Identification of Two Plastid Proteins in the ... - ACS Publications

Identification of Two Plastid Proteins in the Dinoflagellate Alexandrium affine That Are Substantially Down-Regulated by Nitrogen-Depletion Fred Wang-Fat Lee,† David Morse,‡ and Samuel Chun-Lap Lo*,†,§ The Proteomic Task Force, Department of Applied Biology and Chemical Technology, The Hong Kong Polytechnic University, Hong Kong, Institut de Recherche en Biologie Ve´ge´tale, De´partement de Sciences Biologiques, Universite´ de Montre´al, Canada, and State Key Laboratory of Traditional Chinese Medicine and Molecular Pharmacology, Shenzhen, China Received May 29, 2009

The formation of harmful algal blooms (HABs) by dinoflagellates has been correlated with the nitrogen load in coastal waters. Nitrogen is implicated as an important factor in the initiation and maintenance of phytoplankton blooms. To characterize the cellular response to nitrogen, 2DE was used to compare protein expressions from dinoflagellates grown under nitrogen depleted and nitrogen replete conditions. A total of 17 differentially expressed protein spots were found, nine of which showed a roughly 16fold decrease in N-depleted conditions. Five of these nine spots were all identified as isoforms of the plastid Form II ribulose-1,5 bisphosphate carboxylase/oxygenase (Rubisco II), while an additional four protein spots with a molecular weight of 50 kDa were identified as isoforms of a novel protein named nitrogen-associated protein 50 (NAP50). NAP50 was located in the plastids as shown by the presence of an N-terminal plastid targeting leader sequence and by immunohistochemistry. Levels of both Rubisco II and NAP50 decrease sharply between 24 and 36 h following nitrogen depletion and the decrease can be blocked if the N source is replenished before degradation occurs. Both proteins are rapidly resynthesized if the nitrogen source is replenished after degradation has occurred. These results are a first step in the dissection of the behavior of the dinoflagellate proteome under nitrogen stress conditions and may provide new insights into the relationship between dinoflagellate blooms and the nitrogen budget. Keywords: Plastid proteins • Dinoflagellates • Nitrogen stress • proteome • Rubisco II

Introduction Dinoflagellates are one of the major causative agents of Harmful Algal Blooms (HABs) and the mechanism of these sudden algal blooms is still poorly understood. There is increasing evidence that a global increase in nutrient levels in coastal waters through riverine and sewage inputs,1,2 atmospheric deposition3 and bottom layer inputs4 are the major causes of algal blooming. Although the exact extent to which an increase in occurrence of HABs can be attributed to the increase in nutrient levels is not known, a strong relationship exists between frequencies of algal blooming and the nitrogen load of coastal waters. For example, a 5-fold increase in dissolved nitrate levels between 1978 and 1985 in various locations (including Tolo Harbour, Hong Kong, the Japanese * Corresponding author: Prof. Samuel Chun-Lap Lo, The Proteomic Task Force, Department of Applied Biology and Chemical Technology, The Hong Kong Polytechnic University, Hong Kong SAR, China. Tel: (852)-34008669. Fax: (852)-23649932. E-mail: [email protected]. † The Hong Kong Polytechnic University. ‡ Universite´ de Montre´al. § State Key Laboratory of Traditional Chinese Medicine and Molecular Pharmacology.

5080 Journal of Proteome Research 2009, 8, 5080–5092 Published on Web 09/18/2009

and North European coastal waters) coincided with increased incidences of HAB (from 2 cases in 1978 to 17 cases in 1984).2,5,6 Nitrogen is believed to be an important factor in the initiation and maintenance of phytoplankton blooms.7,8 For example, outbreaks of high density Cochlodinium polykrikoides blooms in the coastal seawaters in Korea were correlated with high levels of nitrate initiated by inflows of rainfall.9 Nitrogen in both organic and inorganic forms is generally available for phytoplankton from either endogenous or exogenous nitrogen sources. Recent studies have shown that dinoflagellate blooms tend to associate with high nitrogen concentrations, in particular the reduced forms such as ammonium and urea.10,11 Smayda (1989) proposed that phytoplankton growth in the sea is often limited by nutrient availability,12 and in this view, nitrogen addition would relax nutrient limitation thus causing “blooms” of these cells. High concentrations of nitrogen are usually found in discharge areas such as bays and estuaries,13 which accelerates growth and cellular metabolism. Thus, both the amount and the form of nitrogen supply can be important regulatory factors for growth and cellular metabolism in dinoflagellates. Many studies have already been carried out to address the effects of nitrogen loads on the growth rate, cell density, cell 10.1021/pr900475f CCC: $40.75

 2009 American Chemical Society

Identification of Two Plastid Proteins in A. affine size, pigment composition and toxin production in dinoflagellates.9,14-19 However, very little is known about the biochemical and molecular mechanisms that bring about the dinoflagellate responses to an increase in nitrogen loads. Further, there is no protein expression studies aimed at understanding the cellular responses of dinoflagellates to the availability of nitrogen. In the present study, two-dimensional gel electrophoresis (2DE) and protein sequencing was used to identify Alexandrium affine proteins differentially expressed under nitrogen depleted and nitrogen replete conditions. We found that levels of two plastid proteins, rubisco II and NAP50, decreased dramatically following nitrogen depletion. We also found that this decrease may be regulated by a complex interplay of specific degradation and translational control mechanisms. Nevertheless, our results reveal an unexpected link between nitrogen availability and the levels of specific plastid proteins that may help explain the molecular responses of dinoflagellates to changing nitrogen loads.

Materials and Methods Dinoflagellate Species and Culture Conditions. The unialgal culture of A. affine used in this study was isolated locally from Junk Bay in August 1998. Taxonomic identification of A. affine was confirmed by their ITS sequence (accession no. EF579793) and protein profiling mass spectrum.20 The synthetic seawaterbased f/2 media used for culturing the dinoflagellates21 was prepared from Instant Ocean (USA) using ddH2O to a salinity of 25 parts per thousand (ppt) (salinity was checked routinely with refractrometer) and stored at 4 °C before use. All synthetic seawater was filtered with 0.45 µm nylon membrane (Millipore, Billerica, MA) and autoclaved in Teflon culture bottles prior to subculture. Nutrients required to make up f/2 medium21 were added to the autoclaved synthetic seawater aseptically. Unless stated otherwise, all chemicals were obtained from Sigma (St. Louis, MO). Stock cultures of A. affine cells were kept at exponential growth phase by transferring to new medium every 5 or 6 days in a ratio of 1:10 (v/v). Vegetative cells from cultures in mid- or late-exponential phase of growth were inoculated into freshly prepared culture medium. Possible contamination of algal culture was monitored by regular microscopic examination. The cultures were grown at 22 °C under a 16:8 h light/ dark cycle at a light intensity of 120 µE m-1 s-1 provided by cool white fluorescent tubes in a Conviron growth chamber (Model EF7). Cell Counts. Cell density was measured at the same time each day from a 1 mL aliquot removed and fixed with 10 µL of Lugol’s solution. Cells were counted under a light microscope using a Sedgwick-Rafter cell counter. Effects of Nitrogen on the Growth of A. affine. For nitrogen depletion, roughly 105 cells were harvested from midlog phase culture by centrifugation (1500g for 10 min at room temperature), washed twice with sterile synthetic seawater, and inoculated into 100 mL of f/2-medium without added nitrogen to yield an initial cell density of 500 cell mL-1. For nitrogen replete conditions, the cells were similarly harvested and washed, but were inoculated into culture medium containing different nitrogen sources such as urea (200 µM-N), L-aspartate (10 µMN), glycine (10 µM-N), ammonium (40 µM-N) and nitrate (200 µM-N). In other experiments, the different nitrogen sources were added to nitrogen depleted cultures at day 3, day 8, or day 13. Cell growth was monitored every day. The whole experiment was repeated three times, with triplicates in each

