LIANG Dayong, WANG Xiaodong, HUO Yiping, WANG Yan,*, and LI Shaoshan

1) Research Center for Harmful Algae and Marine Biology, and Key Laboratory of Eutrophication and Red Tide Prevention of Guangdong Higher Education Institutes, Jinan University, Guangzhou 510632, China

2) Key Laboratory of Ecology and Environmental Science in Guangdong Higher Education, School of Life Science, South China Normal University, Guangzhou 510631, China

Abstract Large-scale blooms of Phaeocystis globosa have caused serious damage to marine ecosystems in coastal waters of China.Phaeocystis blooms depend on the competitive advantage of their heteromorphic life history: colony formation has the benefit of resisting herbivory by zooplankton, and solitary cells can absorb nutrients rapidly. In order to better understand the mechanisms underlying the metabolic differences between the two types of cells, morphological observations, rapid light curve analysis, fatty acid profiling, and transcriptome assessment were conducted in the laboratory. The rapid light curve of colonial cells was higher than that of solitary cells, which indicated that colonial cells had higher CO2 fixation capacity. The fatty acid level of colonial cells was evidently lower than that of solitary cells, which is consistent with down-regulated synthesis of fatty acids and up-regulated degradation of fatty acids in the transcriptome. ATP-binding cassette transporters, the TCA cycle, and biosynthesis of exopolysaccharides (EPS)also displayed obvious differences. In summary, colonial cells have stronger carbon fixation capacity.They do not synthesize fatty acids as energy storage materials but secrete EPS, which might be one of the mechanisms of colony formation. Here we present a physiological and molecular overview of the differences between solitary cells and colonial cells and thereby provide further insight to help unravel the mechanisms that help Phaeocystis globosa adapt to different environments.

Key words Phaeocystis globosa; harmful algal bloom; heteromorphic life cycle; transcriptome

1 Introduction

The marine planktonic algal genusPhaeocystishas been observed to form extensive blooms in coastal waters throughout the world, which often lead to serious natural disasters and economic losses (Bretonet al., 2006; van-Leeuweet al.,2007; Pavlovet al., 2017).Phaeocystis globosa,P. pouchetii, andP. antarcticaare common causes of harmful algal blooms, which usually occur through the formation of colonies (Smithet al., 1991; Schoemannet al., 2005).P. globosablooms have occurred along the southeastern coast of China continuously since 1997 (Chenet al., 1999, 2002).Toxin released byP. globosais also a direct cause of fish death when algal blooms erupt (Longet al., 2016). Blooms have also occurred in the Beibu Gulf of Guangxi province,China, and blocked the cold source water intake system of nuclear power stations, causing a potential serious safety hazard (Liet al., 2015). Therefore,P. globosahas come into the public’s attention and attracted widespread interests.

P. globosahas a heteromorphic life cycle involving colony formation with a tegument of exopolysaccharidic matrix and diverse types of solitary cells (Parkeet al., 1971;Rousseauet al., 1994; Peperzaket al., 2000). Solitary cells are generally 3-10 μm in diameter, and colonies can reach up to 3 cm (Rousseauet al., 2007, 2013). The success ofP.globosais considered to be due to its ability to transform into a colonial form or remain as solitary cells. Colony formation is one of the remarkable plastic phenotypic characters ofPhaeocystis(Wanget al., 2015). The large size and tough outer layer of the colony inhibit the ingestion by zooplankton, protecting the continued growth of cells in the colony (Hamm, 2000). Such colonial formation and enlargement can reduce the risk of predation and play a key role in the marine system (Nejstgaardet al., 2007). Although solitary cells are susceptible to ingestion, they grow rapidly and can survive in harsh conditions such as darkness or nutrient restriction (Riegmanet al., 1992). Solitary cells have a competitive advantage when ammonium and phosphate are limited (Rousseauet al., 2007). The existence of a heteromorphic life history thus allowsP.globosato outcompete other algal species in a complex environment. Many studies have documented the physical differences between colonial and solitaryPhaeocystiscells as well as their varying fitness under a myriad of conditions (Benderet al., 2018; Mars-Brisbinet al., 2019). A recent study revealed the mechanism of colony formation and the changes in transcriptome genes during the life cycle transition ofP.globosa(Zhanget al., 2020). However,few studies have isolated cells from different stages of the life cycle in simultaneous culture, and the differences in molecular regulatory mechanisms between two types of cells, each with diverse competitive advantages, are still unknown.

