Characterization of the Proteomic Profiles of the Brown Tide AlgaAureoumbra lagunensis under Phosphate- and Nitrogen-LimitingConditions and of Its Phosphate Limitation-Specific Protein withAlkaline Phosphatase Activity

Ming-Ming Sun,a Jin Sun,b Jian-Wen Qiu,b Hongmei Jing,a and Hongbin Liua

Division of Life Science, the Hong Kong University of Science and Technology, Clear Water Bay, Hong Kong, China,a and Department of Biology, Hong Kong BaptistUniversity, Hong Kong, Chinab

The persistent bloom of the brown tide alga Aureoumbra lagunensis has been reported in coastal embayments along southernTexas, but the molecular mechanisms that sustain such algal bloom are unknown. We compared the proteome and physiologicalparameters of A. lagunensis grown in phosphate (P)-depleted, P- and nitrogen (N)-depleted, and nutrient-replete cultures. Forthe proteomic analysis, samples from three conditions were subjected to two-dimensional electrophoresis and tandem massspectrometry analysis. Because of the paucity of genomic resources in this species, a de novo cross-species protein search wasused to identify the differentially expressed proteins, which revealed their involvement in several key biological processes, suchas chlorophyll synthesis, antioxidative protection, and protein degradation, suggesting that A. lagunensis may adopt intracellu-lar nutrient compensation, extracellular organic nutrient regeneration, and damage protection to thrive in P-depleted environ-ments. A highly abundant P limitation-specific protein, tentatively identified as a putative alkaline phosphatase, was furthercharacterized by enzyme activity assay on nondenaturing gel and confocal microscopy, which confirmed that this protein hasalkaline phosphatase activity, is a cytoplasmic protein, and is closely associated with the cell membrane. The abundance, loca-tion, and functional expression of this alkaline phosphatase all indicate the importance of organic P utilization for A. lagunensisunder P limitation and the possible role of this alkaline phosphatase in regenerating phosphate from extra- or intracellular or-ganic phosphorus.

Aureoumbra lagunensis (Pelagophyceae), the so-called Texasbrown tide alga, is a small single-celled pelagophyte that had

formed a persistent bloom in several coastal embayments alongsouthern Texas from 1989 to 1997 (12). This was the longest con-tinuous harmful algal bloom ever documented in history andcaused great damage to the pelagic and benthic ecosystems (37).As algal blooms require environmental inorganic nutrients to sus-tain, this case has raised questions about the relationship betweenalgal bloom and nutrient supply. A field survey revealed a highlysignificant inverse relationship between ambient phosphate leveland A. lagunensis cell density during the bloom (40). Laboratorybatch culture (49) and chemostate (29) experiments confirmedthe extremely high tolerance of A. lagunensis to phosphate (P)limitation (critical nitrogen [N]:P � 174). These field and labora-tory studies indicated that A. lagunensis is well adapted to low-phosphate environments.

Different from many algal species which bloom when phos-phorus input is high (2), the pelagophytes A. lagunensis and Au-reococcus anophagefferens and the coccolithophore Emiliania hux-leyi bloom at low dissolved inorganic phosphorus (DIP)concentrations. Studies indicated that A. anophagefferens and E.huxleyi employ two main strategies to cope with P deficiency: pro-ducing either more or new phosphate transporters (10, 17, 51) andregenerating P from dissolved organic phosphorus (DOP), a sig-nificant part of the marine total dissolved P pool (6, 24). Alkalinephosphatases (APs) are the key enzymes that marine planktonicmicrobes produce to hydrolyze orthophosphate from phosphorusester, the dominant high-molecular-weight DOP class (13, 15,16). Though DIP is the most significant bioavailable P form for

living organism in oceans, previous studies have shown that DOPcan support the growth of A. lagunensis as the sole P source (34).Nevertheless, the mechanisms of P acquisition in A. lagunensis arelargely unknown, and no studies have been conducted to examinethe relationship among P acquisition, algal growth, and proteinexpression in this species.

Proteomics, a molecular tool that detects the global proteinexpression, is suitable for investigating the molecular mechanismof adaption to specific environmental conditions. In marine algae,proteomics has been applied to understand the mechanisms ofadaptation to salinity (26, 28) and copper (11, 42). Successfulapplication of identification-based proteomics heavily dependson a database of gene or protein sequences for homology search.This reliance on the existing genomic database has limited theapplication of proteomics in many nonmodel marine algae withfew genomic resources. However, the recently developed de novocross-species protein identification strategy has made it possibleto conduct proteomic studies in nonmodel species with limitedgenomic resources (46). This strategy has been successfully ap-plied to a variety of eukaryotic life forms (e.g., pine [4], gastropod

Received 6 June 2011 Accepted 7 January 2012

Published ahead of print 13 January 2012

Address correspondence to Hongbin Liu, liuhb@ust.hk.