research articles run. Each data point on the curve represents the mean of triplicate results in one single run. Results shown are a representative example of the 3 experiments. Proteomic Analysis of A. affine Cells Grown under N-Depleted and N-Replete Conditions. 2DE using proteins isolated from A. affine cells grown under nitrogen depleted and nitrogen replete conditions were used to find proteins whose levels differed in the two conditions. To prepare the nitrogen-depleted samples, A. affine cells were grown to late-log phase (day 6) (10 000-13 000 cells mL-1) and harvested by centrifugation (1500g, 15 min) at room temperature. The pellets were washed twice with sterile synthetic seawater lacking nitrogen to avoid any carryover of nitrogen from the previous medium. The pellets were then inoculated into new medium without nitrogen and the initial cell concentrations determined by counting. Cells harvested at 48 h from these cultures were used as “Ndepleted” samples. For the preparation of nitrogen-replete samples, sodium nitrate (200 µM) was added to the 48 h nitrogen depleted cultures, and the cells were harvested either 24 or 48 h following addition of nitrate. The cell number in all cultures was determined by counting under the microscope before harvesting. The harvested cell pellets were stored at -80 °C until use. Preparation of Protein Extracts. Proteins were extracted according to the optimized method developed previously.22 Briefly, cell pellets using either nitrogen-depleted or nitrogenreplete cultures were resuspended in 1 mL of Trizol reagent (Roche, Switzerland). The cells were lysed by short pulses of sonication for a total of 3 min on ice, and cell lysis was confirmed by light microscopy. Subsequently, 200 µL of chloroform was added to the cell lysate and the mixture was shaken vigorously for 15 s. The mixture was allowed to stand for 5 min at room temperature before being centrifuged at 12 000g for 15 min at 4 °C. The top pale-yellow or colorless layer was removed. Three hundred microliters of ethanol was added to the reddish bottom layer and the mixture was centrifuged at 2000g for 5 min at 4 °C. The supernatant was transferred to a new tube and 1.5 mL of isopropanol was added. The mixture was allowed to stand for at least 20 min for complete precipitation of proteins, then centrifuged at 14 000g for 10 min at 4 °C. The pellet obtained was briefly washed with 95% ethanol and allowed to dry in air. Lysis buffer (40 mM Tris, 7 M urea, 2 M thiourea, 4% CHAPS, 0.2% DTT) was added to solubilize the protein pellet before loading onto the first dimension IEF. Determination of Protein Concentration. Protein quantification in the urea-containing protein samples was performed using a modified Bradford protein assay (Bio-Rad, Hercules. CA) as described previously.23 2-DE and Imaging Analysis. Typically, a 340 µL sample containing 80 µg of protein (for silver staining) or 700 µg of protein (for Coomassie blue staining) in rehydration buffer (containing 7 M urea, 2 M thiourea, 4% CHAPS, 0.2% DTT and 3.4 µL of pH 4-7 or pH 3-10 IPG buffer) was used to rehydrate 18 cm pH 4-7 or pH 3-10 IPG strips (Bio-Rad, Hercules, CA) for 16 h. IEF was performed using a Protean-IEF cell (Bio-Rad, Hercules, CA). Voltage was applied according to the following schedule: 1 h at 100 V, 2 h at 300 V, 2 h at 1000 V, 2 h at 4000 V, and 5 h at 8000 V. Following IEF, the gel strip was equilibrated with equilibration buffer (50 mM Tris, pH 8.8, 6 M urea, 30% glycerol, 2% SDS, 1% DTT, and trace amounts of bromophenol blue) for 15 min. The gel strip was then placed in fresh equilibration buffer containing 1% iodoacetamine Journal of Proteome Research • Vol. 8, No. 11, 2009 5081

research articles (instead of DTT) for a further 15 min. The second-dimension SDS-PAGE was performed using 10% polyarylamide gel running at a constant current of 15 mA/gel until the bromophenol blue dye reached the end of the gel. After electrophoresis, the gel was either stained with silver or with Coomassie blue R250 and scanned using a Perfection 1200U scanner (Epson). The images were analyzed by Melanie III (GeneBio, Switzerland) as described in the user manual. Each sample was analyzed by 2-DE in triplicate and only proteins consistently present in all three gels were considered. A 10-fold difference in spot optical density was taken as a cutoff for differentially regulated proteins. Each protein spot has to be present in all 3 triplicates before it is regarded as real. Each experiment was repeated three times. Gels shown in the Results section are representatives of the triplicates. MALDI-TOF MS Analysis. Differentially expressed proteins were excised from the Coomassie blue stained gel and transferred to a microcentrifuge tube. The gel plugs were washed twice in 25 mM NH4HCO3 in 50% acetonitrile (ACN). Subsequently, the gel plugs were washed with 100% ACN and dried under vacuum for 10-15 min. In-gel trypsin digestion was performed by adding 20 ng/mL of trypsin in 25 mM NH4HCO3 overnight at 37 °C. For MALDI-TOF mass spectrometry analysis, 1 µL of peptide mixture was mixed with 1 µL of matrix solution (HCCA, saturated solution in ACN/0.1% TFA (1:1)) on the target plate before being dried and analyzed with a MALDI-TOF mass spectrometer (Autoflex; Bruker, Germany) in reflectron mode over a mass range of 1000-3000 Da and using external mass calibration with calibration standards from the manufacturer. Spectra from 150 shots at several different positions on the target plate were combined to generate a peptide mass fingerprint (PMF) for bioinformatic database searches. Each PMF was searched against the NCBI nonredundant database using the search engine MASCOT (miscleavages were set at 0 and mass tolerance was set at 150 ppm). N-Terminal Sulfonation and Postsource Decay (PSD) Analysis. De novo peptide sequencing was performed with the aid of N-terminal sulfonation by methodologies described previously.24 Briefly, 10 mg/mL 4-sulfophenyl isothiocyanate (SPITC) was added to the tryptic peptides and incubated at 55 °C for 30 min for the sulfonation reaction to occur. Sulfonated peptides were cleaned by absorption and subsequent elution from zip-tips before being analyzed with MALDI-TOF mass spectrometer using postsource decay (PSD) as described in the user manual (Autoflex, Bruker, Germany). The acquired PSD spectrum was analyzed by the de novo sequencing function in Biotools 3.0 (Bruker, Germany). Amino acid sequences were deduced from the ladder sequences (spectrum) obtained (with mass tolerance within (0.3 Da mass differences between adjacent peaks) and were searched against the NCBI nonredundant database. Liquid-Chromatography Linked Tandem Mass Spectrometry (LC-MS/MS) Analysis. Proteins excised from a Coomassie blue stained 2-D gels were digested by trypsin as described above. Dried peptide extracts were redissolved in 20-60 µL of water, depending on the staining abundance of the protein spots, and 10 µL was loaded using an autosampler on a nano-LC-MS/MS Ultimate 3000 system (Dionex, Netherlands) interfaced online to an ion-trap mass spectrometer HCTultra (Bruker, Germany). The mobile phase was composed of 100% H2O (solvent A) and 20:80 (v/v) H2O/ACN (solvent B). Peptides were first loaded onto a trapping microcolumn C18 PepMAP100 at a flow rate of 20 mL min-1. After 4 min, they were back-flush eluted and 5082