In the present study, the morphology ofP. globosawas distinguished by light microscopy and electron microscopy,respectively. Its physiology was studied by measuring the rapid light curve (RLC) and fatty acid (FA) profiling. We describe the results of high-throughput transcriptomic sequencing, followed byde novoassembly and differential gene expression analyses ofP. globosacells at different stages in the life cycle. This comprehensive analysis allows us to seek differences between the two cell types considering the metabolic pathways of photosynthesis, carbon source allocation, and exopolysaccharide (EPS) secretion.These results provide clear insight into the regulatory profiles of the different cell types and a rich source of genetic information onP. globosa. The heteromorphic life cycle is an important competitive adaptation. Here, we provide significant preliminary findings regarding the metabolic mechanisms underlying rapid algal bloom formation.

2 Methods

2.1 Algae Culture and Sampling

Algae were isolated as colonies from the Beibu Gulf(21°56.83′N, 108°20.80′E) during the bloom on September 10, 2017. A single-cell isolation was established on September 15, 2017 by selecting solitary cells using a micropipette. The phylogenetic analysis of its partial sequence of 18S rDNA indicated that this strain wasP. globosa. A pure strain was conserved in the Research Center for Harmful Algal and Marine Biology of Jinan University.Algae were cultured infiltered and sterile artificial seawater (salinity 30) enriched with f/2 medium as described by Guillard and Ryther (1962). Cultures were maintained in flat-bottomed conical laboratory flasks (2 L) in three parallel rows at 20 - 22℃ under a 12 h:12 h light:dark cycle with approximately 80 - 100 μm s-1m-2of cool white fluorescent illumination. Solitary cells (GX-S) and colonial cells(GX-C) were separated through a filter membrane (20 μm)after 10 days (late exponential growth) of cultivation, and the solitary cells were washed off the membrane with sterile seawater. GX-S and GX-C were centrifuged (5000 r min-1,5 min) and immediately frozen in liquid nitrogen and stored at -80℃ for subsequent use.

2.2 Morphological Observation

Algae samples were stained with 1 mL 0.02% Alcian blue (pH = 2.5, containing 0.06% acetic acid) and observed under an inverted light microscope (LM, Olympus-CKX41,Japan). GX-S and GX-C were centrifuged, and the precipitate was washed with 0.1 mol L-1phosphate buffer (pH 7.2). The cell sediment was fixed in glutaraldehyde (2.5%final concentration) and then refrigerated at 4℃ overnight.Samples were filtered through a 1 μm membrane, and the filter was dehydrated in a graded ethanol series. The filter was then dried to eliminate the dehydrating agent and a small amount of moisture, and the samples were then goldcoated and observed under a scanning electron microscope(SEM, Zeiss SIGMA 500, Germany).

2.3 Rapid Light Curve (RLC) and Fatty Acid(FA) Profiling

Chlorophyll fluorescence measurement was performed by connecting a pulse modulated chlorophyll fluorescence meter (PAM-2100, Walz, Germany) to an excitation-detection unit (ED-101US/M, Walz, Germany). Curve fitting was performed by Statistica software and using the Platt (1980)formula:

wherePis the relative electron transfer rate;Pmis the maximum potential electron transfer rate without photoinhibition;αis the initial slope of theP-Icurve, andβis the light suppression parameter.

