Proteomic response of Phaeocystis globosa to nitrogen limitation*

Haisu LIU, Ruiwang WEI, Qiangyong LEI, Lei CUI,**, Songhui LÜ,2,**

Abstract Phaeocystis globosa is an important unicellular eukaryotic alga that can also form colonies.P.globosa can cause massive harmful algal blooms and plays an important role in the global carbon or sulfur cycling.Thus far, the ecophysiology of P.globosa has been investigated by numerous studies.However, the proteomic response of P.globosa to nitrogen depletion remains largely unknown.We compared four protein preparation methods of P.globosa for two-dimensional electrophoresis (2-DE)(Urea/Triton X-100 with trichloroacetic acid (TCA)/acetone precipitation; TCA/acetone precipitation;Radio Immuno Precipitation Assay (RIPA) with TCA/acetone precipitation; and Tris buffer).Results show that the combination of RIPA with TCA/acetone precipitation had a clear gel background and showed the best protein spot separation effect, based on which the proteomic response to nitrogen depletion was studied using 2-DE.In addition, we identified six differentially expressed proteins whose relative abundance increased or decreased more than 1.5-fold (P＜0.05).Most proteins could not be identified,which might be attributed to the lack of genomic sequences of P.globosa.Under nitrogen limitation,replication protein-like, RNA ligase, and sn-glycerol-3-phosphate dehydrogenase were reduced, which may decrease the DNA replication level and ATP production in P.globosa cells.The increase of endonuclease Ⅲ and transcriptional regulator enzyme may affect the metabolic and antioxidant function of P.globosa cells and induce cell apoptosis.These findings provide a basis for further proteomic study of P.globosa and the optimization of protein preparation methods of marine microalgae.

Keyword: Phaeocystis globosa; nitrogen limitation; proteomic response; two-dimensional electrophoresis

1 INTRODUCTION

Phaeocystisglobosabelongs to thePhaeocystisgenus of prymnesiophytes, and is widely distributed in the ocean.Massive blooms caused byP.globosawere frequently reported in the North Sea in the Atlantic Ocean and the coastal waters of East and Southeast Asia in the Pacific Ocean (Wang et al.,2021; Lavigne et al., 2022; Song et al., 2022; Zhang et al., 2022).DuringP.globosablooms, the formation of gelatinous foam and mucilage could clog the fish gills or harm circulating water filtration facilities (Wang et al., 2022b).Moreover,P.globosablooms produce sulfur compounds and create a low oxygen environment, which results in massive mortality of marine organisms (Wang et al., 2021).Some algal blooms can secrete large amounts of toxins into water not only toxic to aquatic organisms but may also be absorbed by fish and passed up to the food chain to humans through bioaccumulation(Tian et al., 2014).

Phaeocystisglobosaplays an important role in sulfur cycles (Song et al., 2021).In a sulfur cycle,dimethyl sulfide (DMS) is the most important volatile biogenic sulfide in the ocean (Leng et al.,2021).About 1.5×1013g of sulfur in the atmosphere is originated from the release of DMS every year,affecting global climate change and causing acid rain.P.globosaextensively releases 3-dimethylsulphoniopropionate (DMSP) into waters,which can be transformed into dimethyl sulfide(DMS) by marine bacteria (Gage et al., 1997;Mohapatra et al., 2013; Wang et al., 2022a).DuringP.globosablooms,Phaeocystiscan fix atmospheric CO2at a rate of up to 40 gC/m2per month (Chin et al., 2004), which will segregate carbon and influence atmospheric carbon dioxide concentrations and the global carbon cycle.

Nitrogen is an important limiting factor of microalgal growth.Nitrogen stress can strongly influence microalgal metabolism, such as photosynthesis.InNannochloropsisoceanica, nitrogen depletion destroys thylakoids and leads to macroautophagy in chloroplasts, reducing the total protein and carbohydrates by 33% and 12%-13%,respectively (Roncaglia et al., 2021).Previous studies have found that nitrogen limitation can decrease the expression of the photosystem Ⅱprotein, chloroplast light harvesting complex, and chlorophylla/bbinding of marine microalgaeChaetocerosaffinis,Chrysochromulinapolylepis,andGephyrocapsaoceanica, thereby reducing the photosynthetic transformation efficiency in systemⅡ (Harke et al., 2017).Additionally, Qiao et al.(2021) found that nitrogen limitation would reduce the repair ability of photosystem Ⅱ ofThalassiosira weissflogiiandThalassiosirapseudonana, the electron transfer rate of photosystem Ⅱ, and rapidly induce non-photochemical quenching (NPQ),making algal cells more sensitive to photoinhibition.Therefore, nitrogen limitation may lead to reduced photosynthesis in many algal cells.Similarly,duringP.globosablooms, variation of nitrogen concentration in waters clearly influences the photosynthetic efficiency and growth ofP.globosa.Under nitrogen limitation, colonies ofP.globosawere formed (Riegman et al., 1992).Colonies ofP.globosaare the main form of blooms in different marine regions.Notably, natural or anthropogenic nitrogen contributes to the rapid development ofP.globosablooms (Madhu et al., 2020).Therefore, the presence of nitrogen may lead to the frequent occurrence ofP.globosablooms, whereas nitrogen consumption induces the formation of gelatinousP.globosacolonies.Moreover, the decrease inP.globosablooms may be closely related to nitrogen depletion.However, thus far, the responses ofP.globosato nitrogen pulse and limitation remain largely unknown.