Supplemental material for this article may be found at http://aem.asm.org/.

Copyright © 2012, American Society for Microbiology. All Rights Reserved.

doi:10.1128/AEM.05755-11

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[48], and fungi [9]), but to our knowledge it has been appliedto only one eukaryotic alga, Dunaliella salina, before extensivegenomic resources were available for this species (28), and in thesalinity tolerance study of D. salina, de novo cross-species proteinidentification strategy resulted in the identification of more thantwice as many proteins as the MASCOT method and with higherconfidence.

Here, we reported a proteomic study of the responses of the ma-rine alga Aureoumbra lagunensis to P- and P- and N-deficient condi-tions. Using de novo cross-species identification, we identified thedifferentially expressed proteins under P- and N-deficiency. Mean-while, several physiological parameters associated with algal growthunder the different nutrient treatments were also measured to pro-vide a better link between algal physiology and proteomics. Enzymeactivity assay on gel, ELF-97 labeling, and confocal microscopy wereemployed to characterize a highly abundant, P limitation-specificprotein discovered by the proteomic analysis.

MATERIALS AND METHODSAlgal culture. Aureoumbra lagunensis was cultured at 25°C under coolwhite fluorescent light (140 �mol quanta m�2 s�1) with a 14 h:10 hlight:dark cycle. Three modified f/2 media (without silicate and replacingnitrate by ammonium) with autoclaved filtered seawater pumped fromClear Water Bay, Hong Kong, were used in experiments: (i) �N�P me-dium (200 �M extra NH4

�, 20 �M extra PO43�); (ii) �N�P medium

(200 �M extra NH4�, no extra PO4

3�); and (iii) �N�P medium (40 �Mextra NH4

�, no extra PO43�). The �N�P and �N�P treatments repre-

sented the nutrient-replete control and the P-depleted condition, respec-tively. To compare the responses between N and P limitation and theeffect of N limitation on P depletion, we added the �N�P treatment.Algal cells were collected by centrifugation for 10 min at 4,000 � g from 3liters of culture (�N�P medium) at late exponential phase. After beingwashed with autoclaved seawater, algal cells were resuspended in auto-claved seawater, and the same volume of cell suspension was added intoquadruplicate 2-liter glass Erlenmeyer flasks containing 1.5 liters of�N�P medium, �N�P medium, and �N�P medium. Subcultureswere taken daily at the same time point for 10 days and were diluted to 105

to 106 cells per ml for cell density measurement by using a Coulter counter(Beckman, California).

Algal culture physiology. Intracellular phosphate concentration wasmeasured before the inoculation (initial) and on the first 5 days (exceptfor day 2). A subsample of 10 ml algae was collected from each cultureflask, filtered onto a precombusted Waterman GF/C glass fiber filter (25mm), and stored at �80°C until analysis. The intracellular phosphateconcentration was determined using the method described by Solorzanoand Sharp (47). The final results were normalized by cell volume derivedfrom Coulter counter readings. Phosphate concentrations in algal culturewere also monitored daily by using the Molybdate Blue method (21).

Cellular chlorophyll a (Chl a) concentration was determined daily onthe first 5 days (except for day 2) (8). Briefly, a 10-ml subsample from eachflask was filtered onto GF/C. Chlorophyll on the filters was extracted in90% acetone by sonication and overnight incubation at �20°C in thedark. In vitro fluorescence of Chl a was determined using a Turner Designsmodel 7200 fluorometer and calculated and normalized to cellular Chl aconcentration according to a standard curve and cell number.

APA. Cellular and soluble alkaline phosphatase activity (APA) wasmeasured daily on the first 5 days (except for day 2). For cellular APAmeasurement, a 10-ml algal suspension was filtered onto a 1-�m-pore-size polycarbonate filter (25 mm in diameter). The filter was immersed in1 ml AP buffer (50 ml 0.5 M CaCl2, 100 ml 0.5 M Tris [pH 8.0], 800 mlautoclaved seawater, 0.5 mM 4-methylumberlliferyl phosphate [MUP])and then incubated at 25°C under vibration. To determine the time-dependent changes in fluorescent product hydrolyzed from MUP by al-kaline phosphatase, 50 �l AP buffer was removed from the mixture at

15-min intervals within 1 h. After 200 �l 0.2 M Na2CO3 was added andafter centrifugation, the florescence of the supernatant was measured at a360-nm excitation/460-nm emission immediately with a Wallac Victor 3multilabel counter (Perkin Elmer). For soluble APA measurement, 0.5 mlof 0.22-�m-pore-size-filtered algal suspension was added into 0.5 ml ofAP buffer (1 mM MUP), and the following measurement was the same asfor cellular APA. Cellular APA was determined by measuring the linearincrease in fluorescence versus time, and the enzyme activity was normal-ized to cell number, whereas soluble APA was expressed on a volumebasis.