Journal of Proteome Research • Vol. 8, No. 11, 2009

Lee et al. separated on the same nanocolumn at a flow rate of 300 nL min-1 in a mobile phase gradient from 0 to 50% of solvent B over 60 min, from 50-90% B over 10 min, then 100% B for 10 min (% B refers to the solvent B content in an A and B mixture). Peptides were infused into the ion-trap mass spectrometer via a dynamic nanospray probe and analyzed in positive mode. The three most abundant precursor ions detected in the full MS survey scan (m/z range of 350-1500) were isolated within a 4.0 amu window and fragmented by tandem mass spectrometry. MS/MS fragmentation was triggered by a minimum signal threshold of 500 counts and carried out at the normalized collision energy of 35%. MS/MS spectra were analyzed by Data Analysis Software from Bruker (Bruker, Germany) and de novo sequences were predicted with Biotools 3.0 (Bruker, Germany). Edman Microsequencing. The N-terminal of NAP50 was determined by N-terminal Edman protein sequencing. Briefly, NAP50 from the 2-D gels was transferred onto PVDF membrane as mentioned previously. The PVDF membrane-bound proteins were visualized by staining with 0.1% Coomassie blue R-250 in 40% methanol for 1 min, and then destained in 40% methanol and 10% acetic acid. The piece of PVDF membrane containing NAP50 was loaded onto an Applied Biosystems 494 Procise protein sequencer on a pulsed liquid sequencing method as recommended by the manufacturer. At least 7 cycles of N-terminal sequencing were run. In case where internal sequences were needed, the gel plug containing NAP50 was excised and processed before N-terminal sequencing (see below for procedures). These protein spots, after being excised from Coomassie stained 2-D gels, were washed twice with milli-Q water. Subsequently, the samples were reduced and alkylated in-gel with DTT and iodoacetamine, before being digested with trypsin in-gel at 37 °C for 16 h. The peptides were extracted from the gel and fractionated by RP-HPLC using a 1.0 mm diameter column with a 100 µL min-1 flow rate. Two minute fractions were collected into 96-well plates. The eluting fraction that appeared to contain a single peptide was selected for N-terminal sequencing. Ninety microliters of the fraction was loaded onto a biobrene-treated, precycled glass fiber filter and subjected to 13 cycles of Edman N-terminal sequencing using an Applied Biosystems 494 Procise Protein Sequencing System on a Pulsed Liquid sequencing method (according to instructions from the manufacturer). Performance of the sequencer is assessed routinely with 10 pmol β-Lactoglobulin standard. cDNA and Deduced Amino Acid Sequence of NAP50. Two hundred milliliters of A. affine cell culture, grown as above, was harvested during exponential growth by centrifugation at 1500g for 15 min at 4 °C. The cell pellets were resuspended in Trizol reagent (Roche, Switzerland) for RNA isolation according to instructions from the manufacturer. First-strand cDNA was synthesized from approximately 2 µg of total RNA using SuperScript III RT (Invitrogen, Carlsbad, CA) and oligodT primers according to the instructions of the manufacturer. Synthesized cDNA was used as template for PCR, with degenerate primers designed according to the peptide sequences of NAP50 (Table S1). PCR was performed by a denaturing step of 95 °C for 5 min, followed by 35 cycles of 94 °C for 45 s, 55 °C for 45 s, and 72 °C for 2 min, then finished by a final elongation of 72 °C for 10 min. The 3′ ends of NAP50 cDNA were obtained using the 3′ RACE methodology (GeneRacer kit, Invitrogen, Carlsbad, CA). This method was originally designed to synthesize and amplify cDNA selectively from mRNA molecules that have a poly (A) tail on the 3′ end. For the subsequent nested PCR, precisely matched primers (Table S2) were designed based

Identification of Two Plastid Proteins in A. affine

Figure 1. Light microscopic picture of A. affine.

on the partial cDNA sequences obtained. The 5′ extended sequences of NAP50 cDNA were obtained using 5′ RACE method with gene-specific primer NAP_rt_r (Table S3) as described.25 PCR was carried out at 95 °C for 5 min; then, 35 cycles of 94 °C for 45 s, 50 °C for 45 s, and 72 °C for 1 min; and finally, 72 °C for 10 min. All PCR products were cloned into pGEM-T easy vectors (Promega, Madison, WI) and the clones sequenced at a commercial facility using traditional dideoxymethodology. Analysis of mRNA Expression Level by Real-Time PCR. A. affine total RNA was isolated from cells using the Trizol reagent (Roche, Switzerland) according to the manufacturer’s instructions. The RNA preparation was then subjected to on-column digestion with RNase-free DNase I from the RNeasy Mini kit (Qiagen, Germany) to remove contaminating genomic DNA. Samples were checked for residual genomic DNA by standard PCR or by real-time PCR using the corresponding primers (Table S3). RNA samples were deemed to be free of genomic DNA if no amplification product was detected by agarose gel electrophoresis (or by real-time PCR) after at least 30 cycles of amplification. Reverse transcriptase PCR (RT-PCR) reactions were performed with 100 ng of total RNA and iQ SYBR Green Supermix (Bio-Rad, Hercules, CA) according to the manufacturer’s instructions. PCR reactions were performed with the following conditions: denaturation at 94 °C for 45 s, annealing at 55 °C for Rubisco II (60 °C for NAP50) for 45 s., and elongation at 72 °C for 1 min for 35 cycles. For real-time RTPCR, first-strand cDNA was prepared using Superscript III Plus RNase H- Reverse Transcriptase (Invitrogen, Carlsbad, CA) with random hexamer primers as described in the protocol provided by the manufacturer. The resulting cDNA was amplified using the Smart Cycler system (Cepheid, Sunnyvale, CA) in a final reaction volume of 25 µL containing 3 µL of first-strand cDNA, 0.3 µM of each primer, and 12.5 µL SYBR-Green supermix (BioRad, Hercules, CA). The amplification conditions for real-time PCR were as follows: denaturation at 95 °C for 45 s, annealing at 55 °C (60 °C for NAP50) for 45 s, and elongation at 72 °C for 45 s for 45 cycles. The gene-specific primers used in RT-PCR and in the real-time PCRs are listed in Table S3. The threshold cycle for each real-time PCR was determined from a second derivative plot of total fluorescence as a function of cycle number using the software package supplied with the Smart Cycler system. Real-time PCRs were carried out for three

research articles batches of experiments with duplicates in each batch. After some real-time PCRs, the end-point amplification products were visualized by electrophoresis in 1% agarose gels. Determination of real-time PCR efficiencies of reference gene (βactin), target genes (NAP50 and rubisco II) with their corresponding primers and the relative mRNA expressions were calculated according to procedures described previously.26,27 Production of Antibodies against NAP50. Antibodies were raised in female Sprague-Dawley rats. Care of these rats followed the Code of Practice for Care of the Animals of The Hong Kong SAR Government, and the experimental protocol described below was approved by the Animal Subjects Research Ethics Subcommittee of the Hong Kong Polytechnic University. Coomassie blue stained spots of NAP50 were excised from 2-DE gels for preparation as immunogen. Before injection into rats, identities of excised NAP50 spots were validated by comparing their PMFs to that of NAP50. To have enough antigens for immunization, 8-10 gel-plugs of the same protein were pooled. After excision from the 2-DE gels, these gel-plugs were first destained in destaining solution (40% methanol, 10% acetic acid) then washed extensively with PBS (pH 7.4) until the pH of the washing buffer was near 7.4. Subsequently, these gel plugs were homogenized into a slurry-like suspension which was then mixed 1:1 with either Complete or Incomplete Freund’s Adjuvant (Sigma, St. Louis, MO) for initial injections or subsequent booster shoots, respectively. One milliliter of the emulsion was injected into each rat subcutaneously near the hind limbs. The process was summarized in Table S4. To gauge the antibody production process, periodical bleeding was performed to obtain 1 mL of blood from each immunized rat. The blood was allowed to clot at room temperature for 30 min then stored overnight at 4 °C. Subsequently, the blood was centrifuged at 1500g at 4 °C for 20 min. The clear supernatant containing polyclonal antibodies was used as the antiserum and was aliquoted before storage at -20 °C. Transmission Electron Microscopy. Roughly, 100 mL of A. affine cells in their exponential growth phase was harvested and fixed with 1 to 2% glutaraldehyde in 0.3 M phosphate buffer, pH 7.4, for 35-60 min. Anti-NAP50 IgG was purified by HiTrap Protein G HP Columns according to procedures described by the manufacturer (GE Healthcare, Piscataway, NJ). Affinity purified IgG from anti-NAP antiserum was used as the primary antibody. Transmission electron microscopy was performed using a JEOL JEM 100S microscope operating at 80 kV. Immunostaining using thin-sectioned LR White embedded samples was performed essentially as described28 using a 1/50 dilution of the primary antibody in TBS containing 3% BSA and a 1/200 dilution of a 20-nm gold-conjugated secondary antibody in the same buffer (Ted Pella, Redding, CA). No significant difference was observed in cell morphology or labeling intensity using the different fixation treatments, and preimmune serum did not show any significant labeling on the sections. Immunoblotting Analysis. Following either 1D or 2D electrophoresis, proteins were electroblotted onto nitrocellulose membranes at 100 V for 2 h at 4 °C. The membrane was then rinsed with TBS buffer (20 mM Tris-HCl and 137 mM NaCl, pH 7.6) and blocked with 5% BSA in TBS buffer for 2 h at room temperature. After washing six times with TBS buffer containing 1% Tween 20 (TBST buffer), the membrane was probed either with anti-NAP50 (1:4000) or anti-rubisco II (1:3000). After incubation for 1 h at room temperature, the membrane was washed again before a 1 h Journal of Proteome Research • Vol. 8, No. 11, 2009 5083

research articles

Lee et al.