FA profiling was performed on lyophilized cell pellets(Modul YOD-230, Thermo-Fisher, USA)viagas chromatography-mass spectrometry (Agilent 6890 gas chromatograph coupled to an Agilent 5975 mass selective detector,Agilent Technologies, USA). Heptadecanoic acid (C17:0,Sigma-Aldrich, USA) was added as an internal standard for FA quantification. Fatty acid methyl esters (FAMEs)were prepared and analyzed according to the protocol of Luet al. (2012). The degree of lipid unsaturation (DLU)was calculated following the method of Kates and Baxter(1962) which is as follows:

2.4 RNA Extraction, cDNA Library Construction,Sequencing, Data Filtering and Mapping

For RNA extraction, GX-S and GX-C were resuspended, respectively, in 2 mL RNA extraction buffer (1:1 mix of aqua-phenol and buffer L [0.5% SDS, 10 mmol L-1EDTA, 0.2 mol L-1sodium acetate (pH 5), and 1:100 βmercaptoethanol] ) and then incubated with DNase I (Takara, Japan) for 30 min at 37℃ to remove potential genomic DNA pollution. RNA quality analysis, cDNA library construction, sequencing, data filtering and mapping were performed by Novogene Bioinformatics Technology Co.,Ltd. (Beijing, China). The Illumina TruSeq RNA Sample Prep Kit (Cat#FC-122-1001) was used with 1 μg of total RNA for the construction of sequencing libraries. The data discussed in this publication have been deposited in NCBI’s Gene Expression Omnibus and are accessible through GEO Series accession G-SE140985 (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE140985).

2.5 Differential Gene Expression Analysis

Differential gene expression analysis of solitary cells and colonial cells was performed using the DESeq R package(1.10.1). DESeq provides statistical routines for determining differential expression in digital gene expression data using a model based on the negative binomial distribution(Loveet al., 2014). The resultingPvalues were adjusted using the Benjamini and Hochberg’s approach for controlling the false discovery rate. Genes using the criteria as|log2(Fold change)| ＞ 1.5 andP＜ 0.05 found by DESeq were assigned as differentially expressed genes (DEGs). Functional classification of DEGs was conducted according to annotation in gene ontology, and the pathway analysis was carried out according to KEGG.

2.6 Quantitative RT-PCR

To validate the transcriptome results and DEGs, quantitative RT-PCR was performed on a Quant Studio 6 Flex Real-Time PCR System (Applied Biosystems, USA) using Platinum? SYBR? Green qPCR SuperMix-UDG (Invitrogen, USA). The genes encoding ATP-binding cassette, subfamily C (ABCC1), transketolase (TK), aconitate hydratase 2 (ACO2), acetyl-CoA carboxylase (ACC), bubblegum (ACSBG), and UDP-sugar pyrophosphorylase (USP)were selected for validation. The sequences of targeted genes were obtained from the transcriptome database, and primers were designed using the Primer Premier 5.0 software program. The Actin2 gene (At3G18780) was used as an internal control. The thermal cycle parameters used were as follows: 95℃ for 2 min, then 40 cycles at 60℃ for 30 s and 60℃ for 30 s. The relative quantity of each gene transcript was normalized to that of the reference gene Actin2 in each sample. The expression log2(Fold change)was calculated as follows: log2(Fold change) gene of x =(CtGX-S- CtGX-C) of gene x - (CtGX-S- CtGX-C) of Actin2.

2.7 Statistical Analysis

Data were calculated from three replicates per treatment and presented as means ± SE. Significant differences between solitary cells and colonial cells for each of the tests were compared using one-way analysis of variance (ANOVA)by SPSS version 22.0 (IBM, USA). Statistical significance was set atP＜ 0.05 compared with the control.

3 Results

3.1 Solitary Cells and Colonial Cells of P. globosa

The results of LM and SEM (Fig.1) showed thatP. globosaconsisted of solitary cells and colonial cells, which differed in form and lifestyle. GX-S was absolutely dominated by flagellate cells with two equal-length flagella and a haptonema (Fig.1A). They generally had a rounded shape and a smooth, naked cell surface (Fig.1B). The solitary cells continuously released EPS and formed a giant colony through cell mitosis (Figs.1C, F). GX-C cells were randomly distributed at the periphery of the spherical colony, and had a smooth, scaleless surface (Fig.1D). SEM observation showed that flagella and a short haptonema were replaced by two short appendages (0.2 - 0.4 μm in length) at the center of the apical pole (Fig.1E).

