WEN Ruobing, SUI Zhenghong, BAO Zhenmin ZHOU Wei and WANG Chunyan

1) Key Laboratory of Marine Genetics and Breeding of Ministry of Education of China, Ocean University of China, Qingdao 266003, P. P. China

2) North China Sea Environmental Monitoring Center, State Oceanic Administration of China, Qingdao 266033, P. R. China

3) Putian Marine and Fishery Environmental Monitoring Station, Putian Marine and Fishery Bureau, Putian 351100, P. R. China

Isolation and Characterization of Calmodulin Gene of Alexandrium catenella (Dinoflagellate) and Its Performance in Cell Growth and Heat Stress

WEN Ruobing1),2), SUI Zhenghong1),*, BAO Zhenmin1), ZHOU Wei1), and WANG Chunyan1),3)

1) Key Laboratory of Marine Genetics and Breeding of Ministry of Education of China, Ocean University of China, Qingdao 266003, P. P. China

2) North China Sea Environmental Monitoring Center, State Oceanic Administration of China, Qingdao 266033, P. R. China

3) Putian Marine and Fishery Environmental Monitoring Station, Putian Marine and Fishery Bureau, Putian 351100, P. R. China

Harmful algal blooms (HABs) can occur and then disappear quickly, corresponding to consistent growing and declining of heavy biomasses. The molecular mechanism of blooming remains unclear. In this study, calmodulin gene (cam) of HAB causing species Alexandrium catenella was isolated and characterized. The expression of calmodulin gene was profiled at different growth rates and in heat stress. The full cDNA of cam was 597 nucleotides (nt) in length, including a 25 nt 5′ untranslated region (UTR), an 122 nt 3′ UTR, and a 450 nt open reading frame (ORF) encoding 149 amino acids. The deduced calmodulin (CaM) was highly conserved in comparison with those of other organisms. As was determined with real-time RT PCR, the abundance of cam transcript varied in a pattern similar to cell growth rate during the whole growing period. The abundance of cam transcript increased by more than 8 folds from lag growth phase to exponential growth phase, and then obviously decreased from exponential growth phase to stationary/decline growth phase. In addition, the relative abundance of cam transcript significantly declined with time during heat shock. Taking CaM function described in other organisms into account, we believe that Ca2+-involved signal transduction, methylation of DNA and toxin precursors underlined the cell growth of this species. The response of cam gene to heat stress in dinoflagellate suggested restrictions in Ca2+signal transduction and methylation. These findings are helpful to understand the relationships among growth, cell signal transduction, bloom formation and interaction with environmental stimuli in dinoflagellates.

harmful algal bloom; Alexandrium catenella; calmodulin; growth rate; heat stress

1 Introduction

In last two decades, harmful algal blooms (HABs) which are hazardous to human health, occurred more frequently, persistently and/or intensively (Smayda, 1989, 1990; Hallegraeff, 1993; Van Dolah, 2000). Multidisciplinary researches including molecular and cell biology, large-scale field surveys, numerical modeling and remote sensing from space have been conducted on HABs (Anderson, 2009). Dinoflagellates are considered as the most notorious HABs causing organisms. Due to its global distribution and paralytic shellfish poisoning (PSP) toxins producing abilioty, Alexandrium catenella was among the best studied dinoflagellates (Uribe et al., 2008; Toulza et al., 2010; Lin, 2011).

HABs often form and then disappear quickly in an unexpected manner, corresponding to consistent growth and declining of heavy biomasses. Considering the burst growing characteristics of blooming, it is speculated that the process is related to the reaction of individual cells to a common signal substance. Such a reaction subsequently activates the signal transduction cascade and induces the expression of growth regulation-related genes. Aldehydes have been found to be signal substances responsible for the growth control of both diatoms themselves and their predators (Ianora et al., 2004). However, the mechanism of blooming and the signal transduction system remained unclear. Cyclins and cyclin-dependent kinases have been identified in several marine phytoplanktons, such as Dunaliella tertiolecta (Lin et al., 1996; Lin et al., 2000) and Gambierdicus toxicus (Van Dolah et al., 1995). In addition, proliferating cell nuclear antigen (PCNA) was demonstrated to be related to the cell growth of chlorophyta and chrysophyta (Lin and Carpenter, 1998; Lin and Corstjens, 2002) and Prorocentrum donghaiense (Zhao et al., 2009), and Alexandrium catenella (Huang et al., 2010). PCNA displays a highly correlation with the growth of dinoflagellate cells (Zhao et al., 2009; Huang et al., 2010). In addition, homologs of genes known to be involved in programmed cell death (PCD) in other eukaryotes have been identified in some dinoflagellates andfound to be induced by oxidative stress (Okamoto and Hastings, 2003). These studies expanded our knowledge of HABs occurrence mechanism. Considering the function of these proteins, a complex modulation system was expected to exist in growth control. However, roles of other genes related to the signal transduction of cells have not been reported in dinoflagellates.