Figure 2. The effect of nitrogen-depletion and repletion on the growth of A. affine. Different nitrogen sources, (a) nitrate, (b) ammonium, (c) urea, (d) glycine, and (e) aspartate, were used initially or added to a nitrogen-depleted culture at the indicated times (arrows). Closed circles indicate the cell growth under nitrogen-depleted condition. Closed squares indicate the normal cell growth with nitrogen provided. Open symbols indicate nitrogen was added to the nitrogen-depleted culture at either day 3 (triangles), day 8 (circles), or day 13 (squares).

Cruz Biotechnology, Santa Cruz, CA) for anti-NAP50 or with peroxidase-conjugated anti-rabbit secondary antibodies diluted 1:100 000 with 1% BSA in TBS (Santa Cruz Biotechnology, Santa Cruz, CA) for anti-rubisco II. After washing six times with TBST buffer, levels of rubisco II and NAP50 were assessed by a chemiluminescence based method using Supersignal Chemiluminescent Substrate kit (Pierce Chemical, Rockford, IL). The procedures were performed according to instructions from the manufacturer.

Figure 3. 2-DE protein expression profiles of 700 µg of protein extracts of A. affine over a pH range of 4-7. Protein samples from nitrogen-depleted cultures (right panel) and nitrogen-replete cultures taken 48 h after nitrate addition (left panel) were subjected to 2-DE and proteins visualized by Coomassie blue staining. Proteins whose levels vary at least 10-fold between the two treatments are circled and numbered. (Gels shown are the representative of triplicate experiments.)

incubation with either peroxidase-conjugated anti-rat secondary antibodies diluted 1:2000 with 1% BSA in TBS (Santa 5084


Results and Discussion Effects of Nitrogen Stress on the Growth of A. affine. Most marine systems are nitrogen-limited,12 as addition of nitrogen usually causes phytoplankton to grow rapidly or “bloom” until other nutrients are exhausted. Therefore, understanding the cellular responses to the presence or absence of nitrogen is important to understand the “blooming” mechanism. The dinoflagellate genus Alexandrium contains some of the most studied HAB causative agents as some species produce paralytic shellfish toxins. A fast-growing Alexandrium species

Identification of Two Plastid Proteins in A. affine Table 1. Proteins Pinpointed by 2-DE Whose Levels Vary by at Least 10-fold Following Nitrate Depletion

research articles times in nitrogen-depleted medium (i.e., at day 3, day 8 and even day 13). These results indicate that nitrogen-depleted cells maintain a “ready-to-grow” status for a long period as growth can be resumed upon nitrogen addition. The essential features of nitrate-dependent growth are recapitulated when various other nitrogen sources are used (Figure 2b-e). Differential Protein Expressions of A. affine under Nitrogen Repletion and Depletion. Although many studies have been carried out to understand the effects of nitrogen sources on the dinoflagellates,9,14-19 little is known about the biochemical and molecular responses of the cells to nitrogen availability. To address this, we compared two-dimensional electrophoresis protein patterns from A. affine cells grown under nitrogendepleted and nitrogen-replete conditions. Initially, 2-D gels with a pH 3-10 range were used (data not shown), but most of the proteins were located at the acidic side of the gels as described previously by Chan et al.29-32 To improve the resolution on the 2-D electropherogram, the pH range was reduced to 4-7 (Figure 3).

a Fold changes in protein levels detected by comparing optical intensity of spots in N-replete extracts with those of N-depleted extracts after analysis of 2-DE using the Melanie 3 software. *Protein spot with over 15-fold difference and selected for amino acid sequencing.

(Figure 1) was selected as the model strain for the present study and it was identified as A. affine from analysis of the ribosomal gene sequence (accession no. EF579793). Growth of A. affine is clearly dependent on nitrogen, even though many different nitrogen sources can be used (Figure 2). The growth rates measured in three independent batch culture experiments were highly similar for all nitrogen sources at the optimal concentrations (see Supporting InformationTable S5 and Figure S1) (data for experimental runs 2 and 3 were not shown). Nevertheless, the highest maximum growth rate and cell abundance was obtained in nitrate-enriched cultures, suggesting that nitrate is the preferred nitrogen source for this strain. With 200 µM-N nitrate as the sole nitrogen source (Figure 2a), A. affine grew to a maximum cell density of 12 000 cells mL-1 with a maximum growth rate of 0.9 day-1. In the nitrogen-depleted cultures, nitrate concentrations as measured by the Nitrate Test Kit (NECi, Lake Linden, MI) are below the limit of detection (0.05 ppm nitrate-N, equivalent to 3.6 µM nitrate). The small increase in cell numbers observed in the first 2 days may thus be supported by intracellular N pools or other N nutrient reserves in the organism which provide residual substrates for growth.15 After the first 2 days, cell growth started to slow down and cell numbers remained constant at a basal cell density at around 500-700 cell mL-1 throughout the next 14-18 days. When nitrate was added to the nitrogen-depleted cultures, cell growth resumed after a lag phase of about 1 day. The growth patterns, growth rates and the maximum cell density of the nitrogen-replete cultures were highly similar to those of normal cultures. Moreover, cells resumed normal growth when nitrate was added after different

When gels loaded with equal amounts of protein are compared, the staining intensities of several groups of proteins are clearly different in nitrogen-depleted and nitrogen-replete conditions (circled in Figure 3). To locate proteins of the A. affine proteome that were differentially expressed in these conditions, 2-D gel images were analyzed using the Melanie 3 software (Genbio, Switzerland). A total of 1736 and 1853 proteins were detected on 2-D gels from nitrogen-depleted and nitrogen-replete samples, respectively. However, only 17 protein spots with at least a 10-fold difference in protein expression level (Figure 3, spots 1-17) were observed. Of these 17 protein spots, 12 showed at least 15-fold differences in expression level (Table 1). These proteins were named the “Nitrogen Associated Proteins” (NAPs) because of their tight association with the availability of nitrogen, and are thus potentially involved in cellular processes that may facilitate rapid growth at different nitrogen concentrations. Protein Identification by MALDI-TOF Mass Spectrometry. To identify the differentially expressed proteins, in-gel tryptic digestion and MALDI-TOF MS were used initially to produce peptide mass fingerprints (PMFs) (Table 1). However, out of all 17 differentially expressed protein spots, only 5 protein spots were successfully identified. Bioinformatics searches from the NCBInr databases using the Mascot search engine with the PMFs showed that spots 1-5 (see Supporting Information, Figure S2) all corresponded to ribulose 1,5-bisphosphate carboxylase/oxygenase II (rubisco II). The fact that the PMFs of these 5 protein spots were nearly identical, coupled with the observation that they all had the same molecular weight (55 kDa) and slightly different pI points (pI ranged from 5.4-5.7, Figure 3), strongly suggested that these 5 proteins represented different Rubisco II isoforms. To confirm this, two peptides (1000.431 and 1537.916 m/z) from the digested proteins were sulfonated, and the amino acid sequences were analyzed by MALDI-PSD (see Supporting Information, Figure S3). Sequence tags QF[I/L]HYHR and YW[I/L]S[I/L]TEED[I/L][I/L]R were obtained (the isobaric pair isoleucine and leucine are denoted as I/L) (Table 2). These sequence tags were also used to search against the NCBI nr database. The sequence tag QF[I/L]HYHR matched with the sequence QFLHYHR of the Symbiodinium spp. rubisco II (accession no. AAY51977), while sequence tag YW[I/L]S[I/L]TEED[I/L][I/L]R matched with the sequence YADLSLTEEDLIK of the Prorocentrum minimum rubisco II Journal of Proteome Research • Vol. 8, No. 11, 2009 5085

research articles

Lee et al. a

Table 2. Peptide Sequences of the Differentially Expressed Spots spot b

protein

1-5

Rubisco II

6-9b

NAP 50

10c

NAP 40

11c

NAP 58

12c

NAP 25

peptide mass (m/z)