As a supplement to genomics and transcriptomics,proteomics has been used in recent years to study the metabolic pathways of algae because it can more directly understand complex biochemical processes at the molecular level (Chakdar et al., 2021).In algae research, comparative proteomic approaches have been successfully employed to analyze the copper tolerance of the brown algaEctocarpus siliculosus(Ritter et al., 2010) and oxidative damage ofChlamydomonasreinhardtiicaused by cyanobacterial blooms (Chen et al., 2020).They have also been used to further analyze the central metabolic pathways and secondary metabolic pathways of macroalgal cells, as well as the regulation of algal cell lifespans (Root, 2022).In this study, a comparative two-dimensional electrophoresis (2-DE) proteomic approach combined with mass spectrometry analysis was used to investigate the proteome ofP.globosaunder nitrogen limitation.This will lay the foundation for further research on the algal bloom and extinction mechanism ofP.globosain the future.

2 MATERIAL AND METHOD

2.1 Algal cultivation and experiment

Phaeocystisglobosacells were isolated from Dapeng Bay, Guangdong Province, China.Cultures ofP.globosawere maintained in f/2 medium (refer to the National Center for Marine Algae and Microbiota (NCMA)) without silicate prepared using artificial seawater at 20±0.5 °C under a light：dark cycle of 12 h：12 h in light density of 100 µmol/(m·s).The growth of the cultures was monitored daily by cell counting on hemacytometer.For the preparation of nitrogen-depleted cells, cultures at the late exponential phase (1×106cells/mL) were centrifuged at 2 000×gfor 8 min at 20 °C, and the pellets were inoculated into the f/2 medium without and with nitrate (8.82×10-4mol/L), respectively,after they were rinsed with sterile artificial seawater.After 4 days, the cultures were harvested and the pellets were used for protein preparation.

2.2 Optimization of protein preparation for twodimensional electrophoresis (2-DE)

To acquire high-quality 2-DE images, four methods were used for the protein extraction ofP.globosa: (A) this method followed the procedures reported previously with minor modifications (Wang et al., 2009).Algal pellets were suspended in urea/Triton X-100 buffer with 2% carrier ampholytes,and subjected to sonication on ice.Cell lysis was confirmed using a light microscope.Subsequently,this slurry was centrifuged at 15 000×gfor 30 min at 4 °C.The supernatant was transferred to a new tube and precipitated with 20% trichloroacetic acid in acetone (TCA/acetone, 1：4 v/v) for at least 12 h at-20 °C.The mixture was centrifuged at 20 000×gfor 30 min at 4 °C, and the pellets were then rinsed twice with ice-cold acetone and subsequently air-dried.Protein was dissolved in 100 μL of rehydration buffer (7-mol/L urea, 2-mol/L thiourea,4% 3-[(3-Cholamidopropyl) dimethylammonio]-1-propanesulfonate (CHAPS)).(B) Algal pellets were suspended in 10% TCA/acetone solution, and lysed using a sonicator.The slurry was centrifuged at 15 000×gfor 30 min at 4 °C.The pellets were rinsed twice with ice-cold acetone containing 20 mmol/L of dithiothreitol (DTT) and then air-dried.The protein powder was dissolved in 100-µL rehydration buffer.(C) Compared with methods A and B, our method used Radio Immuno Precipitation Assay(RIPA) lysate buffer (containing 50 mmol/L of Tris(pH 7.4), 150 mmol/L of NaCl, 1% Triton X-100,1% sodium deoxycholate, 0.1% sodium dodecyl sulfate (SDS)) for the first time to lyse algal pellets using a sonicator.Subsequently, this slurry was centrifuged at 15 000×gfor 30 min at 4 °C.The supernatant was transferred to a new tube and then precipitated with 20% TCA/acetone (1：4 v/v) for at least 12 h at -20 °C.The mixture was centrifuged at 20 000×gfor 30 min at 4 °C, and the pellets were then rinsed twice with ice-cold acetone and subsequently air-dried.Protein powder was dissolved in 100-µL rehydration buffer.(D) Algal pellets were sonicated in pre-chilled 40-mmol/L Tris buffer.The slurry was centrifuged at 15 000×gfor 30 min at 4 °C, and the supernatant was concentrated using a 3-kD Nanosep centrifugal ultrafiltration device (Pall Life Sciences, USA).The retentate was further purified using a cleanup kit(Bio-rad, USA).