Calcium (Ca2+) mediated signal transduction is known to play a crucial role in almost all intracellular events of eukaryotic organisms. Various extracellular stimuli can promote Ca2+signal pathway, which involve kinase cascades, protein activation and other operation. Calmodulin (CaM) is the best studied and ubiquitous calcium sensor protein. Calmodulin, as a regulator of cell growth, plays an important role in G1/S and G2/M progression (Sasaki and Hidaka, 1982; Eilam and Chernichovsky, 1988; Davidkova et al., 1996) and increases DNA synthesis in a variety of cells (Means, 1994). It is relatively small (e.g. vertebrate CaM only has 148 amino acid residues) and evolutionarily highly conserved and comprises four EF hands, structural motifs binding with Ca2+. By combining with Ca2+, CaM can interact with a heterogeneous population of target proteins which can regulate many physiological activities (Trewavas and Knight, 1994; Sanders et al., 1999; Reddy, 2001; Snedden and Fromm, 2001; Ihara-Ohori et al., 2007; Popescu et al., 2007). The conserved and ubiquitous function of Ca2+/CaM cascade suggests that CaM may exist in phytoplankton metabolism. It has been shown that intracellular Ca2+may be involved in the encystment response of dinoflagellats Alexandrium catenella and Crypthecodinum cohnii (Tsim et al., 1997). Recently, CaM was extracted and purified from marine phytoplankton Prorocentrum donghaiense Lu cells (Liu et al., 2006; Guo et al., 2009). Liu et al. (2006) discovered that the effect of different N:P ratios of nutrient supply on the growth of P. donghaiense Lu involved the Ca2+/CaM signal transduction pathway, which suggested that Ca2+/CaM signal pathway was involved in the regulation of marine phytoplankton interaction with the environment, playing an important role in regulating cell-cell communication. However, the function of cam in regulating cell growth and responding to environmental stresses were still unclear.

The objective of this study is to understand the role of Ca2+/CaM signal transduction during the growth of HABs causing algae and in extracellular stimuli process. In present study, cam gene was isolated from Alexandrium catenella and characterized with its expression analysis carried out during culture period and in heat stress.

2 Materials and Methods

2.1 Algal Culture

Alexandrium catenella was obtained from Culture Collection of National Marine Environmental Monitoring Centre, State Oceanic Administration of People’s Republic of China. Batch cultures of A. catenella were conducted at 20℃ in f/2 medium under a photon flux of 35 μE m?2s?1. A photocycle of 12 h light and 12 h dark was maintained during culture.

2.2 Nucleic Acid Extraction

Total RNA was extracted and purified using Trizol Reagent (Invitrogen, CA) in combination with QIAGEN RNeasy Plant Mini Kit (Qiagen, Hilden, Germany). The extracted was treated with DNase I (2U) to eliminate genomic DNA contamination. Genomic DNA was extracted with CTAB method (Murray and Thompson, 1980; Porebski et al, 2007).

2.3 Primer Designing

Primers used in this study and their detail applications were shown in Table 1.

Table 1 Primers used in this study

2.4 Reverse Transcription (RT) PCR to Obtain cam Partial Sequence

First-strand cDNA was synthesized using 2 μg of total RNA with Oligo (dT) primer. The partial cam cDNA sequence was amplified using degenerate primers cam1 and cam2 (Table 1). The amplification was carried out by predenaturing at 94℃ for 5 min, followed by 30 cycles of denaturing at 94℃ for 30 s, annealing at 63℃ for 30 s and extending at 72℃ for 40 s and a final extension at 72℃for 5 min.