Algal protein preparation. Algal cells in 1 liter of culture were col-lected by centrifugation at 4,000 � g for 10 min from four biologicalreplicates for three treatments at the late exponential phase (on day 5).The algal pellets were washed twice with autoclaved seawater and resus-pended immediately in 200 �l of lysis buffer containing 7 M urea, 2 Mthiurea, 4% CHAPS (3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate), 20 mM dithiothreitol (DTT), and 2% Bio-Lyte 3/10ampholyte. Samples were frozen in liquid nitrogen and then immediatelytransferred into a 37°C water bath to break the algal cells. After sonicationand centrifugation at 13,000 rpm for 20 min, the protein in supernatantwas purified using a two-dimensional (2D) cleanup kit (Bio-Rad, Hercu-les, CA) and then dissolved in 150 �l of rehydration buffer (7 M urea, 2 Mthiourea, 2% CHAPS, 40 mM DTT, 0.2% Bio-Lyte 3/10 ampholyte).

2D electrophoresis. Proteins in the purified samples were separatedby 2D electrophoresis as described by Sun et al. (48). Briefly, 0.8 mgprotein from each biological sample was loaded onto a 17-cm immobi-lized pH gradient (IPG) strip (Bio-Rad, Hercules, CA), pH 4 to 7 (linear),to allow for a 16-h active rehydration, followed by isoelectric focusing(IEF) in a Protean IEF cell at 20°C. IPG strips, pH 4 to 7, were used,because a preliminary experiment with pH 3 to 10 IPG strips showed thatmost protein spots were resolved in this pH range. The electrophoresiswas run at 250 V for 20 min, followed by a linear increase from 250 to8,500 V in 2.5 h, and at 8,500 V for a total of 60,000 volt hours (Vh). Priorto the second dimensional run, the strips were equilibrated for 15 min inequilibration buffer I (6 M urea, 2% SDS, 0.05 M Tris-HCl, 50% glycerol,and 2% [wt/vol] DTT) followed by 15 min in equilibration buffer II (thesame buffer as equilibration buffer I but containing 2.5% iodoacetamideinstead of DTT). The IPG strips were placed on the top of 12.5% SDS-PAGE gels (18 cm by 18 cm) and sealed with 0.5% [wt/vol] agarose. Thesecond dimension separation was performed with a constant current of 16mA per gel for 12 h. The gels were stained with colloidal Coomassie G250solution.

Gel image analysis. The 2D gels were scanned with a calibrated trans-parency densitometer (GS-800; Bio-Rad, Hercules, CA). Gel images wereanalyzed using PDQuest version 8.0 (Bio-Rad, Hercules, CA) and visuallyconfirmed. Total spot densities were normalized across gels. For quanti-tative analysis, spots showing at least a 2-fold difference and that had a Pvalue of �0.01 in Student’s t test were considered up- or downregulated,and only spots presented in at least 3 replicates were analyzed. For quali-tative analysis, spots showing at least 20-fold changes were consideredabsent or present. The comparisons were conducted between �N�Ptreatment and �N�P treatment and between the �N�P treatment and�N�P treatment to find the differentially expressed protein under P andN depletion, respectively.

Electrospray ionization (ESI) QqTOF analysis. The most abundantup- or downregulated protein spots were subjected to mass spectrometryanalyses. Protein spots were cut from the gels and digested as described byShevchenko et al. (46). Briefly, the gel pieces were destained with 50 mMNH4HCO3 in 50% methanol, washed with Milli-Q water, and then driedwith 100% acetonitrile (ACN) followed by rehydration with 100 mMNH4HCO3 twice. After adding 15 �l of 12.5 ng/�l sequencing-grade tryp-sin (Promega, Madison, WI) in 10 mM NH4HCO3 and 10% ACN, the gelpieces were incubated at 37°C overnight. Formic acid (FA; 5%) in 50%ACN was used to extract peptides from the gel pieces. Extracts were driedwith SpeedVac (Thermo Electron, Waltham, MA), dissolved in 5% FA,

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desalted with C18 ZipTip, and analyzed by using a quadrupole time offlight (QqTOF) mass spectrometer (QSTARXL; Applied Biosystems/Sciex, Ontario, Canada) equipped with a nanoelectrospray ion source.Peptides with �2 to �3 charge states were selected for tandem massspectrometry (MS/MS).