3.2 RLCs, PS II Electron Transport Rates, and Fatty Acid Profiling

Fig.1 LM and SEM photographs of cells and a colony. Solitary cells showing the two flagella with a tapered end and a haptonema (A, B); colonial cells showing the two short appendages on their apical side (D, E); a 700-μm-diameter, spherical colony containing some 1400 colonial cells and tegument of exopolysaccharidic matrix was dyed by Alcian Blue (C, F). 1,flagella; 2, haptonema; 3, appendage.

The relative electron transport rate (rETR) measured by fluorescence reflects not only the photosynthetic capacity but also the photoinhibition curve, which is an effective alternative to traditional methods. After cultured for 10 d,the α value (initial slope of the RLC) showed no significant differences between GX-S and GX-C (Fig.2,P＞ 0.05,ANOVA). However, the point of light saturation (Ik) and the maximal relative electron transport rate (Pm, rETRmax)increased by 26.48% and 34.07% in the GX-C group, respectively (Fig.2,P＜ 0.01, ANOVA). The RLC of the slope was larger in GX-C, and the maximum value was higher than that of GX-S, which indicated that colonial cells had higher photosynthetic activity (Fig.2).

The fatty acid methyl ester (FAME) content and profile were further analyzed on the 10th day of culture, and the FA content was compared between GX-S and GX-C (Table 1). The FA profiles indicated that C14, C16, C18 and C24 were the main FA components inP. globosa.C16,C18 and C24 were the major FA components, accounting for more than 90% of the total FA. The FA content of GXC (2.249 mg g-1) was evidently lower than that of GX-S(10.222 mg g-1), which accounted for only 22% of GX-S(Table 1,P＜ 0.05, ANOVA).

Fig.2 Changes in photosynthetic coefficients α, Pm, and Ik and rapid light curves for the two cell types (black represents GX-C; orange represent GX-S). Each bar represents the mean of three biological replicates, and error bars signify the standard deviation of those means.

Table 1 Quantity (mg g-1) and proportion (%) of fatty acid components in different P. globosa cells

3.3 Transcriptome Analysis

In order to study the differences between solitary cells and colonial cells at the transcriptome level, GX-S and GXC (10 d) were subjected to RNA extraction and transcriptome sequencing analysis. GX-S was set as the control group, and different genes were identified by comparing GX-S and GX-C. The analyses were conducted with triplicates. Pearson correlation analysis was conducted on each sample, and R2 values ranged from 0.944 to 0.984. Raw data ranged from 25608632 to 35516726 reads per sample. After more than 24 million clean reads were obtained,low-quality sequences and adapter sequences were removed.As the results, 4386 genes were differentially expressed at statistically significant levels, which included 2268 up-regulated genes and 2118 down-regulated genes (Fig.3a). Genes with significantly differential expressions were involved in several pathways, including starch and sucrose,phagosome, inositol phosphate metabolism, and FA degradation (Fig.3b). Exploring the variations of these pathways revealed the variation in metabolism between solitary cells and colonial cells in the life cycle ofP. globosa.

3.3.1 Enhanced carbon fixation in the Calvin cycle

Under conditions of elevated CO2, genes associated with photosynthetic CO2fixation, known as the Calvin cycle,are up-regulated. Genes encoding transketolase (TK), multifunctional protein 2 (MFP2), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and triosephosphate isomerase(TPI) were significantly up-regulated in GX-C (Fig.4b). TK(40138.19899, 41388.20196) is a very important enzyme in the process of carbon metabolism and plays a key role in the Calvin cycle of photosynthesis. TPI (40138.36029,56561.0) catalyzes reversible exchange between dihydroxyacetone phosphoric acid (DHAP) and D-glyceraldehyde-3-phosphate (GAP) isomers. No significant change was found in RuBisCo (Ribulose-1, 5-bisphosphate carboxylase/oxygenase), which is a key enzyme catalyzing the initial step of carbon immobilization, and might be regulated post-transcriptionally. Up-regulation of Calvin cycle genes implied that more carbon was fixed through the Calvin cycle in GX-C, consistent with the stronger photo-synthetic activity.

