Cell cultures
Axenic P. tricornutum cells were obtained from the Institute of Hydrobiology, Chinese Academy of Sciences, and grown at 20 °C in a photoperiod 16 h:8 h, light:dark. Cells were grown under 80 μmol photons m−2 s−1 and collected during the exponential growth phase.
Phylogenetic analysis and prediction of Pt2015 localization in P. tricornutum
Phylogenetic analysis of Pt2015 proteins was performed using the MEGA v.7.0 platform using the neighbor-joining method. In total, 34 protein sequences were used to perform the phylogenetic analysis. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (500 replicates) was shown next to the branches in Fig. 1. Moreover, a comparison of the conserved regions in the Pt2015 protein from diatoms, chrysophytes, dinoflagellates, and other red-tide algae was performed using the MEGA v.7.0 platform. In addition, the cloned Pt2015 sequences were analyzed for the presence of N-terminal signal peptides and cleavage sites using the SignalP v.4.1 Server (www.cbs.dtu.dk/services/SignalP/). The TMHMM Server v.2.0 (www.cbs.dtu.dk/services/TMHMM/) was used to predict transmembrane domains.
Construction of Pt2015 knockout strains using CRISPR/Cas9
Pt2015 knockout strains were constructed according to a previous method in which the Cas9 system was delivered into P. tricornutum via conjugation of plasmids from a bacterial donor cell [7, 8]. Briefly, three guide RNAs (gRNAs) targeting Pt2015 (Additional file 1: Table S1) were designed on the website (http://crispor.tefor.net/). To generate gRNA cloning inserts, the designed oligonucleotide corresponding to the predicted gRNA binding site and the complementary oligonucleotide were ordered, and then phosphorylated and annealed. Using Golden Gate assembly (New England Biolabs), cloned gRNA was inserted into the pPtGE35 plasmid obtained from Addgene (107999). The product from the Golden Gate reaction was transformed into Epi300 E. coli cells using heat shock. Then, the properly cloned gRNA plasmid was transformed into DH10B E. coli cells containing the pTA-Mob plasmid [35] obtained from Dr. Rahmi Lale (Norwegian University of Science and Technology, Trondheim, Norway).
Subsequently, according to the published protocol [8], the pPtGE35 plasmid containing gRNA inserts was transferred into P. tricornutum via conjugation from E. coli. Screening for Pt2015 knockouts in P. tricornutum induced by Cas9 was performed as previously described [8] with minor modifications. After 2–3 weeks, ten P. tricornutum exconjugants were randomly selected, inoculated into liquid F/2 medium with 50 μg mL−1 zeocin (Invitrogen), and grown for 5 d. The P. tricornutum exconjugant cells were lysed and used as the template for polymerase chain reaction (PCR) amplification of the gRNA target site using the specific primers shown in Additional file 1: Table S2. The PCR products were sent for Sanger sequencing to verify the deletion length. Subsequently, liquid cultures of P. tricornutum exconjugants that were identified to be edited were diluted to 10–4 and plated onto F/2 medium plates containing 50 μg mL−1 zeocin and grown for 10–14 d to obtain sub-clones.
Construction of vectors for Pt2015 overexpression
The coding sequence (444 bp) of the Pt2015 gene was amplified for overexpression using the primers Pt2015oe-F and Pt2015oe-R, and further digested with EcoRI and KpnI, and then inserted into pPha-T1 vector [36]. Additionally, the coding sequence (444 bp) of the Pt2015 gene for overexpression was amplified with Pt2015eGFP-F and Pt2015eGFP-R, and further digested by EcoRI and KpnI, and then inserted into pPHat_eGFP vector. The vectors containing coding sequences of the Pt2015 gene or the Pt2015-eGFP were introduced into WT P. tricornutum using biolistic transformation with the Biolistic PDS-1000/He Particle Delivery System (Bio-Rad, CA, USA) [37], respectively. For the selection of overexpression clones, bombarded cells were plated onto 50% fresh seawater agar plates (1% agar) supplemented with 100 μg mL−1 zeocin (Invitrogen). The plates were placed under conditions of 80 μmol photons m−2 s−1 for 2–3 weeks, and resistant colonies were inoculated into liquid F/2 medium containing 50 μg mL−1 zeocin. The transformants were screened using primers (YzF and YzR, Additional file 1: Table S2).