Mass spectrum interpretation. The data were submitted through on-line MASCOT to the NCBI nonredundant database with taxon restrictionin Viridiplantae (green plants). Tolerances for precursor and fragmentmasses were set at 0.3 Da and 0.5 Da, respectively. The following param-eters were used in database searching: fixed modification, carbamidom-ethyl (cysteine); variable modification, oxidation (methionine). Up to onemissed trypsin cleavage was allowed. Hits were considered significantwhen at least three peptides of a protein showed MASCOT scores of over50 simultaneously.

For the samples that cannot be identified by using MASCOT, auto-mated de novo peptide sequencing was performed using PepNovo as de-scribed by Waridel et al. (50). After manual interpretation of the candidatesequences deduced from PepNovo with the help of the Analyst 1.1 soft-ware (Applied Biosystems), the peptide sequences from one protein spotwere merged and submitted to MS-BLAST at http://genetics.bwh.har-vard.edu/msblast/ (4) for searching against the NCBI nonredundant da-tabase (nrdb95). Hits with a total BLAST score above 100 or with at leastone high-scoring segment pair above 60 were considered positive. Themolecular weight and pI of the matched proteins were deduced by usingthe ExPASy Proteomics Server (www.expasy.org).

Nondenaturing electrophoresis and APA in gel assay. Whole-cellprotein was extracted from the late-exponential culture using a nondena-turing extraction buffer (0.02 M Tris [pH 8.0], 1 mM DTT, 0.4% TritonX-100). After centrifugation at 16,000 � g for 30 min, the supernatant wasrun on a 12.5% nondenaturing gel. APA in gel assay was performed withELF-97 (the endogenous phosphatase detection kit; Molecular Probes),which can generate fluorescent precipitation (excitation/emission: 345/530 nm) after AP hydrolysis. Briefly, each gel was soaked in AP buffer(0.02 M Tris [pH 8.0], 0.01 M CaCl2, 0.1 M NaCl) and incubated in 20�diluted ELF-97 in the dark. After 30 min, AP buffer was added to stop thereaction. ELF-97 fluorescence of the gel was examined with 350 nm UVexcitation, and the emission filtered by EtBr filter was collected. To sepa-rate the subunits of native-labeled protein complex and determine themolecular weight of each subunit, the band labeled by ELF-97 was excisedand soaked in 1% SDS and 1% mercaptoethanol for 2 h. After being rinsedwith Milli-Q water, the gel band was loaded on the top of the SDS-PAGEgel and the second dimensional electrophoresis was run. To determine themetal dependence of the AP in A. lagunensis, after running the nondena-turing gel, the gel lanes were sliced and incubated separately in bufferscontaining metal ions of different concentrations (Ca2� [10 mM to 0.01mM], Mg2� [10 mM to 0.01 mM], Zn2� [0.1 mM to 0.1 �M], and Co2�

[10 mM to 1 �M]) instead of 0.01 M CaCl2.Confocal microscopy. The localization of AP in cells was determined

under a confocal microscope after labeling with ELF-97. Cells grown un-der �P conditions and �P conditions were labeled with ELF-97 as de-scribed by González-Gil et al. (20), except for shortening the 70% ethanolfixing step to 5 min. Microscopic slides prepared with 5 �l of labeled cellseawater suspension and 5 �l of mounting medium were observed byusing a Leica TCS-SP5 spectral laser scanning confocal microscope. Todetect the florescence of ELF-97, a 510- to 600-nm emission was collectedwith a DPSS 405-nm laser excitation. Images were obtained with a �100magnification, 1.4-numerical-aperture oil objective.

Correlation between P limitation-specific protein and cellular APA.To confirm the relationship between the appearance of a P limitation-specific protein band on nondenaturing gel and cellular alkaline phospha-tase, A. lagunensis was cultured under P-depleted conditions for 5 days.Cellular APA and the percentage of ELF-97-labeled cells were measureddaily. Algal cells with green ELF-97 signal were identified using an Olym-pus BX51 microscope with a DAPI (4=,6-diamidino-2-phenylindole)long-pass filter set. At least 5 views were randomly chosen for each sample,

and the numbers of total and ELF-97-labeled cells were both counted.Protein was extracted from algal cells harvested from 150 ml of cultureevery day, and the same amount of protein was loaded on a nondenatur-ing gel.