2.3 Two-dimensional electrophoresis

The protein content was measured using the Bicinchoninic Acid Assay (BCA) assay kit(Promega, USA).For each sample, 30 µg of protein was added into the rehydration buffer, and immobilized pH gradient (IPG) strips of linear pH gradient 4-7 were used.Isoelectric focusing was conducted in the strip holder.Active rehydration was run under 50 V for 12 h before the following procedures were used: 0.5 h at 250 V, 0.5 h at 1 000 V,4 h at 4 000 V, and 40 000 Vhr at 8 000 V.After isoelectric focusing, these strips were immersed into 10 mL of equilibration buffer 1 and a trace amount of bromophenol blue for 20 min.Subsequently, the strips were transferred to equilibration buffer 2(same as the equilibration buffer 1 except DTT was replaced with 2.5% iodoacetamide) for another 20 min.The strips were subsequently loaded on top of 12.5% acrylamide gels and run at constant current.After electrophoresis, the gels were stained with silver nitrate as described by a previous study(Chan et al., 2004).

2.4 Gel analysis

Gel image were captured and gel comparisons were conducted according to the method of Dong et al.(2015).A software was used to automatically conduct spot detection and matching before conducting manual editing and normalization.To reduce possible staining differences between gels,spot quantification was normalized, and the percentage of each spot volume to the total spot volumes of the gel was obtained.Matching errors produced by the software were checked manually.Three gels from biological replicates were analyzed for the control and treatment groups.Statistically significant difference (P＜0.05) and ratio ＞1.5 were used for cutoffs.These clear spots with significant variation were selected for mass spectrometric analyses.

2.5 Tryptic in-gel digestion

In-gel digestion of protein spots was conducted according to a procedure from a previous study with minor modifications (Wang et al., 2011).Differential protein spots were excised and then transferred into 0.2-mL microcentrifuge tubes, and rinsed according to the method used by Wang et al.(2011).Subsequently, they were reduced with 10 mmol/L of DTT in ammonium bicarbonate before alkylation with 55 mmol/L of iodoacetamide in ammonium bicarbonate.All gel pieces were incubated with 12.5×10-3-μg/μL sequencing grade trypsin (Promega,USA) in 20 mmol/L of NH4HCO3overnight at 37 °C, and the supernatant was dried in a SpeedVac centrifuge.The dried peptides were then dissolved in 0.5% trifluoroacetic acid, and spotted on the target plate for mass spectrometry analysis.

2.6 MALDI-TOF/TOF analysis and database search

Matrix-Assisted Laser Desorption/Ionization Time of Flight (MALDI-TOF) mass spectrometry and tandem TOF/TOF mass spectrometry were studied on the Applied Biosystems Sciex 4800 MALDI TOF/TOF mass spectrometer using the method of Ma et al.(2010) and the GPS Explorer software to analyze mass spectrometry (MS) and tandem mass spectrometry (MS/MS) raw data (Ma et al., 2010).The MS and MS/MS spectra were combined and searched against the National Center for Biotechnology Information (NCBI) non-redundant database using an in-house Mascot server using following parameters: one missed cleavage site,significance threshold ofP＜0.05, peptide mass tolerance of 50×10-6, MS/MS tolerance of 0.2-0.3 Da,carboamidomethylation of cysteine as fixed modification, and methionine oxidation as variable modification.Known contaminant ions (keratin)were excluded.Mascot protein scores greater than 61 were considered to be statistically significant(P＜0.05).