2.5 Rapid Amplification of cDNA 3’ Ends (RACE)

The 3’ end of cam cDNA was obtained with 3’ Full-RACE (Rapid Amplification of cDNA Ends) Kit (Takara, Dalian, China). First-strand cDNA was synthesized using 2 μg of total RNA with 3’site primer (Table 1). Then nested PCR amplification was performed using primers cam1-3AP as the outer pair and calf-3AP as the inner pair (Table 1). PCR was carried out by predenaturing at 94℃for 5 min, followed by 30 cycles of denaturing at 94℃ for 30 s, annealing at 60℃ for 30 s and extending at 72℃ for 30 s and a final extension at 72℃ for 5 min.

2.6 Amplification of 5’ End

The 5’ end of cam cDNA was obtained using the In Vitro Cloning Kit (Takara) according to the manufacturer’sinstruction. In brief, genomic DNA was extracted with CTAB method (Murray and Thompson, 1980; Porebski et al., 2007). After digested with Pst I, the DNA fragment was linked with Pst I cassette. By using adapter-ligated fragments as template, nested PCR was performed. Primers cami and C1 (Table 1) were used in the first round PCR as the outer pair, and the annealing temperature was 55℃. The second round PCR was then carried out by predenaturing at 94℃ for 5 min, followed by 30 cycles of denaturing at 94℃ for 30 s, annealing at 60℃ for 1 min and extending at 72℃ for 1 min and a final extension at 72℃ for 5 min. Primer calr and C2 served as inner pair (Table 1).

2.7 Cloning and Sequencing of PCR Products

The PCR products were purified using Agarose Gel DNA Purification Kit (TakaRa) and cloned into pMD18-T vector following the manufacturer’s instructions. Recombinants with appropriate inserts as screened with PCR were sequenced commercially in Invitrogen (Biotehnology Co., Ltd). Sequences obtained were analyzed using BLAST against other calmodulin sequences deposited in GenBank. Most dinoflagellate genes lack introns and a TATA box or consensus sequences in their promoters (Moreno Díaz de la Espina et al., 2005). The BLAST results confirmed that the DNA sequence of 5’ end of cam obtained from In Vitro cloning was highly similar to the calmodulin cDNA of other diniflagellates (A. fundyense (gi:189081555), Karlodinium micrum (gi:189081808) and Pfieteria piscicida (gi:189081809)) in Genebank. After assembling 3’ and 5’ ends of cam，the ORF was used to deduce amino acid sequence. The functional domains of the deduced protein were predicted through NCBI conserve domain search service (http://www.ncbi.nlm.nih.gov/ Structure/cdd/wrpsb.cgi). Multiple amino acid sequence alignments were performed with CLUSTALW 1.8.

2.8 Expression Analysis of cam

A. catenella (initial density 1000 cells mL?1) was cultured under conditions mentioned in 2.1 for 24 d. Cell growth was monitored with microscopy counting which was carried out on a hemacytometer plate every day. The following formula was adopted to calculating specific cell growth rate (μ):

where Nt1and Nt2were the cell density (mL?1) at time t1and t2, respectively. In order to determine the abundance of cam transcript, approximately 106algal cells were collected every four days and preserved in liquid nitrogen until RNA extraction.

2.9 Determination of cam Gene Response to Heat Stress

A. catenella was cultured as described in 2.1. For heat shock experiment, 600 mL of A. catenella at late exponential/early stationary phase was transferred from a 20℃to 30℃. After 1, 4 and 8 h, 200 mL algal culture was collected by centrifuging at 8000 r min-1for 10 min, respectively.

2.10 Real-Time Quantitative RT-PCR

The abundance of A. catenella cam gene transcript was determined with real-time quantitative RT-PCR. The reaction was carried out in a volume of 20 μL containinig 3 μmol L?1primers (calf and calr each, Table 1) and 2 μL of cDNA, 10 μL of FastStart Universal SYBR Green Master (Roche, Germany), generating a 212-bp product of cam cDNA. The amplification was carried out by denaturation at 95℃ for 10 min followed by 40 cycles of denaturing at 95℃ for 15 s and annealing and extending at 60℃ for 60 s. The cytochrome b gene (cob) cDNA fragment (140 bp) was simultaneously amplified with primers cob1 and cob2 (Table 1) as an internal control.

