantity One with background subtracted. The ratio for Rubisco was calculated as [14.8 KDa abundance]/([~55 kDa abundance] + 1) and that for PCNA as [115.5 kDa]/([55 kDa abundance] + 1); “–“ indicates that no protein was detected.

S. Zheng et al. / Journal of Experimental Marine Biology and Ecology 438 (2012) 100–108

% of cells

55kDa

A

100 90 80 70 60 50 40 30 20 10 0

Day 9

%G1 %S %G2M

0

6

12

18

24

Circadian time (hr)

Rubisco/5,000 cells

Day 12 55kDa

105

18000 16000 14000 12000 10000

B

8000 6000 4000 2000 0

Day 33

0

6

12

18

24

Circadian time (hr) 7000

55kDa

C

PCNA/5,000 cells

6000 5000 4000 3000 2000 1000

Day 37

0 0

55kDa

6

12

18

24

Circadian time (hr) Fig. 6. (A) Cell cycle profiles in the 4 °C culture during the diel cycle on day 39. Cells were sampled on hours 0, 6, 12, 18, and 24. Dark period lasted from the 4th hour to the 14th hour (black bar). Abundances of Rubisco (B) and PCNA‐like protein (C) in 5,000 cells were measured by Quantity One (with background subtracted) and presented in arbitrary units.

Fig. 5. Expression of α-tubulin on days 9, 12, 33 and 37 in the 4°, 10° and 15 °C cultures. The same western blots as in Figs. 3 and 4 reprobed with the monoclonal anti-α-tubulin antibody (Sigma) after stripping off PCNA antibodies.

4. Discussion 4.1. Growth and cell cycle of P. glacialis at 4 °C and the effect of temperature rises If growth rate is lower than one doubling per day (i.e. b 0.69 d −1), the generation time, i.e. period of a complete cell cycle, must be 100

%G1

90

%S

80

%G2M

70

% of cells

18 while %G2M had its minimum (in the light period). Unfortunately, our attempt to extract the proteins from samples collected in this diel cycle was unsuccessful. At 15 °C, %G2M maintained the lowest among the three temperature treatments (P b 0.05) and lower than 10% during the diel cycle on day 35 (Fig. 8A). %G1 remained higher than the 10 °C-culture (P b 0.01). These results indicated the cell cycle arresting effect of 15 °C on P. glacialis. Western blot of Rubisco of the 15 °C-culture showed a major band at ~ 55 kDa in the diel cycle on day 35. Its abundance increased abruptly at hour 18 and hour 24 (both in light phase), with the highest abundance at hour 18 (Fig. 8B). Minor bands of ~ 19.4 kDa and ~ 14.8 kDa were present except hour 18 and hour 24. For PCNA, major bands of ~ 55 kDa and ~ 115.5 kDa were identified in the western blot. The abundance of the ~ 115.5 kDa protein kept higher than that of ~ 55 kDa (Fig. 8C). Their abundances also peaked at hour 18 and hour 24. The diurnal patterns of these two proteins' abundances were distinctly different from that in the 4 °C-culture (Fig. 6B, C).

60 50 40 30 20 10 0 0

6

12

18

24

Circadian time (hr) Fig. 7. Cell cycle profiles in the 10 °C culture on day 37. Cells were sampled on hours 0, 6, 12, 18, and 24. Dark period lasted from the 4th hour to the 14th hour.

% of cells

106

S. Zheng et al. / Journal of Experimental Marine Biology and Ecology 438 (2012) 100–108

100 90 80 70 60 50 40 30 20 10 0

A

%G1 %S %G2M

0

6

12

18

24

Circadian time (hr)

Rubisco/5,000 cells

14000

B

12000 10000 8000 6000 4000 2000 0 0

6

12

18

24

18

24

Circadian time (hr) 5000

C

PCNA/5,000 cells

4000 ~115.5 kDa ~55 kDa

3000 2000 1000 0 0 -1000

6

12

Circadian time (hr)

Fig. 8. (A) Cell cycle profiles in the 15 °C culture on day 35. Cells were sampled on hours 0, 6, 12, 18, and 24. Dark period lasted from the 4th hour to the 14th hour. Abundances of Rubisco (B) and PCNA‐like protein (C) in 5,000 cells were measured by Quantity One (with background subtracted) and presented in arbitrary units.