RESULTSAlgal growth. Among the three treatments, the two P-depletedtreatments (i.e., �N�P and �N�P treatment) showed very sim-ilar growth tendencies with a shorter lag phase and faster growththan the P-replete treatment (i.e., �N�P treatment) in the first 2days. But the P-replete treatment reached to a higher final cellconcentration than the P-depleted treatments at the stationaryphase (Fig. 1A).

Cellular phosphate concentration. Cellular P concentrationin the �N�P treatment increased abruptly 1 day after inoculationto approximately five times the initial cellular P concentration andthen gradually decreased to the initial level during the following 4days. For the two P-depleted cultures, the initial DIP of the culturemedium was about 0.05 �M and then became undetectable fromday 1 to day 5. Cellular P concentration decreased to approxi-mately 2/3 of the initial level on the first day and decreased furtherfor the rest of the experimental period to reach only 10% of theinitial value in the �N�P treatment and 7% in the �N�P treat-ment on day 5 (Fig. 1B). For each day, one-way analysis of vari-ance (ANOVA) revealed significant treatment effects (P � 0.01),and the Duncan’s test showed that the P-replete (�N�P) treat-ment had significantly higher cellular P levels than two P-depleted(�N�P and �N�P) treatments from day 1 to day 5.

Cellular Chl a. Cellular Chl a content in the �N�P and�N�P treatments did not show a significant change from day 1 today 5, but it declined progressively with time in the �N�P treat-ment (Fig. 1C). One-way ANOVA followed by Duncan’s testshowed that the cellular Chl a levels of N-depleted (�N�P) treat-ment were significantly lower than those of the control (�N�P)and P-depleted (�N�P) treatments on day 5, which indicatedthat chlorosis occurred only in the N-depleted treatment.

APA. Both cellular and soluble APAs were measured, but sol-uble APA was undetectable throughout the experiment. CellularAPA of the three treatments did not differ significantly in the first3 days, and the cellular APA level in the �N�P treatment was verylow throughout the whole experiment (Fig. 1D). On days 4 and 5,the cellular levels of APA in the �N�P and �N�P treatmentswere significantly higher than that of the �N�P treatment; andon day 5, the �N�P treatment had significantly higher cellularAPA than the �N�P treatment (one-way ANOVA, Duncan’stest, P � 0.01), which indicated a reverse effect of persistent Ndepletion on APA when P is limited.

2D electrophoresis pattern. The 2D electrophoresis resolved262 to 326 spots in each gel (Fig. 2). When using the �N�Ptreatment (Fig. 2A) as the background, the �N�P treatment (Fig.2B) resulted in 18 downregulated spots, 7 upregulated spots, 9absent spots, and 9 novel spots. When using the �N�P treatmentas the background, the �N�P treatment resulted in 5 downregu-lated spots, 18 upregulated spots, 7 absent spots, and 9 novel spots.

MS analysis and protein identification. Of the 38 most abun-dant and differentially expressed spots subjected to ESI QqTOF,22 were successfully identified (labeled in Fig. 2), of which 5 wereidentified by using MASCOT directly and 17 by de novo sequenc-ing and MS-BLAST similarity database search (see Tables S1 andS2 in the supplemental material). The differentially expressed pro-

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teins between the �N�P and �N�P treatments belonged to sixfunctional assemblages: nucleotide metabolism, amino acid me-tabolism, cellular signaling, oxidative stress defense, utilization oforganic phosphorus, and hypothetical protein. The differentiallyexpressed proteins between the �N�P and �N�P treatments

fell into five functional groups: chlorophyll synthesis, amino acidmetabolism, carbohydrate metabolism, oxidative stress defense,stress response, and other functions.

Nondenaturing electrophoresis and APA in gel assay. TheAPA in gel assay revealed a protein band with APA only in the �P

FIG 1 Changes in physiological parameters of A. lagunensis under three different treatments during experiments. (A) Algal density (cells ml�1). Graphs includespositive control (�N�P) (�), P-depleted (�N�P) (Œ), and N- and P-depleted (�N�P) (’) cultures; (B) cellular P content (fmol cell�1); (C) cellular Chl aconcentration (pg cell�1); (D) cellular alkaline phosphatase activity (APA) (fmol Pi min�1 cell�1). Bars in panels B, C, and D represent positive-control (�N�P)(black bars), P-depleted (�N�P) (white bars), and N- and P-depleted (�N�P) (gray bars) cultures and initial cellular P content at day 0 (hatched bars).

FIG 2 Representative 2D electrophoresis gel images of A. lagunensis under the three nutrient treatments. The successfully identified differentially expressedproteins are labeled on the images.