Cell preparation for electron microscopy
The procedures used for scanning electron microscopy sample preparation were as previously described, with minor modification [30]. The cells were fixed with 5% glutaraldehyde in phosphate buffered saline (PBS, pH 7.4) for 2 h and dehydrated in a series of increasing ethanol concentrations (30%, 50%, 70%, 80%, 90%, and 100%) for 10 min at 4 °C. The cells were treated with isoamyl acetate for 10 min, critical point dried (Hitachi-HCP, Hitachi, Japan), sputter coated with platinum (MC1000, Hitachi), and examined with a scanning electron microscope (S-3400 N, Hitachi).
The ultrastructure of the different samples was examined using transmission electron microscopy as previously described [30]. Cells were fixed with 2.5% glutaraldehyde (Hushi, Shanghai, China) in PBS for 2 h and washed three times for 30 min in 0.1 M PBS at 4 °C. The cells were then post-fixed with 1% osmium tetroxide (Ted Pella, CA, USA) in PBS for 1.5 h and washed three times with 0.1 M PBS at 4 °C. Next, the samples were dehydrated in alcohol (Hushi), infiltrated with acetone (Tieta, Laiyang, China) and an epoxy resin (SPI-CHEM, USA) mixture, embedded, and polymerized in epoxy resin. Ultrathin sections were obtained using a Leica EM UC7 ultramicrotome (Leica Microsystems, Germany) and transferred onto copper grids covered with a Formvar membrane (Electron Microscopy China, Beijing, China). Two-percent uranyl acetate and lead citrate (Ted Pella Inc.) were used for contrast staining. The sections were photographed using a transmission electron microscope (HT7700, Hitachi).
Fluorescent microscopy
Subcellular localization analysis of chlorophyll autofluorescence and the green fluorescent protein (GFP) fusion of Pt2015 was performed using laser-scanning confocal microscopy (LSM710, Carl Zeiss, 40 ×). Chlorophyll autofluorescence was excited at 633 nm and detected at a bandpass filter of 675–740 nm. GFP fluorescence was excited using an excitation wavelength of 488 nm and detected with a bandpass filter of 510–570 nm.
Lipid analysis
For lipid body labeling, the cells (1 × 106 cells/mL) of different strains were cultured for 7 d and incubated with BODIPY 505/515 (Thermo Fisher Scientific) at a concentration of 0.5 mg/mL for 7 min in the dark [38]. For cell imaging, BODIPY 505/515 was excited at 488 nm and fluorescence was collected from 510 to 550 nm. Gravimetric means, a conventional lipid quantification method, was used for total lipid analysis. Algal cultures were harvested after cultivation for 7 d by centrifugation at 2500 × g for 5 min. Algal cells were frozen in liquid nitrogen and then dried in a freeze dryer. Lipids were extracted from 50 mg dried algal powder using a modified chloroform–methanol system [39]. Crude samples were dried under a N2 flow until a constant weight was obtained.
Plastid purification, membrane solubilization, and immunoblotting
Plastids were purified as previously described [40]. Briefly, cells were centrifuged and re-suspended gently in isolation buffer (0.5 M sorbitol, 50 mM HEPES–KOH, 6 mM ethylenediaminetetraacetic acid (EDTA), 5 mM MgCl2, 10 mM KCl, 1 mM MnCl2, 1% (w/v) polyvinylpyrrolidone 40, 0.5% bovine serum albumin (BSA), and 0.1% cysteine; pH 7.2–7.5) and slowly passed through a French Press at 90 MPa. The mixture of broken cells was centrifuged at 300 × g for 8 min to remove intact cells and cell debris. The supernatant was collected and then centrifuged at 2000 × g for 10 min at 4 °C. The pellet containing the plastids was gently re-suspended in washing buffer (0.5 M sorbitol, 30 mM HEPES–KOH, 6 mM EDTA, 5 mM MgCl2, 10 mM KCl, 1 mM MnCl2, 1% polyvinylpyrrolidone 40, 0.1% BSA; pH 7.2–7.5) and loaded onto a discontinuous Percoll gradient (10%, 20%, and 30%) in the same buffer. After centrifugation (SW40Ti rotor, Beckman Coulter, Indianapolis, IN, USA) at 10,000 × g for 35 min, the plastid fraction was recovered in the 20% Percoll layer of the gradient, diluted in washing buffer (without BSA), and subjected to centrifugation at 14,000 × g for 10 min at 4 °C. To separate thylakoids and the stroma, plastids were subjected to osmotic shock (induced by incubation for 5 min in the washing buffer without sorbitol), and centrifugation. The supernatants contained the stroma and the pellets comprised the thylakoids, which were re-suspended in washing buffer without sorbitol.