longer than one day. Thus, the cell cycle of P. glacialis at 4 °C seemed to take over 24 h to complete as shown by the growth rate data (Fig. 1). The maximum growth rate observed at this temperature was 0.45 d −1. The longer than 24-h cell cycle is also inidcated by the flow cytometric result (Fig. 6A). On day 39, only 2.32% of the cell population progressed through all the four phases of the cell cycle, which is consistent to the observed growth rate. Nevertheless, the time period between the peak of G1 phase and G2+ M phase was about 24 h (Fig. 6A), indicating that the major cell cycle stages are still phased to the natural 24-h day and exhibited a circadian rhythm. As the cycle length is more than 24 h, the cell division cycle of P. glacialis can only be “gated” by the circadian program (Roenneberg and Mittag, 1996). Its circadian clock opens a window of opportunity for mitosis to occur at a particular time of day through which cells in an appropriate phase of the cell cycle can pass (Dagenais-Bellefeuille et al., 2008). Thus, there was always a small portion of P. glacialis cells passing through the S and G2+ M phases to carry out division in a natural and asynchronized culture. Organisms may respond to sudden temperature changes differently: from a quick adaptation, surviving with compromised metabolic efficiencies, experiencing stress, to death if the temperature anomaly is extreme (Fogg, 2001). Since P. glacialis with the cell density of 5.1 × 104 cells mL−1 was in the exponential stage, the initial reduction of cell concentrations in the two populations cultured at 10° and 15 °C indicated heat shock effect of this species to a sudden temperature change by over 5 degrees.

No research has been done to determine P. glacialis's optimal growth temperature. The recommended culturing temperature for P. glacialis is 4 °C by CCMP. Increases in temperature will result in increased metabolic activity and growth of microalgae, provided that the increased temperature is still below the optimal temperature of the algae, and that growth is not limited by other factors (Beardall and Raven, 2004). On the contrary, temperature elevation above the optimum level has been found to result in thermal shock in the form of membrane deformation and rapid cell death, which is due to fast depletion of reserved photosynthetic product and increase in respiration rate (Agrawal, 2003). The population decline (cell death) in the beginning of the heat shocked cultures indicated that 10 and 15 °C were above P. glacialis' optimal temperature. In response to adverse external environments such as the absence of key nutrients or growth factors, cell cycle may slow down or be arrested (Slater et al., 1977). Adverse environmental conditions often result in an increase in the duration of G1 phase of the cell cycle. G1 phase lengthened, when Amphidinium carteri grew under limited light (Olson and Chisholm, 1986; Olson et al., 1986). The durations of the S and G2 phases can also vary, but generally not as much as G1 (Guiget et al., 1984). Temperature has been shown to affect the length of the cell cycle stages, particularly G1 and G2 (Vaulot, 1995). Suboptimal temperature has been shown to cause increases in the duration of all phases of the cell cycle but not equally in Hymenomonas carterae and Thalassiosira weissflogii, a coccolithophore and a centric diatom, respectively (Olson et al., 1986). During about 40 days of our growth study, the %G2M of 10 °C were the highest among the three temperatures examined (P b 0.05), as the %G1 being the lowest among the three temperatures (P b 0.05). In the 15 °C-culture, %G2M decreased and %S increased. Especially in the DNA histograms of the 15 °C-culture on day 37 (Fig. 2), most cells stopped at G1 and S phase, and barely any G2 peak was observed in the steady-cell-concentration stage. During the diel sampling on days 35, 37 and 39, %G2M in the 15 °C-culture (Fig. 8A) remained the lowest among three temperatures (Fig. 6A, Fig. 7) (P b 0.05). In the 10 °C-culture, the diel %G2M was the highest among three temperatures and the %G1 stayed in the lowest among three temperatures (Fig. 7) (P b 0.01). It was conceivable that G1 and S phases were prolonged when the culture was grown at 15 °C; however, increased %G2M and decreased %G1 in the 10 °C-culture indicated that P. glacialis cells that survived this temperature were likely to have higher metabolic activity and more active cell cycle progression, since G1 phase is usually lengthened by environmental stress in most studies.

4.2. Damaged cell cycle and photosynthesis apparatus after temperature rises PCNA protein is highly conserved across a wide phylogenetic range (Kelman and O'Donnell, 1995), the molecular mass of which is estimated to be 29 kDa (Krishna et al., 1994) but typically appeared to be 36 kDa on the SDS–PAGE gel (Lin et al., 1994; Matsumoto et al., 1987). Three monomers combine to form the active trimeric complex with a ring like structure. Although mass spectrum analysis or DNA sequencing might be needed to confirm its identity in this study, a protein band of ~55 kDa was the major band detected by the PCNA antibody in the western blot, and its diel abundance varied in correspondence to %S and %G2M (Figs. 6 and 8). Unusually large PCNA-like proteins have also been detected in other dinoflagellates. A PCNA-like protein of 55 kDa was found in Crypthecodinium cohnii Biechelerand and Gymnodinium catenatum Bravo (Leveson and Wong, 1999). It has been suggested that the anomalous size of the PCNA-like protein observed in dinoflagellates could be related to the peculiar and varied dinoflagellate chromosomal structure (Leveson and Wong, 1999); however, PCNA gene from P. piscicida (Zhang et al., 2006) and P. donghaiense (Zhao et al., 2009), representing the orders of Peridiniales and Prorocentrales,