3.3.2 FA biosynthesis and degradation

Suppressed FA biosynthesis and enhanced FA degradation were found in GX-C (Fig.4d). The expression of acetyl-coenzyme carboxylase (ACC, 46738.0), a key regulatory enzyme in the FA synthetic pathway, was significantly down-regulated 5.28 fold. The reaction catalyzed by ACC is regarded as the rate-limiting step in many organisms. Genes coding nearly all the enzymes throughout the FA degradation pathway were consistently up-regulated compared with GX-S, including acyl-CoA synthetase (ACS),acyl-CoA dehydrogenase (ACADM), and acyl-CoA oxidase(ACOX). ACS homologous proteins comprise the longchain acyl-CoA synthetase (ACSL, 46738.21248), and bubblegum (ACSBG, 41388.21236) subfamilies, which activate long-chain and very-long-chain FAs to form acyl-CoAs,and the genes that encode these proteins were up-regulated. Suppression of FA biosynthesis and enhancement of FA degradation in GX-C led to a sharp decrease in FA content, which was consistent with our physiological data.

3.3.3 Accelerated TCA cycle

Impressively, genes of GX-C coding enzymes throughout the TCA cycle were consistently up-regulated, including pyruvate dehydrogenase (PDH), aconitase 2 (ACO2),2-oxoglutarate dehydrogenase (OGDH), and succinyl-CoA synthetase (SCS) (Fig.4c). Plant mitochondrial ACO2(41388.884) reacts asymmetrically with citric acid to catalyze the formation of isocitric acid, which enables the TCA cycle to proceed smoothly and provides energy and various intermediate metabolites for plants. OGDH(41388.30069), as a metabolic regulator of alpha-ketoacid in microbial cells, is composed of three different enzymes.Synergistic up-regulation of expression accelerated the TCA cycle and produced more NADH and GTP/ATP. Upregulation of TCA cycle genes implied that more metabolic energy and intermediates were generated through the accelerated TCA cycle to sustain anabolism in GX-C.

3.3.4 EPS biosynthesis

Polysaccharide production pathways are conserved across prokaryotes and eukaryotes, with monosaccharides converting to nucleotide sugars and assembling into polysaccharides by the activity of glycosyltransferases (GTs) (Gügiet al., 2015). Mannosyl-oligosaccharide alpha-1,3-glucosidase (MOGS, 41388.31660) has the property of glucosidase, which converted oligosaccharides to glucose (López-Romeroet al., 2004b), and we found that MOGS was downregulated during this process. UDP-sugar, as a sugar donor of glyconucleotides, is synthesizedin vivoand catalyzed by UDP-sugar pyrophosphorylase (USP, 41388.15749)(Fig.4e). In our study, the genes encoding USP and PGM were generally up-regulated. This indicated that the synthesis of nucleotide sugars was increased in GX-C, among which glucose and galactose were the main glycosyl donors.

3.4 Validation of DEGs by qRT-PCR

Fig.3 Diagram of mRNA sequence analysis in P. globosa from solitary cells (GX-S) and colonial cells (GX-C). (a), The identification of differentially expressed genes; (b), KEGG pathway enrichment analysis.