Thylakoid membrane complexes were solubilized and separated as previously described, with minor modification [41]. The thylakoids were solubilized in a buffer (25 mM HEPES–KOH [pH 7.0], 10 mM NaCl, and 5 mM MgCl2) containing 1% n-dodecyl-β-D-maltoside (β-DM) with a chlorophyll concentration of 0.5 μg/μL for 10 min on ice. After centrifugation at 15,000 × g for 10 min at 4 °C, the supernatant (200 μg chlorophyll) was loaded onto the top of a sucrose density gradient (1.6 M, 1.3 M, 1 M, 0.7 M, 0.4 M, and 0.1 M) containing 25 mM HEPES–KOH (pH 7.0), 10 mM NaCl, 0.02% β-DM, and 5 mM MgCl2. Thylakoid protein complexes were separated by ultracentrifugation for 22 h at 220,000 × g at 4 °C in a SW40 rotor (Beckman Coulter). Ten fractions were extracted with syringes and equal amounts of each fraction were used for further tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting analysis.
Immunoblotting analysis was performed as previously described [41]. Antibodies to PsbA, PsbD, PsaA, CytF, Actin, ribulose bisphosphate carboxylase large subunit, and LhcX (LhcSR of moss) were purchased from Agrisera (Vännäs, Sweden). Peptides were designed to match unique sections of Pt2015 (CREWRCKFEGDKSDSE), chemically synthesized, and then injected into rabbits to generate polyclonal antibodies (PhytoAB, San Jose, CA, USA). Horseradish peroxidase-conjugated secondary antibody and an enhanced chemiluminescence detection kit (Tian Gen, Beijing, China) were used for detection.
Measurement of chlorophyll fluorescence and P700
In vivo chlorophyll a fluorescence and P700 oxidation signals were measured simultaneously at 25 °C using a Dual-PAM-100 (Heinz Walz, Germany) in P. tricornutum WT, 2015KO, and oeT strains. Before measurements, cells were centrifuged (1500 × g, at 20 °C, 3 min) and concentrated 40-fold, and then dark acclimated for 10 min. A saturating pulse of light (300 ms, 10,000 μmol photons m−2 s−1) was used to determine the maximal fluorescence levels in the dark-adapted state (Fm) and during actinic light illumination (Fm′). The steady-state fluorescence level (Fs) was recorded during actinic light illumination (80 μmol photons m−2 s−1). The quantum yield [Y(II)] of photosystem II (PSII) was calculated as (Fm–Fs)/Fm′. The Y(NO) and Y(NPQ) were calculated using the Dual-PAM software and saved in a report file. Photosystem I (PSI) was measured in the dual-wavelength mode (photodetector set to measure 875 nm and 830 nm pulse-modulated light). The maximum P700 signal, Pm, was determined by the application of the saturation pulse in the presence of far-red light. The P700 signal, P, was determined just before the saturation pulse. Pm′ is the maximum P700 signal induced by combined actinic illumination with the saturation flash. Y(I) was calculated as (Pm′–P)/Pm. The acceptor-side limitation of PSI (Y(NA)) was calculated as (Pm–Pm′)/Pm, while the donor-side limitation of PSI (Y(ND)) was calculated as 1–Y(I)–Y(NA)[42, 43].
Measurements of photosynthetic O2 evolution
Rates of photosynthetic O2 evolution of the WT, 2015KO, and oeT strains under different light conditions and dark respiration were measured using a Clark-type oxygen electrode (Hansatech, Norfolk, UK). Cells were concentrated via centrifugation at 1500 × g for 3 min. After centrifugation, cells were re-suspended in fresh F/2 culture medium. The OD730 value was adjusted to 1, and 2 mL was placed into the electron chamber. Before measurement under different light conditions, the samples were dark acclimated for 10 min. After the dark pre-treatment, the rate of O2 evolution in the chamber was measured over a range of light intensity (50–2800 μmol photons m−2 s−1), provided by an LED light source peaking at 630–640 nm. In addition, after the dark pre-treatment, the rate of O2 evolution of the cells was measured under a light intensity of 100 and 2000 μmol photons m−2 s−1, respectively.