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respectively, are similar to the typical PCNA gene in both sequence and length. PCNA was found to express more abundantly in actively dividing cells in many species (Celie and Celis, 1985; Negishi et al., 1990; Ng et al., 1990). In Pleurochrysis carterae, for example, PCNA was found highly transcribed and translated during the exponential stage relative to the stationary stage, with a positive correlation between gene expression and growth rate (Lin and Corstjens, 2002). Despite smaller magnitude, similar pattern has been found for PCNA in the heterotrophic dinoflagellate P. piscicida (Zhang et al., 2006). Higher %S and %G2M levels and PCNA abundance in the exponential growth stage of P. glacialis when cultured at 4 °C suggests more active cell cycle progression in this stage than in the early stationary growth stage. For Rubisco, its abundance in the 4 °C culture increased in the exponential growth phase and decreased when the culture entered the stationary stage. This result suggests contrasting photosynthetic rates between the two growth phases that demand different levels of Rubisco. Given that Rubisco functions in photosynthesis, a high level of expression in the light period and low level in the dark period would be expected. That seems to be true for Rubisco abundance in chromophyte algae (Steinbib and Zetsche, 1986; Steinmuller and Zetsche, 1984). Yet the light-dependent expression pattern does not seem to apply to chlorophytes and dinoflagellates examined so far. In Dunaliella tertiolecta Butcher, Rubisco is not cell cycle dependent (Lin and Carpenter, 1997). Similarly, Rubisco abundance was found to be constant over time in L. polyedrum (Nassoury et al., 2001). Consistent to these findings, our result from the current study also indicated no diel cycle rhythm in Rubisco abundance. Our western blot analyses clearly showed remarkable alteration of Rubisco and PCNA as the result of the culture's exposure to elevated temperatures. And as the temperature rose higher, the proteins changed sooner (Figs, 3 and 4, Table 2). The ~14.8 kDa protein detected by Rubisco antibody on the western blot on days 33 and 37 for the 10° and 15 °C cultures were likely the degradation fragments of the typical ~55 kDa Rubisco protein. The typical ~55 kDa Rubisco declined over time in these thermal stressed cultures, and finally was replaced by the degraded protein fragment (Fig. 3), resulting in increasing ratio of the degraded to normal protein abundances (Table 2). Photosynthesis inhibition is one of the symptoms of thermal stress. Symbiodinium microadriaticum, for example, when shifted from the original temperature of 26 °C to 20, 25, 30 and 35 °C, photosynthesis increased from 20 °C to 30 °C, then decreased above 30 °C, and ceased completely at 35 °C. Photosynthesis was inhibited at temperatures above 30 °C, because of an uncoupling of energy absorption and photochemistry, probably resulting from changes in lipid characteristics of the thylakoid membranes (Iglesias-Prieto et al., 1992). The respiration showed a similar trend, but did not cease at 34–36 °C, suggesting that the cells were not dead. At high temperatures deficiency of oxygen, which is much less soluble in hot than in cold water, may be another cause of stress (Brock, 1978). The degradation of Rubisco at the elevated temperatures as observed in our experiment would result in photosynthesis inhibition of P. glacilis. Similarly, while the 4 °C culture expressed a ~ 55 kDa PCNA-like protein, it was replaced by a ~ 115.5 kDa protein in 10° and 15 °C cultures, which might be a dimeric aggregate of the ~ 55 kDa PCNA-like protein (Fig. 4). The quantity of this protein aggregate to the normal protein increased from day 9 to day 12, and then stayed stable as the culture was maintained at 15 °C (Table 2). This result indicates damage of the cell cycle regulatory machinery by the heat stress. The absence of α-tubulin in 10° and 15 °C culture was another result of thermal stress (Fig. 5). Despite all the growth inhibition and molecular damages, P. glacialis seems to be able to survive the thermal stress. It is noteworthy that in the present study the cultures were directly transferred from 4 °C to 10 and 15 °C without progressive intermediate steps. In response to such sudden temperature shift, the cultures first experienced a period