Fig.4 Responses to changes in the transcriptional abundance of genes involved in metabolic pathways and biological processes. A, ATP-binding cassette transporters; B, Calvin cycle pathway photosynthesis; C, TCA cycle; D, fatty acid biosynthesis and degradation; E, EPS biosynthesis. Key enzymes are included in the map and presented as their names (in red up-regulated; in blue down-regulated; in black relatively unchanged). Gene IDs and levels of fold changes are as indicated by the color boxes. Chemical compound abbreviations: ACAC1, acetyl-CoA acyltransferase 1; ABCC, ATP-binding cassette, subfamily C; AMT, ATP-binding cassette, subfamily B, mitochondrial transporter; PEPCK, phosphoenolpyruvate carboxykinase (ATP); PDH, pyruvate dehydrogenase E2 component; ACO2, aconitate hydratase 2; OGDH, 2-oxoglutarate dehydrogenase E1 component; SCS, succinyl-CoA synthetase beta subunit; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; GAPA, glyceraldehyde-3-phosphate dehydrogenase; TK, transketolase; TPI, triosephosphate isomerase; MFP2,multifunctional protein; ACACA, acetyl-CoA carboxylase; ACC, acetyl-CoA carboxylase; KAS II, 3-oxoacyl-[acyl-carrier-protein] synthase II; ACSL, long-chain acyl-CoA synthetase; ACSBG, bubblegum; ACOX, acyl-CoA oxidase; ACADA,acyl-CoA dehydrogenase; ALG3, alpha-1,3-mannosyltransferase; MOGS, mannosyl-oligosaccharide alpha-1,3-glucosidase;USP, UDP-sugar pyrophosphorylase; PGM, phosphoglucomutase.

Fig.5 Quantitative RT-PCR analyses at the later stage. Expression log2 (Fold change) (GX-S/GX-C) of six genes were evaluated by quantitative RT-PCR.

The six DEGs in the transcriptome data were validated by using qRT-PCR (Fig.5). From solitary cells to colonial cells, the expression levels of the gene encoding TK related to the Calvin cycle, ACO2 related to the TCA cycle,ACSBG related to FA degradation, and USP related to EPS biosynthesis were increased (P＞ 0.05), while the following unigenes were decreased (P＞ 0.05): ABCC1 related to environmental tolerance and ACC related to FA biosynthesis. Although the log2(Fold change) of all six DEGs between the qPCR and RNA-Seq results were not identical, the variation trends for these DEGs were consistent. This implies that our transcriptome data are accurate and reliable.

4 Discussion

Some algae that form blooms have complex life cycles,characterized by a series of transitions between several different life stages. Organisms may adopt different forms in different environments and have different functional roles in the ecosystem, even though they are genetically identical (Von-Dassow and Montresor, 2011). The specific factors that trigger transitions between the different stages in algae in nature are mostly unknown, but it is clear that an understanding of this process provides the key to effectively forecast bloom recurrence, maintenance, and decline (Figueroaet al., 2018). High-biomass-forming species of the genusPhaeocystisare well-known harmful species, and their life cycle has not been fully elucidated, especially in terms of the function of different cell types.

P. globosahas an unusual heteromorphic life cycle that includes gelatinous colonies composed of thousands of nonmotile cells embedded in a mucilaginous matrix under certain conditions (Lancelotet al., 1998; Rousseauet al.,2007). We found that the solitary cells (GX-S) was dominated by flagellate cells with two equal-length flagella and a haptonema, and the colonial cells (GX-C) only had two short appendages at the apical pole. The results of the morphological differences between the two cell types described here confirmed the findings of Schoemannet al. (2005)and Rousseauet al. (2013). The cells formed a mosaic in the colony, and few solitary cells were found in samples during algal blooms ofP. globosa. Smithet al. (2014) found thatP. globosacells existed in the colonial form, and their diameters ranged from 0.07 to 1.3 cm along the coast of Vietnam. Many researchers believe that the success ofP.globosais mainly due to colony formation. As described in the introduction, zooplankton are unable to effectively feed on colonies due to their relatively large volume and tough tegument, and the colonies are less vulnerable to viral infection (Brussaardet al., 2007); however, the process of colony formation requires more energy (Jakobsen and Tang, 2002; Brussaardet al., 2005). We found that GXC cells have stronger photosynthetic activity, suggesting that they have a greater carbon fixation function, which may be used for the formation of EPS. In addition, the content of FAs is obviously low in colonial cells, which indicates that the metabolic mechanism is shifting from FA synthesis to EPS formation. Although colony formation can resist feeding pressure, the growth rate of colonial cells is reduced, and nutrient diffusion is restricted by the heavy tegument (Plouget al., 1999). Flagellates are also an important stage in their life cycle. Verityet al. (2007)argued that solitary cells had higher resource competitiveness and therefore a higher survival rate under light or nutrient constraints, which was one of the advantages of the heteromorphic life cycle patterns ofP. globosa. The morphologies and physiological differences suggest that there may be a large difference in the molecular regulatory mechanisms to resist environmental stress.