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treatment (Fig. 3A, arrow). This band was excised, and SDS-PAGEwas performed without (Fig. 3B, �P Non) or with (Fig. 3B, �PD)equilibration in denaturing buffer. The same position of this bandin the �P treatment was also included in Fig. 3B (�PD) to show

the background of the nondenaturing gel. After SDS-PAGE, a pro-tein band of 200 kDa appeared in the �P Non lane and a proteinband of approximately 100 kDa appeared in the �PD lane (Fig.3B), and 100 kDa corresponded to the position of the identifiedputative AP on the 2D gel. This P limitation-specific band on the�PD lane (Fig. 3B) was also subjected to the ESI QqTOF. Fromthe MS profile, two peptides with the same peptide mass as spot 10(Fig. 2) were found. The same peptide sequence derived from denovo sequencing (Fig. 4) further confirmed that this P limitation-specific protein band with APA on denaturing gel and spot 10 (Fig.2) of the 2D gel were the same protein. The APA assay in thenondenaturing gel using buffer containing Ca2�, Mg2�, Zn2�, orCo2� showed that this A. lagunensis AP was activated only by Ca2�

(Fig. 5).Localization of alkaline phosphatase. To further determine

the location of AP, we labeled it with ELF-97 and detected thefluorescence of ELF-97 by using confocal microscopy. WhenELF-97 labeling was applied to A. lagunensis, the detectable punc-tuate fluorescence was specific to �P-treated algal cells (see Fig. S1in the supplemental material). Confocal images with differentdepths (Fig. 6) showed that the labeled AP was cytoplasmic andadjacent to the inner cell membrane.

Correlation between P limitation-specific protein and cellu-lar APA. Under P-depleted conditions, cellular APA increasedgradually from day 1 to day 4 and reached a plateau afterward, butELF-97-labeled algal cells were detected from day 2, with values of59% in day 2 and 65 to 83% in days 3 to 5. The P limitation-specific protein band also appeared from day 2 (Fig. 7, arrow),corresponding to the increases in the percentage of ELF-labeledcells in the A. lagunensis culture and 1 day later than the appear-ance of cellular APA.

DISCUSSION

Our direct submission of the MS/MS from the 38 differentiallyexpressed protein spots resulted in the identification of only 5

FIG 3 Characterization of P limitation-specific in A. lagunensis. (A) Nondena-turing gels stained by Coomassie blue and ELF-97. The whole-cell protein samplesfor nondenaturing electrophoresis came from P-replete culture (�P) andP-depleted culture (�P). Arrow indicates position of the band used in SDS-PAGE.(B) SDS-PAGE using the band cut from the position indicated by arrow in panel Aon the nondenaturing gel. Lane M, protein standard; lane �P Non, band in �Ptreatment without equilibration in denaturing buffer (1% SDS, 1% mercaptoeth-anol); lane �PD, band in �P treatment with equilibration in denaturing buffer;lane �PD, band in �P treatment with equilibration in denaturing buffer.

FIG 4 MS/MS spectrum of one peptide from spot 10 on the 2D gel. The peptide sequence FLAFSGGPYLR is derived from de novo peptide sequencing. Peakscorresponding to the y and b ions from this peptide are labeled on the spectrum. This peptide was also found in the MS result of ELF-labeled protein on thenondenaturing gel.

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proteins (13%), whereas de novo cross-species identificationhelped to identify another 17 proteins, raising this ratio to 22 of 38(58%). The much higher rate of identification demonstrates theusefulness of de novo sequencing and the MS-BLAST similaritydatabase search in proteomic studies of nonmodel marine phyto-plankton.

Proteomic profile changes. To understand the differential ef-fects of N and P limitation on algal cell, and the interplay betweenP and N limitation, we examined the differences between controland nutrient-deficient treatments. Under N- and P-colimitedcondition (�N�P treatment), besides the proteins related to thegeneral metabolism, several enzymes evolved in Chl a synthesis(see Table S2 in the supplemental material) were downregulated,and an endopeptidase (spot 19) that might play an important rolein cellular N recycling through protein decomposition was up-regulated. Specifically, as photosynthetic pigments and protein-rich chloroplast have a high N:P ratio and are a good N storagesource in algal cells (1, 3), the degradation of chloroplast proteinsand a decline in cellular pigment content has been observed widelyin N-limited marine algae (5, 18, 51). As damaging the photosyn-thetic system and interrupting the function of the electron trans-port chain will increase the amount of electrons accepted by O2