1000.4(1+) 1537.9(1+) 1152.5(1+) 626.2(2+) 672.2(2+) 654.2(2+) 1237.5(1+) 952.4(1+) 1076.4(1+) 1819.2(1+) 886.4(1+) 1682.8(1+) 1814.1(1+)

amino acid sequences

sources

QFI/LHYHR YWI/LSI/LTEEDI/LI/LR YHFATMNI/LR QVETEASNMK AFADWNAEYEd FFEAESADY GFKDDFDAWRd,e GIWEELPKd AXAI/LNGFGR NXYDNEWGYSXR YFAGXI/LR EXADI/LXAADHFR NXYSQI/LTYNQVR

Sulfonated-MALDI-PSDf

LC MS/MSg

Edman microsequencing Sulfonated-MALDI-PSDf

a

X represents a gap in the peptide sequence which contains one or more amino acids. b Isoforms of the same protein. PMF of protein spots are shown in Supporting Figures S2 and S4. c PMF of protein spots 10, 11, 12 are shown in Supporting Figures S8, S9 and S10, respectively. d Amino acid sequences selected for design of degenerate primers for NAP50 amplification. e N-terminal sequence of NAP50 as confirmed by N-terminal Edman sequencing. f Sulfonated MALDI-PSD spectrum are shown in Supporting Figures S3, S4, S8, S9 and S10. g LC-MS/MS spectra are shown in Supporting Figures S5, S6 and S7;

(accession no. AAO13049.1). Protein sequencing thus indicates spots 1-5 most likely represent A. affine rubisco II. De Novo Protein/Peptide Sequencing. Because of the limited dinoflagellate DNA and protein sequence information available in the NCBI database, it is not surprising that bioinformatic searches using PMFs obtained from the other 12 proteins were not successful. We therefore attempted de novo sequencing with sulfonation using MALDI-PSD. Protein spots 6-9 were of particular interest because they were present in large amounts in nitrogen-replete cultures and because the decrease in protein amount upon nitrogen-depletion was particularly large. For the remaining proteins, because of their relatively low expression levels, attempts are currently in progress either to obtain a sufficient enough amount for MALDI-PSD analysis, or to perform other amino acid sequencing methods such as Nterminal sequencing and LC MS/MS. PMFs of spots 6-9 were highly similar with almost identical peptide masses (see Supporting Information, Figure S4). These four spots all have a molecular weight of around 50 kDa and similar pI values (ranging from 5.4-5.7; Figure 3; Table 1). These results suggested that these four protein spots could be isoforms of the same protein, which was named nitrogenassociated protein 50 (NAP50). Sequence of A. affine cDNA Encoding NAP50. To determine the complete amino acid sequence of A. affine NAP50, degenerate primers were first designed from the peptide sequences obtained for NAP50 (Table S1). Three PCR products with around 1100 bp (with primers NAP1F + NAP3R), 800 bp (with primers NAP1F + NAP2R) and 300 bp (with primers NAP2F + NAP3R) were obtained, with the sequence of the 1100 bp product including those of the 800 bp and 300 bp products (Data not shown). This indicated that all three PCR products were likely derived from the same gene. To determine the sequence at the 3′ end of the cDNA, 3′ RACE was performed using gene-specific primers close to the 3′ end (Table S2). PCR products of around 400 bp were obtained and the sequence was combined with that of the 1.1 kb fragment to produce a total sequence of around 1.5 kb (Figure 4). This fragment has a single open reading frame that includes the 6 peptide sequences (single- and double-underlined in Figure 4) obtained from the de novo sequencing (Table 2). However, it is likely that the cDNA sequence is incomplete, as the 5′ end of the cDNA encodes the sequence of the N-terminal end of the 5086


protein as determined by Edman sequencing (Figure 4, double underlined amino acids), and there is no N-terminal methionine. Therefore, we speculated a potential subcellular targeting information in the form of an N-terminal leader may be present. To determine the extended leader sequence at the N-terminal, 5′ RACE was performed using gene-specific primer (Table S3). A PCR product of around 550 bp was obtained (data not shown). The derived protein leader sequence was found to contain 57 amino acids (Figure 4, dot underline). The first ATG (methionine) is likely to be the actual translational start site, as it has a perfect Kozak context with an A (adenine) at position -3 and a G (guanine) at position +4.33 The derived protein leader sequence was also observed to contain two hydrophobic regions (Figure 5), which reveals a similar characteristic to other dinoflagellate plastid-targeting leader sequences that direct nuclear-encoded proteins to the plastids.34 Therefore, NAP50 is expected to be a nuclear-encoded plastid protein. In addition, when the N-terminal sequence of NAP50 was used to search against the dinoflagellate EST database from the NCBI, we found that it hits some clones of Alexandrium catenella (see Supporting Information, Figure S11). The 5′ end sequence was also observed to contain the 22-nt spliced leader (SL) sequence that is conserved in all nuclear-encoded mRNAs of dinoflagellates (Figure 4, boxed sequence).35,36 This suggests that NAP50 is indeed derived from the dinoflagellate rather than from a bacterial contaminant in our unialgal (but not axenic) cultures. The whole cDNA sequence of NAP50 was submitted to GenBank (NCBI accession no.: GQ480795), The entire derived protein sequence was used in BLAST searches, but no significant homology to known proteins was found. Thus, NAP50 seems to be a novel protein with an as yet undetermined function (see below). Antiserum Raised against NAP50. To investigate the changes in NAP50 under different nitrogen conditions, rat polyclonal antibodies were raised against gel purified A. affine NAP50. When used at a titer of 1/4000 on Western blots, only one band with an apparent molecular weight of 50 kDa was observed to react with the anti-NAP50 (Figure 6a). Control experiments either in the absence of primary antibody or using preimmune serum showed no reaction with A. affine protein extracts (data not shown). Likewise, a previously produced anti-rubisco II antibody28 was shown to react with a single band albeit with the slightly higher apparent


research articles

Figure 4. cDNA sequences that encode NAP50. The derived amino acid sequence of NAP50 contains all peptide sequences (Table 2) obtained from de novo sequencing (single and double underlined). The peptide sequence obtained from N-terminal Edman sequencing is double underlined. Dot underlined sequence is the N-terminal leader sequence of NAP50 and 22-nt in box is the spliced leader (SL) that is conserved in all nuclear-encoded mRNAs of dinoflagellates.

molecular weight of 55 kDa (Figure 6a). 2D-Western blots of total protein extracts from A. affine after 2-DE (Figure 6b) show that the anti-rubisco reacted with the five rubisco II isoforms previously identified as spots 1-5, while the antiNAP50 antibodies reacted with the four isoforms previously identified as spots 6-9. Western blots thus confirm the identity of spots 1-9 as isoforms of either rubisco II or NAP50. Subcellular Localization of NAP50. To confirm the subcellular localization of NAP50, immunohistochemical localization experiments using the specific anti-NAP50 were performed using A. affine cells fixed and sectioned for the

transmission electron microscope (TEM). The position of anti-NAP50 binding was then visualized using gold-labeled secondary antibodies. As shown (Figure 7B), more gold particles were observed over chloroplasts than over other regions of the cells. Counts of the number of gold particles in different subcellular compartments indicated that chloroplast labeling is at least 2.5-fold more intense than labeling of other compartments (Table 3). This plastid-specific labeling is not observed if the specific anti-NAP50 antiserum is replaced with preimmune serum (Figure 7A, Table 3). Together with the N-terminal leader sequence containing two hydrophobic regions found (Figures 4 and 5), we Journal of Proteome Research • Vol. 8, No. 11, 2009 5087

research articles

Lee et al.