ATP-binding cassette transporters (ABCs) are a large family of membrane proteins with many functions that exist in most organisms (Reeset al., 2009). ABCA, AMT, and ABCC are obviously down-regulated in GX-C compared to GX-S. Among the three identified ABC transporters, the annotation of cluster (41388.21133, 41388.32967) indicated a characteristic of ABCB, and the annotation of cluster (41388.11087, 41388.8273, 41388.26714, 51150.0) indicated a characteristic of ABCC1. Multidrug resistance(MDR, P-glycoprotein, ABCB) and multidrug resistanceassociated protein (MRP, ABCC) are proteins with good anti-chemotherapy and self-toxicity characteristics (Stukkenset al., 2005; Nuruzzamanet al., 2014). AtMRP6 expression was significantly up-regulated inArabidopsis thalianaunder cadmium stress, thereby playing a role in cadmium resistance (Gaillardet al., 2008). Heavy metals are generally considered to be the major anthropogenic pollutants in global coastal and marine environments. They also affect the growth of dinoflagellates (Couetet al., 2018).A high concentration of Cu2+was found to up-regulate ABCB1, ABCC1, and ABCG2 transcripts inProrocentrum lima, suggesting a potential defensive role of ABC transporters against metal ions in surrounding waters (Guet al.,2019). Down-regulation gene encoding of ABCB and ABCC1 implies that the GX-C form is less tolerant to harmful substances in the environment, which may also be due to encapsulation of the colony in mucus to provide better protection. However, Plouget al. (1999) suggested that mucus is highly permeable to the inorganic substances, which may contradict the possibility of limiting heavy metal ions by diffusion. It is generally believed that any physiological advantage must be balanced in terms of metabolic costs(Wardet al., 2011). Colony formation is the response to major feeding pressures, while it appears to potentially weaken the algae’s resistance to heavy metals in the ocean. Because different defenses are energy demanding, limiting resources could be allocated to growth or reproduction under predation-free conditions to optimize energy expenditures as seen in the solitary cell form ofP. globosa.

Although only ATPF1B was up-regulated in photosynthesis, ATPase is a necessary enzyme for photosynthetic electron transport and photophosphorylation in plant photosynthesis (Youvanet al., 1987). In addition, the gene encoding transketolase (TK) was significantly up-regulated in GX-C, which is the rate-limiting factor for photosynthesis in plants by the Calvin cycle. In tobacco transformed with a construct containing an antisense TK sequence, a 20%-40% reduction of TK activity inhibited ribulose-1, 5-bisphosphate regeneration and photosynthesis (Henkeset al.,2001). The Calvin cycle must be responsive to changes in the supply of products from electron transport and demand for carbon skeletons (Howardet al., 2011). The Calvin cycle of CO2fixation is modulated by the PRK/GAPDH/CP12 complex and a high-molecular-weight oligomeric form of GAPDH, allowing the flux of carbon assimilation to keep pace with the production of chemical energy by light reactions of photosynthesis (Buchananet al.,2005). Such regulatory complexes are popular in higher plants. Boggettoet al. (2007) found that the complex also exist inThalassiosira pseudonanaand considered that CP12 is ubiquitous in diatoms. This suggests that GAPDH may play an important role in the Calvin cycle ofP. globosa,and up-regulation of the gene encoding GAPDH may regulate the Calvin cycle to accommodate more carbon fixation. Generally, up-regulation of ATPF1B and the Calvin Cycle indicates that GX-C has higher RLCS, fixes more carbon, and produces more organic compounds.