3 RESULT

3.1 Comparison of extraction methods

Evaluation of 2-DE image quality obtained using the four methods is presented in Table 1.Using extraction method A, the protein content was 14.0×10-3μg/cell, the number of spots was 256, and the protein separation and background sharpness was“+”.Using extraction method B, the protein content was 13.5×10-3μg/cell, the number of spots was 233,and the protein separation and background sharpness was “-”.Using extraction method C, the protein content was 13.6×10-3μg/cell, the number of spots was 236, and the protein separation andbackground sharpness was “++”.Using extraction method D, the protein content was 13.4×10-3μg/cell,the number of spots was 242, and the protein separation and background sharpness was “-”.No significant difference was observed in the protein contents and spot numbers obtained using the four methods.To further compare the differences of the four protein extraction methods, we used 2-DE to compare the extracted protein qualities.As shown in Fig.1, the electrophoretic effects of different protein extraction methods were different.The gel background interference of groups A and B is serious, and the protein spots cannot be separated.Gel background of group D is clear, but the protein spot observation effect is low, whereas the gel background of group C is clear, and the separation of protein spots is better.

Table 1 Comparison of protein contents and quality of two-dimensional electrophoresis (2-DE) images obtained using the four methods

3.2 Proteomic response to nitrogen depletion

Protein synthesis is closely related to biological metabolism and can accurately reflect the metabolic status of cells under different conditions, thereby facilitating the detailed study of cell metabolic pathways.Here, the proteomes ofP.globosacultures under nitrogen limitation were compared with those of nitrogen-replete cultures using 2-DE.By filtering the speckling and background of 2-DE,we detected 18 distinct protein spots (a1/b1 to a6/b6) for further analysis (Fig.2), among which 4 proteins were up-regulated in the nitrogen-replete group (increased by more than 1.5-fold) and 8 proteins were decreased in the nitrogen-depleted group (decreased by more than 1.5-fold) (P＜0.05,based on thettest).These proteins were picked from the gel and analyzed using MALDI-TOF/TOF mass spectrometry.Six proteins were identified,among which the mago nashi-like protein (a1/b1),sn-glycerol-3-phosphate dehydrogenase (a4/b4),replication protein-like (a5/b5), and RNA ligase (a6/b6) decreased under nitrogen limitation, whereas endonuclease Ⅲ (a2/b2) and putative transcriptional regulator (a3/b3) increased.Most proteins could not be identified, which might be due to the lack of genomic sequences ofP.globosa.Protein annotation and function are presented in Table 2.

4 DISCUSSION

Fig.2 Two-dimensional electrophoretograms of Phaeocystis globosa under nitrogen-replete and nitrogen-depleted conditions obtained using method C (Radio Immuno Precipitation Assay (RIPA) with trichloroacetic acid (TCA)/acetone precipitation)

Table 2 Comparison in differently expressed proteins of Phaeocystis globosa cultured under nitrogen poor and nitrogenrich cultures

Large-scale outbreaks ofP.globosahas occurred in many sea areas around the world.The outbreaks endanger seriously the marine environment and marine life, and also cause harm to human health via food chain.Studies have found that nitrogen is an important limiting factor for the growth of microalgae, and its presence may promote the outbreak of microalgae.Therefore, it is necessary to understand the proteomic response mechanism ofP.globosaunder nitrogen limitation.In this study, we found that under nitrogen limitation conditions, the protein expressions of mago nashi-like protein,replication protein-like, RNA ligase, and snglycerol-3-phosphate dehydrogenase inP.globosadecreased, whereas the expressions of endonucleaseⅢ and putative transcriptional regulator increased,indicating that nitrogen limitation would significantly affect the protein expression ofP.globosa.Additionally, we also optimized the 2-DE protein preparation method ofP.globosa, and found that the combination of RIPA lysis buffer and TCA/acetone precipitation was the best for protein extraction.

Notably, sample preparation is the most crucial step in proteomic analysis.Several protein extraction methods have been used for proteomic studies of marine dinoflagellates.For example, the sequential extraction in combination with desalting by BioSpin chromatography has been successfully applied to the protein extraction ofProrocentrum triestinum(Chan et al., 2002).Alternatively, the optimal method forAlexandriumsp.was the extraction of urea/Triton X-100 buffer followed by TCA/acetone precipitation (Wang et al., 2009).However, these methods were not suitable forP.globosa.The Tris and RIPA lysis buffers used in this study are commonly used in the experiments of protein extraction.For example, the RIPA lysis buffer is used in proteomic analysis of human cancer cells for protein extraction, and the Tris buffer is often used for protein extraction of higher plants cells (Liu et al., 2019; Subedi et al., 2019;Soares et al., 2020).In this study, the Tris and RIPA buffers were used for the protein extraction of algal cells for the first time.We found that the effects of Tris buffer extraction and RIPA buffer extraction combined with TCA/acetone precipitation were superior to those of previously reported algal cell protein extraction methods because the former methods provided a clear gel background, which is essential for the comparison among gels (Wang et al., 2009).Considering the image quality, method C was the best among the four methods; it not only had a clear background, but also showed good separation effect between protein spots (Wang et al.,2009).Method C may have the best extraction effect possibly because RIPA buffer contains 0.1% SDS,which may promote protein dissolution and separation to improve the quality of protein extraction (Kopec et al., 2017).Although the combination of RIPA buffer and TCA/acetone precipitation could improve the protein extraction efficiency ofP.globosa, different marine microalgae require different specific methods of protein extraction for conducting 2-DE, which might be due to the differences in the pigment, compounds, and polysaccharides in different microalgae.