Figure 5. Hydrophobicity plots of leader sequence of NAP50. Two hydrophobic regions (underlined) were observed in the predicted leader of NAP50 using four different algorithms including (A) Kyte-Doolittle (window: 11 amino acid), (B) Eisenberg (window: 7 amino acid), (C) Engelman (window: 7 amino acid), and (D) Janin (window: 7 amino acid). Values above the midline in the four graphs represent hydrophobic regions, and values below the midline represent hydrophilic regions.

conclude that NAP50, like rubisco II, is a nuclear-encoded plastid-directed protein in A. affine. Degradation of Rubisco II and NAP50 under NitrogenDepletion. In both prokaryotes and eukaryotes, most cellular proteins are stable in vivo.37 However, normally stable proteins could become unstable when cells are stressed or are exposed to abnormal environments.38 More interestingly, some rapidly degraded proteins have important regulatory roles in metabolic and developmental processes.37-39 To investigate and confirm the changes in rubisco II and NAP50 levels under N-depletion, 5088


immunoblotting experiments were performed (Figure 8). Levels of both proteins were relatively stable in nitrogen-replete conditions (+0, +24, and +48 h) and for short times following nitrogen-depletion (-0, -24 h), whether samples were standardized by equal protein loading (Figure 8a) or by equal cell number (Figure 8b). However, levels of both rubisco II and NAP50 were drastically reduced after 48 h in nitrogen-depleted conditions (Figure 8, -48 h). Results from these immunoblots confirmed early observations from 2-DE that expression of these two plastid proteins was decreased upon nitrogen-


research articles

Figure 6. Western blotting analysis of total protein extracts of A. affine with antibodies raised against NAP50. Protein extracts from nitrogen replete cultures resolved by either (a) 1-D or (b) 2-D electrophoresis and visualized by either Coomassie blue staining (left panels) or immunoblotting with anti-NAP50 and anti-rubisco II antisera. Arrows indicate the position of NAP50 and rubisco isoforms.

Figure 7. Subcellular localization of NAP50 in A. affine cell sections. Cell sections of A. affine incubated first with either (A) preimmune rat serum or (B) a polyclonal anti-NAP50 antibody, then incubated with a 20 nm gold-conjugate rabbit anti-rat secondary antibody, were examined by transmission electron microscopy. Specific anti-NAP labeling is concentrated over chloroplasts (chl) with little labeling associated with cytoplasm (cyt), vacuoles (vac), or trichocysts (tric). Scale bars are 1 µm. Table 3. Anti-NAP50 Labeling 2

average number of gold particles/µm (number of particles) preimmune

Plastid Nucleus Vacuole Cytoplasm

1.3 ( 0.7 (49) 2.5 ( 1.1 (37) 0.4 ( 0.5 (11) 1.3 ( 1.7 (22)

immune serum

immune/preimmune

4.8 ( 3.4 (207) 1.7 ( 3.9 (22) 0.4 ( 1.7 (67) 1.8 ( 2.9 (51)

3.6 0.7 1.0 1.4

depletion. Curiously, Western blotting analysis of samples taken from nitrogen-depleted cells every 2 h suggests levels of both proteins decrease concurrently and rapidly roughly between 30 and 48 h following nitrogen-depletion. This result is indicative of rapid and regulated degradation of the two proteins, and further studies are underway to examine this in more detail. To assess the possibility that the decreased levels of rubisco II and NAP50 protein might correlate with a decrease in their respective transcript levels, real time PCR was performed using RNA extracted during nitrogen-depleted and nitrogen-replete conditions (Figure S12). These results show that mRNA levels for both rubisco II and NAP50 are constant independent of the conditions. This observation thus excludes transcriptional

Figure 8. Western blotting of protein extracts from nitrogenreplete and nitrogen-depleted A. affine with anti-NAP50 and antiRubisco. A. affine cells grown under either nitrogen-depleted conditions (-) or nitrogen-replete conditions (+) for 0, 24, or 48 h, were incubated with either anti-NAP50 or anti-Rubisco II antiserum. Protein loads were standardized either (a) by protein amount, with R-tubulin used as control for protein load, or (b) by cell number; (c) A. affine cells were grown under conditions of nitrogen-depletion for various times (-24, -36, and -48 h), in nitrogen-replete media (+24, +36, and +48 h), in cultures grown in nitrogen-depleted conditions for 24 h after which nitrogen was added for a further 12 h (24 + N) or in cultures grown in nitrogen-depleted conditions for 36 h after which N was added for a further 12 h (36 + N). Western blots used either antiNAP50 or anti-rubisco II antibodies.

control as a possible mechanism for regulating the amount of both rubisco II and NAP50 proteins. To test if other experimental conditions could produce decreases in rubisco II and NAP50 similar to those caused by nitrogen depletion, the levels of both proteins were monitored over the daily cycle, over the entire exponential growth period, and under conditions of phosphate limitation (Figure 9). Immunoblotting results showed that levels of both rubisco II Journal of Proteome Research • Vol. 8, No. 11, 2009 5089

research articles

Lee et al. 41

Figure 9. Protein levels of rubisco II and NAP50 are usually constant. Protein extracts were prepared from cells growing under nitrogen replete conditions at various times over (a) the course of the daily light dark cycle, (b) the growth curve, or (c) conditions of phosphate depletion. Closed and open rectangles in (a) represent dark and light periods of the daily cycle, respectively, with numbers corresponding to the time (hours) at which protein samples were taken. The growth curve in (b) shows the number of cells in the cultures at the times (days) protein samples were taken. Samples in (c) were taken under conditions of phosphate depletion (-P) between 0 and 72 h (-0 to -72) and from phosphate-replete cultures (+P) with the same times (+0 to +72).

and NAP50 are constant during the daily cycle as well as during the growth period. Constant level of rubisco II expression during the daily cycle had been reported in the dinoflagellate Gonyaulax.28 Furthermore, no change in the amounts of both proteins was observed in either phosphate-depleted or replete conditions (Figure 9c). These results support the idea that the effects of nitrogen depletion on rubisco II and NAP50 levels are a specific consequence of aberrant nitrogen metabolism. Interestingly, levels of rubisco II and NAP50 respond rapidly to addition of nitrogen sources to nitrogen-depleted cultures (Figure 8c). When nitrate is supplied for a 12 h period to cells grown in nitrogen-depleted medium for 24 h, the decrease in levels of both proteins normally seen by 36 h is not observed. Addition of nitrate thus blocks the expected degradation of the two proteins. In addition, when nitrate is supplied for 12 h to cells grown in nitrogen-depleted medium for 36 h, normal levels of both proteins are observed. This recovery of high protein levels from the low levels normally found after 36 h in nitrogen-depleted conditions indicates that there is rapid and concurrent synthesis of both proteins, and that this new synthesis is induced by nitrogen availability. It is generally believed that the different proteins found in living organisms can have different and characteristic lifetimes. Therefore, turnover rates and quality control of proteins are physiologically important for the organism.40 However, these normal turnover rates may be radically affected by developmental state or environmental conditions. For example, protein degradation occurs in plants during senescence,39 and this process is thus a complex and highly regulated developmental phase. For higher plants, the breakdown of specific proteins such as rubisco may provide amino acids that can be mobilized to other organs of the plant. In addition, protein degradation often occurs in response to stress. For example, degradation of light-harvesting phycobiliproteins has been implicated in 5090