GX-C can also fix more carbon, which may not be used to synthesize energy storage materials. It was found that the gene encoding ACC, which is a key enzyme in the FA synthesis pathway, was significantly down-regulated. The ACC enzyme in the plant was investigated, and overproduction of ACC inBrassica napuscaused the lipid content to increase by 6% (Nakamuraet al., 1979). Xinget al.(2018) found that up-regulation of ACC in the oleaginous microalgaAuxenochlorella protothecoidesproteome led to an increase of FA under high temperature. If only FA synthesis pathways were down-regulated, sometimes algae did not display lower FA levels. We also found that the FA metabolism pathway of GX-C was up-regulated.ACS is the key enzyme in the process of FA decomposition, including ACS, ACADM, ACOX, and MFP2. The acyl-carrier-protein synthase is highly homologous to ACS.After the acyl-carrier-protein synthase was knocked out fromSynechoccussp. andSynechccoccus elongatus, free FAs increased significantly in cells (Kaczmarzyk and Fulda, 2010). The FA synthesis pathway was significantly down-regulated, and the FA decomposition pathway was significantly up-regulated in GX-C. This indicates that the excess fixed carbon is not used for FA synthesis.

Microbial production of polysaccharide-rich extracellular polymers is ubiquitous in many environments. EPS production is a particular feature of the pennate diatoms that are dominant in autotrophic biofilms and sea ice microbial assemblages (Underwoodet al., 2003). Hamm (2000)found that the main components of the colony are polysaccharides, which are tough, elastic, and transparent, with a large pore size on the surface. When Alcian blue was used to stain the colony, it was confirmed that the main component of the colony was polysaccharide. However,we did not find many differences in gene expression in the EPS synthesis pathway through our transcriptome data analysis. Colony expansion and the formation of a colony by solitary cells all require the release of large quantities of EPS, and the genes that lead to EPS synthesis were over expressed, thus we could not detect many differences gene between the two cell forms. Interestingly, transcriptomic analysis revealed that the expression of the gene encoding USP increased compared with that of GX-S.UDP-sugar-producing pyrophosphorylases are important enzymes in the process of glucose metabolism, which exist widely in organisms and are responsible for the synthesis and metabolism of UDP-sugar (Okazakiet al., 2009).With the decrease in temperature, the EPS secretion of the diatomFragilariopsis cylindrusincreased, and the results of its proteome showed that USP was significantly up-regulated (Aslamet al., 2018). This indicates that more glycosyl groups can be provided for the synthesis of nucleotide sugars. In addition, we found that the gene GANAB (mannosyl-oligosaccharide alpha-1, 3-glucosidase,41388.31660), which has the property of glucosidase and can convert oligosaccharide to glucose (López-Romeroet al., 2004a), was down-regulated. The increased glycosyl supply and reduced degradation of polysaccharides accelerated the synthesis of polysaccharides in GX-C.

5 Conclusions

This study combined physiology with transcriptome sequencing to study two different types of cells in the heteromorphic life cycle ofP. globosaand the results allow us to study the differences in the molecular regulatory mechanisms of these cells. Colonial cells (GX-C) have stronger photosynthetic activity and carbon fixation ability, a lower FA content, EPS secretion consistent with the metabolic pathways including an enhanced Calvin cycle, suppression of FA biosynthesis, enhanced FA degradation, an accelerated TCA cycle, and increased EPS biosynthesis.GX-C can fix more carbon to produce more energy, which is used to secrete EPS instead of synthesizing FAs as a reserve energy source. Though the results seem to fall short of providing a comprehensive understanding of the complete physiology of the two different types ofP. globosacells, the results do provide a strong basis for further research.

Acknowledgements

This work was supported by the National Key R&D Program of China (No. 2017YFC1404300), the National Natural Science Foundation of China (Nos. 41976082,41676144 and 31670266), the Natural Science Foundation of Guangdong Province (No. 2017A030313115). We thank Prof. Paul Giller (University College Cork, Ireland)for English editing of the manuscript.