The abundance was calculated with 2?ΔΔCTmethod (Livak and Schmittgen, 2001). The 2?ΔΔCTvalue reflects the difference of cam relative transcription normalized to cob. Equation

was used to calculate ΔΔCT, where CT is threshold cycle for amplification.

3 Results

3.1 Isolation and Characterization of Full Length cam Gene

The cam cDNA was 597 nt in length (Fig.1), which contained a untranslated 5’ (25 nt) and 3’ (122 nt) region and an ORF of 450 nt. The cDNA encoded a polypeptide of 149 amino acids. In 3’-untranslated region, no polyadenylation signal was identified with functional analysis software (http://www.dna.affrc.go.jp/PLACE/signalscan. html), which is consistent with the finding in A. tamarense (Hackett et al., 2005).

Conserved domain searching against NCBI data bank disclosed that the 149 amino acids of CaM from A. catenella comprised of 4 E-F hands, a structural motif binding with calcium and being essential for CaM function. The deduced amino acids were compared with those of other species deposited in GenBank with Clustal W package (Fig.2). The CaM sequence is highly conserved among the species compared. CaM of A. catenella was identical to those of A. fundyense, Pfiesteria piscicid and Karlodinium micrum (Zhang et al., 2007), 91.3%–92.6% homologous to those of high plant and animals, and 80% homologous to those of Chlamydomonas reinhardtii and Chlamydomonas incerta (Schleicher et al., 1984; Lukas et al., 1985). The central helix of CaM, a flexible structure helping interaction of downstream enzymes with CaM, is highly conserved among animals and high plants. All 20 species, 3 animals (18–19) and 5 high plants (13–17), shared the same sequences in central helix. However, sequences of central helix of 12 algal species were different, indicating the diverse interaction between CaM and its target proteins.

Fig.1 Nucleotide sequence of cam of A. catenella. The ORF was the region in which nucleotides were in italic. Initiation and termination codons were in bold. The sequence and direction of primers (cam1, cam2, calf and calr) are underlined and directed with arrows.

Fig.2 The alignment of A. catenella CaM sequence with those of other species. Sequences in the frames indicate the binding sites of Ca2+; the sequences in grey frame indicate the central helix of CaM. 1, A. catenella; 2, A. fundyense (gi:189081555); 3, Karlodinium micrum (gi:189081808); 4, Pfieteria piscicida (gi:189081809); 5, Heterocapsa triquetra (gi: 1890811556); 6, Noctiluca scintillans (gi:157093363); 7, Saccharina japonica (gi:157888809); 8, Macrocystis pyrifera (gi:728609); 9, Thalassiosira pseudonana (gi:209585787); 10, Rhodomonas sp. CCMP768 (gi:255966042); 11, Chlamydomonas reinhardtii (gi:225024); 12, Chlamydomonas incerta (gi:74272635); 13, Zea mays (gi:195605834); 14, Glycine max (gi:170070); 15, Oryza sativa (gi:115452695); 16, Hordeum vulgare Barley (gi:167008); 17, Arabidopsis thaliana (gi:166655); 18, Drosophila melanogaster (gi:49037468); 19, Mus musculus (gi:6753244); 20, Homo sapiens (gi:5542035).

3.2 Correlation Between cam Expression and Algal Growth Rate

The cell density of cultured A. catenella was shown in Table 2 and Fig.3 when the cells entered the decline growth phase at day 16. The peak growth rate appeared at day 12 after inoculation (Table 2 and Fig.3). The cam transcription level normalized to the cob transcription was shown in Table 2 and Fig.3.

A similar level of variability was revealed between the growth rate and transcription level of cam (Fig.3). The relative expression level of cam increased from lagging phase, reaching the peak at day 12 synchronically with growth rate, and then decreased with the same trend as that of growth.

Table 2 Expression level of cam gene in A. catenella at different growth phases

Fig.3 Variation of CaM relative transcription level, growth rate, and the cell density of A. catenella at different growth phases.

3.3 Response of cam Gene to Heat Stress

The variation of cam expression level at different times after heat shocking was shown in Fig.4. The relative expression level of cam exhibited a significant decline trend with the increase of heat-stress persistence. The peak was reached immediately after heat treatment, and decreased by about 80% in 8 h at 30℃.

Fig.4 Variation of relative transcription level of cam in A. catenella under heat stress.