nutrient-deprived cyanobacteria, and here, decreased levels of components to the energy generation machinery accompanies the decrease in nutrient availability. These two examples may serve as a basis for understanding the decrease in both rubisco II and NAP50 levels observed during nitrogen-depletion. A decrease in the amount of rubisco II would decrease the photosynthetic capacity of the cells due to an inability to use carbon dioxide as an electron sink. Similarly, even though the function of NAP50 is still unknown, we know that it is a plastid protein and decreased levels of NAP50 may also impair plastid function. Clearly, a decrease in plastid function would correlate with the decreased cell growth and metabolism observed during nitrogen-depletion. Furthermore, if the final breakdown products of rubsico II and NAP50 degradation are amino acids, breakdown of these proteins may act to replenish the nitrogen sources inside the cells.39 Rubisco II and NAP50 are both abundant plastid proteins and may thus be targeted for degradation during nitrogen-limitation to provide emergency amino acids for other essential cellular activities. The above analysis predicts that NAP50 may play a role in the photosynthetic ability of cells. A plastid location for NAP50, as indicated by the plastid targeting sequence motifs and immunolocalization results, would support this idea, despite the lack of sequence homology of NAP50 to known proteins. It is perhaps not surprising that NAP50 is so far an unknown protein, given that the dinoflagellate form II rubisco shares only limited sequence homology with the form I Rubisco found in all other chloroplasts.42,43 The finding that most cellular proteins are not degraded during conditions of nitrogen limitation suggests several questions. Why are rubisco and NAP50 proteins selected for degradation? How does this degradation take place, and how is the process regulated? Does NAP50 act as an N storage protein or does it fulfill an important catalytic role? What are the functions of the degraded products? All these questions remain open, yet may provide valuable clues to the ability of dinoflagellates to form algal blooms. Concluding Remarks. Nitrogen is believed to be an important factor in the initiation and maintenance of phytoplankton blooms. However, very little is known about nitrogen-related changes in the cells at molecular level, in particular the proteome. A fast-growing dinoflagellate species A. affine was chosen as a model system for examining the potential molecular correlates between nitrogen metabolism and the grow process. The experiments described here show that, when nitrogen was depleted, cell growth stops and resumes only after nitrogen is added back to the system. By comparing the 2-DE patterns and immunoblots of nitrogen-depleted and nitrogenreplete samples, two plastid proteins, rubisco II and NAP50, were identified. Both are plastid proteins whose levels decreased by more than 16-folds in the nitrogen-depleted conditions. This decrease for both proteins can be blocked by the replenishment of the nitrogen sources, suggesting the degradation of both is highly reversible and regulated by nitrogen. There is no relation of their mRNA abundance with the availability of nitrogen, indicating the degradation processes were not controlled at the transcriptional level. Very little is known about nitrogen metabolism and chloroplast protein degradation in microalgae, especially in dinoflagellates. In particular, plastid protein degradation during nitrogen stress in dinoflagellates, as well as the novel plastid protein NAP50 found here has never been reported. The precise functions and mechanisms of the plastid protein degradation

research articles

Identification of Two Plastid Proteins in A. affine during nitrogen depletion remain unknown. This study provides an important first look into the relationship between nitrogen economy and plastid proteins in dinoflagellates.

Acknowledgment. We would like to acknowledge the contributions of the Protein Structure Core Facility of the University of Nebraska, Omaha, NE, as well as The Australian Proteome Analysis Facility (APAF) in Macquarie University, Australia for performing some of the Edman microsequencing of the peptides of NAP50. Supporting Information Available: Tables of degenerate primers designed for performing PCR of NAP50 cDNA, primers used in RACE of NAP50 and real-time PCR, schedule of anti-NAP50 production, and summary of optimal concentration of different nitrogen sources and the corresponding growth rate and maximum cell density of the growth of A. affine. Figures of cell growth of A. affine with different nitrogen sources and concentrations; PMFs of spot 1-5 obtained using MALDITOF mass spectrometry; PSD spectra of sulfonated peptides with 1215.55 and 1753.05 m/z; PMFs of spots 6-9 and PSD spectrum obtained using MALDI-TOF mass spectrometry with sulfonated peptide ion 1367.684 (m/z); MS and MS/MS spectra of peptides (QVETEASNMK), (AFADWNAEYE), and (FFEAESADY) obtained from LC-MS/MS ion trap mass spectrometry; PMFs of spot 10 and PSD spectra of peptides 1076.4 and 1819.2 m/z, PMFs of spot 11 and PSD spectra of peptides 886.4 and 1682.8 m/z, and PMF of spot 12 and PSD spectrum obtained using MALDI-TOF mass spectrometry; alignment of the leader sequence of NAP50; real-time PCR analysis of mRNA levels for rubisco II and NAP50. This material is available free of charge via the Internet at http://pubs.acs.org.

(12) (13) (14) (15)

(16)

(17)

(18)

(19)

(20) (21) (22)

(23)

References (1) Middelburg, J. J.; Nieuwenhuize, J. Uptake of dissolved inorganic nitrogen in turbid, tidal estuaries. Mar. Ecol.: Prog. Ser. 2000, 192, 79–88. (2) Hodgkiss, I. J.; Ho, K. C. Are changes in N:P ratios in coastal waters the key to increased red tide blooms. Hydrobiologia 1997, 352, 141–147. (3) Howarth, R. W.; Billen, G.; Swaney, D.; Townsend, A.; Jaworski, N.; Lajtha, K.; Downing, J. A.; Elmgren, R.; Caraco, N.; Jordan, T.; Berendse, F.; Freney, J.; Kudeyarov, V.; Murdoch, P.; Zhu, Z. L. Regional nitrogen budgets and riverine N&P fluxes for the drainages to the North Atlantic Ocean: Natural and human influences. Biogeochemistry 1996, 35 (1), 75–139. (4) Mccarthy, J. J.; Kamykows., D. Urea and other nitrogenous nutrients in La-Jolla Bay during February, March, and April 1970. Fish. Bull. 1972, 70 (4), 1261–1274. (5) Hodgkiss, I. J.; Chan, B. S. S. Pollution studies on Tolo Harbor, Hong-Kong. Mar. Environ. Res. 1983, 10 (1), 1–44. (6) Hodgkiss, I. J.; Chan, B. S. S. Studies on 4 streams entering Tolo Harbor, Hong-Kong in relation to their impact on marine waterquality. Arch. Hydrobiol. 1986, 108 (2), 185–212. (7) Paerl, H. W. Coastal eutrophication and harmful algal blooms: Importance of atmospheric deposition and groundwater as “new” nitrogen and other nutrient sources. Limnol. Oceanogr. 1997, 42 (5), 1154–1165. (8) Smayda, T. J. Novel and nuisance phytoplankton blooms in the sea: Evidence for a global epidemic. In Toxic Marine Phytoplankton; Elsevier Science: New York, 1990; pp 29-40. (9) Lee, Y. S. Factors affecting outbreaks of high-density Cochlodinium polykrikoides red tides in the coastal seawaters around Yeosu and Tongyeong, Korea. Mar. Pollut. Bull. 2006, 52 (10), 1249–1259. (10) Lomas, M. W.; Glibert, P. M.; Clougherty, D. A.; Huber, D. R.; Jones, J.; Alexander, J.; Haramoto, E. Elevated organic nutrient ratios associated with brown tide algal blooms of Aureococcus anophagefferens (Pelagophyceae). J. Plankton Res. 2001, 23 (12), 1339– 1344. (11) Glibert, P. M.; Magnien, R.; Lomas, M. W.; Alexander, J.; Fan, C. L.; Haramoto, E.; Trice, M.; Kana, T. M. Harmful algal blooms in the

(24)

(25) (26) (27) (28)

(29)

(30)

(31)

(32)