A mago nashi-like protein, replication proteinlike, and an RNA ligase were found to decrease significantly under nitrogen limitation.Mago nashilike protein is involved in RNA localization and cell differentiation (He et al., 2007), whereas replication protein-like and RNA ligase are involved in DNA replication and pre-tRNA splicing (Englert and Beier, 2005; Shultz et al., 2007), respectively.Snglycerol-3-phosphate dehydrogenase, which is a key enzyme in glycerol metabolism and converts glycerol 3-phosphate into dihydroxyacetone, interacts with nitrate reductase as a signal (Uribe-Alvarez et al., 2016; Koga et al., 2019).Moreover, sn-glycerol-3-phosphate dehydrogenase can promote the oxidation of NADH to NAD+, which contributes to ATP production in the mitochondria (Zhang et al.,2018; Ishihama et al., 2021).These results demonstrated that nitrogen limitation reduced the ATP production, DNA replication abilities, and transcription in cells ofP.globosa, and sn-glycerol-3-phosphate dehydrogenase might be responsible for transmitting this signal.

Previous studies have suggested that nitrogen limitation may inhibit the proliferation and growth of microalgae, which is further supported by the decreased expression of proteins involved in transcription and replication found in this study (Fan et al., 2019).Simultaneously, our study also found that endonuclease Ⅲ and a transcriptional regulator increased significantly under nitrogen depletion.Endonuclease Ⅲ belongs to the Helix-hairpin-Helix(HhH) DNA glycosylase superfamily, which is common in eukaryotes (Zhang et al., 2021).Endonuclease Ⅲ possesses endonuclease, hydrolase,and DNA lyase activity and is involved in the baseexcision repair, which can help repair cells during oxidative stress, and is used to monitor oxidative DNA damage in cells (Choulet et al., 2006; Zhang et al., 2021; Gupta and Imlay, 2022; Lee et al., 2022).In eukaryotes, transcription regulators play an important role in the life cycle of the cell by integrating cellular and environmental signals and controlling cell division (Liao et al., 2021).Changes in the expression of transcription regulators affect the physiological reactions, such as cell growth,apoptosis, and antioxidant, metabolism (Hernández et al., 2017; Roy et al., 2018; Liao et al., 2021).In this study, the increased expressions of endonucleaseⅢ and transcriptional regulator indicate that nitrogen deficiency may cause oxidative DNA damage ofP.globosa, leading to metabolic disorders of algal cells and resistance to nitrogen depletion by triggering cell apoptosis.Therefore,nitrogen limitation not only inhibitedP.globosaproliferation and growth but also caused oxidative stress, DNA structure damage, and the promotion ofP.globosacell apoptosis.

5 CONCLUSION

In recent years, the eutrophication of the seawater environment has increased the frequency of harmful algal blooms (HABs) around the world.P.globosacauses massive HABs around the world,and plays an important role in the global carbon or sulfur cycling.In this study, we found that the combination of RIPA lysis buffer and TCA/acetone precipitation was the best protein preparation method because of the clear gel background and the best separation effect of protein spots.Additionally,we found that under nitrogen limitation, levels of DNA replication in the cells ofP.globosamight decrease, metabolic and antioxidant functions might be affected, and apoptosis might be triggered against nitrogen limitation.However, owing to the limitation of genomic sequences ofP.globosa,fewer proteins were identified.Through the above results, we proposed a new protein preparation method forP.globosa, and obtained a preliminary understanding of the mechanism thatP.globosarespond to nitrogen depletion.This study provided a reference for upcoming proteomic analyses ofP.globosaand the optimization in preparation of marine microalgae protein in the future.

6 DATA AVAILABILITY STATEMENT

The authors declare that all data in the present study are available upon request.