(35), the decomposing of pigments and photosynthesis proteinsunder N-deficient conditions may lead to an increase in reactiveoxygen species (ROS). Though under both �N�P and �N�Ptreatments typical antioxidative enzymes, such as manganese su-peroxide dismutase (SOD) (spot 7), glutathione S-transferase(GST) (spot 6), and mutator mutT related to repairing oxidativelydamaged DNA (31), were upregulated (see Tables S1 and S2 in thesupplemental material), the upregulation level of SOD was much

higher in �N�P treatment than in �N�P treatment. In addi-tion, ATP synthase gamma subunit (spot 21) and beta subunit(spot 22) were only greatly upregulated in the �N�P treatment.Since superoxide anion radical has been reported to greatly dam-age ATP synthase (7), the increase in ATP synthase indicated thatalgal cells may accumulate more ROS in �N�P treatment than in�N�P treatment. The overexpression of antioxidative enzymesmay endow A. lagunensis cells with the ability to limit oxidativedamage, protect the photosynthesis apparatus, and adjust the cel-lular redox state (36).

In the proteomic profile of the �N�P treatment, 40S ribo-somal protein-like protein (spot 1) was downregulated 2.3-fold,and a protein identified as a putative alkaline phosphatase (AP)(spot 10) was novel compared to the nutrient-replete control. Ri-bosomal protein was reported to respond to P depletion under thecontrol of the P metabolic regulation system (PhoBR) in the ma-rine cyanobacteria Synechococcus (38), and the downregulation ofribosomal protein on transcriptional level in �P treatment wasalso reported in a study of the coccolithophore E. huxleyi (17). AsP-rich ribosome is a significant intracellular P reservoir inside thecell (3), the downregulation of ribosomal protein in the �N�Ptreatment might facilitate the intracellular P recycling in A. la-gunensis. Besides the intracellular nutrient retrieval migrationstrategy, enzymes such as AP might provide a means to access theextracellular DOP pool to increase P availability in A. lagunensis.Studies at the RNA level (17, 51) and protein level (53) confirmedthat the DOP scavenging enzymes can be induced under P limita-tion in marine eukaryotic algae. In our study, one putative alkalinephosphatase was identified, and its upregulation in A. lagunensisunder P limitation was also confirmed by the cellular alkaline

FIG 5 Cofactor characterization of AP. APA assay on nondenaturing gel with buffer containing Ca2�, Mg2�, Zn2�, and Co2� of different concentrations andbuffer containing 10 mM Ca2�, 0.1 mM Mg2�, 0.7 �M Zn2�, and 0.1 �M Co2� as the control (�).

FIG 6 Confocal images with depths, showing intracellular labeling of AP with ELF-97. Images were taken with an interval of 1.09 �m along the z axis. The upperseries of images show the green florescence emitted by ELF-97, and the bottom series of images show the shape of algal cell.

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phosphatase activity assay. Though due to the limitation of the 2Delectrophoresis technology we could not detect hydrophobicphosphate transporters found by suppression subtractive hybrid-ization (SSH) (10) and long serial analysis of gene expression(SAGE) (17, 51), the proteomic data provided direct evidence ofgene expression.

Characterization of the putative AP. The putative AP that ap-peared in both �N�P and �N�P treatments showed a drasticchange (�20-fold change) on 2D gels. The de novo cross-speciesidentification of AP in our study derived from a 10-amino-acidpeptide. Because eukaryotic algal AP may be difficult to identifydue to the high sequence divergence (33), we confirmed the func-tion of this P limitation-specific protein by conducting the APAassay on a nondenaturing gel. The same molecular weight and thesame peptides derived from the ELF-labeled protein band on thenondenaturing gel and spot 10 on the 2D gel confirmed that theyare the same protein, and this P limitation-specific protein is anAP. The presence of this protein as a band of 200 kDa (Fig. 3B, �PNon lane) under nondenaturing conditions indicated that thisalkaline phosphatase may function as a homodimer in A. lagunen-sis, which is the same as the PhoA-type AP in E. coli (44). However,unlike the Zn2�-dependent PhoA, AP in A. lagunensis used Ca2�

as enzyme cofactor (Fig. 5), which was also the case for a morewidespread marine PhoX-type AP first reported in Pseudomonasfluorescens (32) and then in several cyanobacteria, such as Trichodes-mium, Synechococcus, Cyanothece, and Microcystis aeruginosa (25,45). Distinct trace metal cofactor used by algal cell enzymes canconfer successful niche occupancy and advantages in competitionwith other species. For example, selenium and metal-requiringenzymes confer a great advantage on the pelagophyte A.anophagefferens to dominate the trace-metal-enriched shallow es-tuaries (19). As Zn2� and other trace metals may be colimited withP (23), relying on Ca2� for activation may favor the populationgrowth of A. lagunensis in P-limited environments.