Chesapeake and coastal bays of Maryland, USA: Comparison of 1997, 1998, and 1999 events. Estuaries 2001, 24 (6A), 875–883. Smayda, T. J. Primary production and the global epidemic of phytoplankton blooms in the sea: a linkage? In Novel Phytoplankton Blooms; Elsevier Science: New York, 1989; pp 449-483. Matsuoka, K. Eutrophication process recorded in dinoflagellate cyst assemblagessa case of Yokohama Port, Tokyo Bay, Japan. Sci. Total Environ. 1999, 231 (1), 17–35. Flynn, K.; Jones, K. J.; Flynn, K. J. Comparisons among species of Alexandrium (Dinophyceae) grown in nitrogen- or phosphoruslimiting batch culture. Mar. Biol. 1996, 126 (1), 9–18. Boyer, G. L.; Sullivan, J. J.; Andersen, R. J.; Harrison, P. J.; Taylor, F. J. R. Effects of nutrient limitation on toxin production and composition in the marine dinoflagellate Protogonyaulax tamarensis. Mar. Biol. 1987, 96 (1), 123–128. John, E. H.; Flynn, K. J. Growth dynamics and toxicity of Alexandrium fundyense (Dinophyceae): the effect of changing N: P supply ratios on internal toxin and nutrient levels. Eur. J. Phycol. 2000, 35 (1), 11–23. Leong, S. C. Y.; Murata, A.; Nagashima, Y.; Taguchi, S. Variability in toxicity of the dinoflagellate Alexandrium tamarense in response to different nitrogen sources and concentrations. Toxicon 2004, 43 (4), 407–415. Leong, S. C. Y.; Taguchi, S. Response of the dinoflagellate Alexandrium tamarense to a range of nitrogen sources and concentrations: growth rate, chemical carbon and nitrogen, and pigments. Hydrobiologia 2004, 515 (1-3), 215–224. Parkhill, J. P.; Cembella, A. D. Effects of salinity, light and inorganic nitrogen on growth and toxigenicity of the marine dinoflagellate Alexandrium tamarense from northeastern Canada. J. Plankton Res. 1999, 21 (5), 939–955. Lee, F. W.; Ho, K. C.; Lo, S. C. Rapid identification of dinoflagellates using protein profiling with matrix-assisted laser desorption/ ionization mass spectrometry. Harmful Algae 2008, 7 (4), 551–559. Keller, M. D.; Selvin, R. C.; Claus, W.; Guillard, R. R. L. Media for the culture of oceanic ultraphytoplankton. J. Phycol. 1987, 23 (4), 633–638. Lee, F. W.; Lo, S. C. The use of Trizol reagent (phenol/guanidine isothiocyanate) for producing high quality two-dimensional gel electrophoretograms (2-DE) of dinoflagellates. J. Microbiol. Methods 2008, 73 (1), 26–32. Ramagli, L. S.; Rodriguez, L. V. Quantitation of microgram amounts of protein in two-dimensional polyacrylamide-gel electrophoresis sample buffer. Electrophoresis 1985, 6 (11), 559–563. Wang, D.; Kalb, S. R.; Cotter, R. J. Improved procedures for N-terminal sulfonation of peptides for matrix-assisted laser desorption/ionization post-source decay peptide sequencing. Rapid Commun. Mass Spectrom. 2004, 18 (1), 96–102. Sambrook, J.; Fritsch, E.; Maniatis, T. Molecular Cloning: A Laboratory Manual; Cold Spring Harbor Laboratory Press: Cold Spring Harbor, NY, 1989. Livak, K. J.; Schmittgen, T. D. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods 2001, 25 (4), 402–408. Pfaffl, M. W. A new mathematical model for relative quantification in real-time RT-PCR. Nucleic Acids Res. 2001, 29 (9), e45. Nassoury, N.; Fritz, L.; Morse, D. Circadian changes in ribulose1,5-bisphosphate carboxylase/oxygenase distribution inside individual chloroplasts can account for the rhythm in dinoflagellate carbon fixation. Plant Cell 2001, 13 (4), 923–934. Chan, L. L.; Hodgkiss, I. J.; Lam, P. K.; Wan, J. M.; Chou, H. N.; Lum, J. H.; Lo, M. G.; Mak, A. S.; Sit, W. H.; Lo, S. C. Use of twodimensional gel electrophoresis to differentiate morphospecies of Alexandrium minutum, a paralytic shellfish poisoning toxinproducing dinoflagellate of harmful algal blooms. Proteomics 2005, 5 (6), 1580–1593. Chan, L. L.; Hodgkiss, I. J.; Lu, S.; Lo, S. C. Use of two-dimensional gel electrophoresis proteome reference maps of dinoflagellates for species recognition of causative agents of harmful algal blooms. Proteomics 2004a, 4 (1), 180–192. Chan, L. L.; Hodgkiss, I. J.; Wan, J. M.; Lum, J. H.; Mak, A. S.; Sit, W. H.; Lo, S. C. Proteomic study of a model causative agent of harmful algal blooms, Prorocentrum triestinum II: the use of differentially expressed protein profiles under different growth phases and growth conditions for bloom prediction. Proteomics 2004b, 4 (10), 3214–3226. Chan, L. L.; Lo, S. C.; Hodgkiss, I. J. Proteomic study of a model causative agent of harmful red tide, Prorocentrum triestinum I: Optimization of sample preparation methodologies for analyzing with two-dimensional electrophoresis. Proteomics 2002, 2 (9), 1169–1186.

Journal of Proteome Research • Vol. 8, No. 11, 2009 5091

research articles (33) Kozak, M. The scanning model for translation: an update. J. Cell Biol. 1989, 108 (2), 229–241. (34) Nassoury, N.; Cappadocia, M.; Morse, D. Plastid ultrastructure defines the protein import pathway in dinoflagellates. J. Cell Sci. 2003, 116 (14), 2867–2874. (35) Zhang, H.; Hou, Y.; Miranda, L.; Campbell, D. A.; Sturm, N. R.; Gaasterland, T.; Lin, S. Spliced leader RNA trans-splicing in dinoflagellates. Proc. Natl. Acad. Sci. U.S.A. 2007, 104 (11), 4618– 4623. (36) Lidie, K. B.; van Dolah, F. M. Spliced leader RNA-mediated transsplicing in a dinoflagellate, Karenia brevis. J. Eukaryotic Microbiol. 2007, 54 (5), 427–435. (37) Mosteller, R. D.; Goldstein, R. V.; Nishimoto, K. R. Metabolism of individual proteins in exponentially growing Escherichia coli. J. Biol. Chem. 1980, 255 (6), 2524–2532. (38) Thiel, T. Protein-turnover and heterocyst differentiation in the cyanobacterium Anabaena variabilis. J. Phycol. 1990, 26 (1), 50– 54.

5092


Lee et al. (39) Hortensteiner, S.; Feller, U. Nitrogen metabolism and remobilization during senescence. J. Exp. Bot. 2002, 53 (370), 927–937. (40) Gottesman, S.; Wickner, S.; Maurizi, M. R. Protein quality control: triage by chaperones and proteases. Genes Dev. 1997, 11 (7), 815– 823. (41) Collier, J. L.; Grossman, A. R. A small polypeptide triggers complete degradation of light-harvesting phycobiliproteins in nutrientdeprived cyanobacteria. EMBO J. 1994, 13 (5), 1039–1047. (42) Morse, D.; Salois, P.; Markovic, P.; Hastings, J. W. A nuclearencoded form-II Rubisco in dinoflagellates. Science 1995, 268 (5217), 1622–1624. (43) Rowan, R.; Whitney, S. M.; Fowler, A.; Yellowlees, D. Rubisco in marine symbiotic dinoflagellates: Form II enzymes in eukaryotic oxygenic phototrophs encoded by a nuclear multigene family. Plant Cell 1996, 8 (3), 539–553.

PR900475F

Identification of Two Plastid Proteins in the ... - ACS Publications

Recommend Documents