4 Discussion

Calmodulin is a highly conserved and well characterized Ca2+sensor in eukaryotes (Zielinski, 1998), and also an essential component of the ryanodine receptor and other cation channels (Saimi and Kung, 2002). It has been biochemically purified and characterized in dinoflagellate Crypthecodinium cohnii (Sako et al., 1989). In another dinaflagellates, Prorocentrum donghaiense Lu, CaM was verified by immunoassay (Liu et al., 2006; Guo et al., 2009). The deduced protein CaM (149aa) isolated from A. catenella showed 98.7%–100% similarity to those of other 5 dinoflagellates, and 91.3%–92.6% similarity to those of high plants and animals (Fig.2). The most variable sequence is the central helix connecting two EF-hands, which can benefit the reaction of CaM with downstream target protein (Babu et al., 1988). The CaM from Chlamydomonas reinhardtii and Chlamydomonas incerta showed the least similarity to tjhose from other species. The unique sequences of Chlamydomonas resulted in the increasing maximal activation of NAD kinase (Lukas et al., 1985). From this point, Chlamydomonas species has developed a different route for CaM evolution. In high plants, CaM is encoded by multiple genes that have been shown to be expressed differently (Ling et al., 1991; Lee et al., 1995; Yang et al., 1996; Reddy et al., 2002). However, further study is needed to verify the existence of different CaM isoforms in dinoflagellates and see if they follow the plant model.

In this study, cam transcription increased more than 8 folds from the lag phase to the exponential phase, andobviously decreased from exponential phase to exponential/decline phase in A. catenella. The expression level of cam showed a similar trend with the cell growth rate through the whole growing stage. The result suggested that CaM acts as a regulator of cell growth in dinoflagellates as has been demonstrated in high plants and animals (Sasaki and Hidaka, 1982; Eilam and Chernichovsky, 1988; Davidkova et al., 1996; Means, 1994). Considering CaM function mentioned previously, the cell growth in this species may be related to Ca2+involved signal transduction process. In addition, CaM has also been suggested to be involved in methylation of DNA and toxin precursors (Lin, 2011), thus the possibility of methylation process during cell growth cannot be excluded. However, CaM is not the only gene involved in growth control. As Toulza (2010) reported, up to 25% of genes identified by expressed sequence tags (ESTs), including 21236 clustered sequences represented enzymes and proteins that participate in a variety of cellular regulatory mechanisms, may characterize proliferating cells.

Recent studies indicated that CaM is also a key Ca2+sensor for organisms’ response to heat stress (Liu et al., 2007; Zhang et al., 2009). In plants, such as maize (Zea mays) and tobacco seedlings, Gong et al. (1997, 1998) observed that heat shock led to a transient increase in Ca2+concentration and the up-regulation of the protein level in CaM. In the present study, the expression level of cam in A. catenella was down-regulated under heat stress, which is different from the findings of other studies in high plants (Zhou et al., 2009). This is the first report about the response of CaM gene to heat stress in dinofalgellate, which suggested the restricted Ca2+-involved signal transduction and methylation events. This study can help to understand the mechanisms underlying growth and stress-response in Alexandrium species. However, the mechanism of down-regulated transcription profile in A. catenella needs further study.

5 Conclusions

We reported full length of calmodulin cDNA sequence of A. catenella. The deduced amino acid sequence showed a high degree of similarity with other CaMs. The variation of expression level of cam was consistent with growth rate during the whole growth process. Moreover, the expression level of cam in A. catenella was downregulated under heat stress. Considering CaM function in other organisms, the cell growth in this species may be related to Ca2+-involved signal transduction process and the methylation of DNA and toxin precursors. The response of CaM gene to heat stress in dinofalgellate suggested the existence of restricted Ca2+signal transduction and methylation events.

Acknowledgements

This work was supported by Specialized Research Fund for the Doctoral Program of Higher Education, China (SRFDP 20100132110007) and Shandong Provincial Natural Science Foundation, China (ZR2011DZ002).

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(Edited by Qiu Yantao)

(Received June 15, 2012; revised July 31, 2012; accepted December 4, 2012)

? Ocean University of China, Science Press and Springer-Verlag Berlin Heidelberg 2014

? Corresponding author. E-mail: suizhengh@ouc.edu.cn