The fact that soluble APA was undetectable in the P-deficientculture of A. lagunensis indicated that this AP could be cytoplas-mic or cell membrane bounded. The confocal microscopy indi-cated that the ELF-97-labeled AP of this alga is located inside thecell and very close to the cell membrane. Though the traditionalopinion was that AP in marine eukaryote phytoplankton is locatedon the cell surface to hydrolyze organic P (14, 27, 53), ELF labelingof many eukaryotic marine microalgae did not support this opin-ion (15, 19). It was possible that due to the instability and weak

signal of the surface-associated labeling, intracellular AP was la-beled more clearly than the surface-associated AP. This phenom-enon has been reported in eukaryotic marine microalgae Isochrysissp. and Amphidinium carterae (19). A previous study also showedintracellular distribution of ELF-97-labeled AP in Prorocentrumminimum, but the AP was evenly dispersed in the cytoplasm (15).High abundance is another characteristic of the P limitation-specific protein reported in our study. It accounted for approxi-mately 5% of the total intensity of proteins detected on the 2D gelin the �N�P treatment and 8% in the �N�P treatment, and ithad the highest intensity among all the protein spots detected onthe 2D gel. These high percentages indicated highly dispropor-tional resource utilization to produce this specific protein under Plimitation by the algal cell, even under P and N colimitation, whennitrogen, a raw material of protein, is scarce.

The abundance and location of this P limitation-specific pro-tein indicated its two possible functional responses to P limitation.One is the utilization of extracellular dissolved organic phospho-rus (DOP). A recent in silico analysis of bacterial AP found that upto 41% AP are cytoplasmic (30). Intracellular hydrolysis of DOP ismore economical than releasing the valuable enzyme into the am-bient environment to hydrolyze extracellular DOP, and the dis-solved organic matter (DOM) can also be utilized by algal cells(24). The membrane-adjacent location of the AP in A. lagunensismay provide clues for its associated transporters that transportDOP into the cell to enable cytoplasmic AP function inside thealgal cell. This P limitation-specific protein may be also involvedin the utilization of cellular P storage. Consuming intracellular Pstorage to relieve environment P stress is one of the most impor-tant strategies used by P limitation-adapted marine microalgae.For example, E. huxleyi can vary its cellular P content from 82 to2.6 fmol P cell�1 (41). In our study, the cellular P content of A.lagunensis changed from 83 to 1.1 fmol P cell�1, which indicated astrong ability of storing excessive P under P-replete conditionsand lowering P demand under P-deficient conditions. Storedpolyphosphate has been extensively reported in many marine mi-crobes, and the P limitation-specific protein has been thought tobe an internal phosphatase for hydrolysis of intracellularly storedP in Dunaliella tertiolecta (22). It is therefore possible that thecytoplasmic AP of A. lagunensis is related to the recycling of intra-cellular P.

By examining the relationship between the changes in this Plimitation-specific protein and the increase in APA during the

FIG 7 Cellular alkaline phosphatase activity (APA), percentage of ELF-labeled cells, and the appearance of P limitation-specific protein detected on nondena-turing gel during the 5-day culture under P-depleted conditions. The bars represent cellular APA, and lines present the percentage of ELF-labeled cells. The Plimitation-specific protein bands are indicated by an arrow.

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experiment, we found that the cellular APA was detected 1 dayearlier than the appearance of the P limitation-specific protein inA. lagunensis. This result indicates that this P limitation-specificprotein is not the only AP in A. lagunensis. Different kinds of APshave been detected in one genome of the cyanobacteria Synechoc-occus (39) and the coccolithophore E. huxleyi (52). Three differentenzyme Km values were detected in the dinoflagellate Pyrocystisnoctiluca, which might be caused by APs with different activitiesand characters (43). It is possible that the P limitation-specific APin A. lagunensis is a high-efficiency AP which is induced only un-der severe P limitation.

The results showed that P and N limitations have great effectson several groups of proteins that function in chlorophyll synthe-sis, oxidative stress protection, and metabolism. Through thefollow-up investigation of the 2D electrophoresis proteomicstudy, we found an AP that is likely important in phosphate re-generation from extracellular DOP or intracellular P storage,which is indicated by the abundance and location of this AP insidethe algal cell under P limitation. Further studies should examinehow this AP works with P-related transporters to cope with Plimitation.

ACKNOWLEDGMENTS

We thank Hudson Robert DeYoe (University of Texas-Pan American) forproviding the algal culture and Yiji Xia (Hong Kong Baptist University)for helpful discussion.

This study was supported by the University Grants Council of HongKong AoE project (grant no. AoE/P-04/04), the Research Grants Councilof Hong Kong RGF grant (no. 661809 and 661610), and the TUYF Char-itable Trust (grant no. TUYF10SC08).

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