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of declination, then cell density tended to become stable (Fig. 1), a sign that a part of the cell population survived. In the 10 °C-culture, there was still a small proportion of cells successfully completed the cell cycle (Fig. 7), indicative of active growth of a subpopulation under this elevated temperature. At the biochemical level, the abundance of the two key proteins, Rubisco and PCNA, first reduced in abundance in the declining cultures; then damages occurred to them, causing the impairment of the photosynthetic and cell cycle machinery (Figs. 3 and 4), and hence a non-growing (steady) population (Fig. 1). But there was a sign of recovery, in terms of cell abundance and biochemistry. The bands of typical Rubisco expressed diurnally (Fig. 8B) in the late stage of the experiment. And PCNA-like protein appeared to increase (Fig. 4) and expressed diurnally (Fig. 8C). All the results suggest that P. glacialis may be able to survive temperature rises to as high as 15 °C, at least for 45 days. If the species can survive such heat shock in the long term, there is good opportunity that it can be transported from polar region to temperate or even warmer waters. Perhaps this explains that taxa closely related to this species occur in temperate aquatic environments (Lin et al., 2009, 2010). Acknowledgments We wish to thank Dr. Huan Zhang and Dr. Yubo Hou for their advice on the experiments, Dr. Carol Norris (Flow Cytometry and Confocal Microscopy Facility, University of Connecticut) and Dr. Lilibeth Miranda for their assistance with flow cytometric analyses. Prof. Jianfeng He (Polar Research Institute of China) and Prof. Dazhi Wang (College of The Environment and Ecology, Xiamen University) kindly provided access to some of the experimental equipment. We are also grateful to the China Scholarship Council for providing financial support for Shuxian Zheng to visit the University of Connecticut to conduct most of the work reported here. [SS] References Agrawal, S.C., 2003. Marine Plants Ecology. Bishen Singh Mahendra Pal Singh, Allababad. Alexandrov, V.Y., 1994. Functional Aspects of Cell Response to Heat Shock. Academic Press, Inc., San Diego. Beardall, J., Raven, J.A., 2004. The potential effects of global climate change on microalgal photosynthesis, growth and ecology. Phycologia 43 (1), 26–40. Berry, J.A., Björkman, J.K., 1980. Photosynthetic response and adaptation to temperature in higher plants. Annu. Rev. Plant Physiol. 31, 491–543. Brock, T.D., 1978. Thermophilic Microorganisms and Life at High Temperatures. SpringerVerlag, New York Heidelberg Berlin. Cachon, J., 1989. Analysis by polarising microscopy of chromosomal structure among dinoflagellates and its phylogenetic involvement. Biol. Cell 65, 51. Celie, J.E., Celis, A., 1985. Cell cycle-dependent variations in the distribution of the nuclear protein cyclin proliferating cell nuclear antigen in cultured cells: subdivision of S phase. Proc. Natl. Acad. Sci. 82, 3262–3266. Dagenais-Bellefeuille, S., Bertomeu, T., Morse, D., 2008. S-Phase and M-phase timing are under independent circadian control in the dinoflagellate Lingulodinium. J. Biol. Rhythm. 23, 400–408. Estrada, M., Berdalet, E., 1998. Effects of turbulence on phytoplankton. In: Anderson, D.M., Cembella, A.D., Hallegraeff, G.M. (Eds.), Physiological Ecology of Harmful Algal Blooms. Springer, Berlin, pp. 601–618. Feinstein, T.N., Traslavina, R., Sun, M.-Y., Lin, S., 2002. Effects of light on photosynthesis, grazing, and population dynamics of the heterotrophic dinoflagellate Pfiesteria piscicida (Dinophyceae). J. Phycol. 38, 659–669. Fogg, G.E., 2001. Algal Adaptation to Stress—Some General Remarks. Springer, Berlin Heidelberg New York . (1–20 pp.). Guiget, M., Kupiec, J.J., Valleron, A.J., 1984. A Systematic Study of the Variability of Cell Cycle Phase Durations in Experimental Mammalian Systems. Marcel Dekker, New York . (97–111 pp.). Guillard, R.R., 1973. Methods for microflagellates and nannoplankton. In: Stein, J.R. (Ed.), Handbook of Phycological Methods. Culture Methods and Growth Measurements. Cambridge University Press, Cambridge, pp. 69–85. Guillard, R.R., Hargraves, P., 1993. Stichochrysis immobilis is a diatom, not a chrysophyte. Phycologia 32, 234–236. Hallegraeff, G.M., Bolch, C.J., 1992. Transport of diatom and dinoflagellate resting spores in ships' ballast water: implications for plankton biogenography and aquaculture. J. Plankton Res. 14, 1067–1084. Iglesias-Prieto, R., Matta, J.L., Robins, W.A., Trench, R.K., 1992. Photosynthetic response to elevated temperature in the symbiotic dinoflagellate Symbiodinium microadriaticum in culture. Proc. Natl. Acad. Sci. 89, 10302–10305. Kelman, Z., O'Donnell, M., 1995. Structural and functional similarities of prokaryotic and eukaryotic DNA polymerase sliding clamps. Nucleic Acids Res. 23, 3613–3620.

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