Cultivation of amoebae
The amoebae used in this study belongs to the Heterolobosea class which was isolated from large-scale cultivation of P. tricornutum. The spores of amoebae were centrifugated at 3500 × g for 5 min, and then diluted with WT and oeT cells with the same cell density (5 × 106 cells/ml), respectively. The diluent was cultured in culture plates (6 wells) at 25 °C under the dark condition. After 24 h, the cells concentrations of the WT and oeT were counted.
Salinity and temperature experiments of the oeT strain
For the salinity experiments, three salinity conditions (100%, 50%, and 30% seawater) were tested using F/2 medium. Nutrient trace metals and vitamins were the same as in the F/2 medium, except the salts were diluted to 50% or 30% seawater. The oeT strain, which was cultured in artificial seawater (100% seawater) for at least 2 months, was moved to three salinity conditions (100% (control), 50%, and 30%) at a starting density of 1 × 106 cell mL−1, followed by weekly cell counting for 60 d. All three salinity treatments were performed at 20 °C. Meanwhile, two temperature conditions (20 °C and 10 °C) with the same salinity (100% seawater) were tested at a starting density of 1 × 106 cell mL−1, followed by cell counting weekly for 60 d. During this period, the F/2 medium was supplemented every 3 weeks and the percentage of abundance of different cell morphologies (triradiate and fusiform) was quantified by counting in a counting chamber.
RNA extraction, sequencing, and bioinformatics analysis
Total RNA was extracted using the TRIzol reagent kit (Invitrogen), according to the manufacturer’s protocol, and was assessed using an Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA, USA) and electrophoresis. The enriched mRNA was fragmented into short fragments and reverse transcribed into cDNA. The cDNA fragments were purified, end repaired, and a poly(A) tail was added. Then the fragments were ligated to Illumina sequencing adapters. The ligation products were size selected using electrophoresis, PCR-amplified, and sequenced using an Illumina Novaseq 6000 by Gene Denovo Biotechnology Co. Ltd. (Guangzhou, China).
To obtain high quality clean reads, reads were further filtered using FASTP (version 0.18.0) (version 0.18.0) [44]. Ribosome RNA reads were removed after alignment with Bowtie2 (version 2.2.8) [45]. The remaining clean reads were assembled and mapped to the P. tricornutum genome (National Center for Biotechnology Information—Assembly: ASM15095v2) using HISAT2 2.4 under default parameters [46]. The mapped reads of each sample were assembled using StringTie v1.3.1 [47]. For each transcription region, a fragment per kilobase of transcript per million mapped reads value was calculated to quantify its expression abundance and variation. Principal component analysis was performed using R package gmodels (http://www.r-project.org/), which is a statistical procedure that converts hundreds of thousands of correlated variables (gene expression) into a set of values of linearly uncorrelated variables called principal components. Differential expression analysis of RNA was performed using DESeq2 software between two different groups (false discovery rate (FDR) < 0.05, absolute fold change ≥ 2) [48]. Gene function classification was annotated using Gene Ontology (GO) and KEGG enrichment (FDR ≤ 0.05) [49].
Tandem Mass Tag (TMT)-based quantitative proteomic analysis
Proteomic analysis of the samples (WT, 2015KO, and oeT strains) was performed by PTM-Bio labs Co. Ltd. (Hangzhou, Zhejiang, China). The samples were ground in liquid nitrogen, and the powder was transferred to a 5 mL centrifuge tube and then sonicated in a lysis buffer (8 M urea, 1% Triton X-100, 10 mM dithiothreitol, 1% protease inhibitor cocktail, 3 μM Trichostatin A, 50 mM N-(9-acridinyl)maleimide, and 2 mM EDTA). The precipitate was then reconstituted with 8 M urea, and the protein concentration was measured using a bicinchoninic acid kit. For trypsin digestion, the protein solution was reduced with 5 mM dithiothreitol at 56 °C for 30 min and alkylated with 11 mM iodoacetamide at room temperature for 15 min in the dark.
After trypsin digestion, the peptides were desalted using a Strata X C18 PSE column (Phenomenex, USA) and freeze dried under vacuum. For TMT labeling, the samples were reconstituted in 0.5 M triethylammonium bicarbonate and processed according to the TMT kit instructions (Thermo-Scientific). The detailed procedures were performed in accordance with previous methods [50, 51].
For high-performance liquid chromatography fractionation, the peptides were fractionated using high-pH reverse-phase high-performance liquid chromatography with an Agilent 300Extend C18 column (Agilent, Santa Clara, CA, USA) (5 mm particles, 4.6 mm internal diameter). The peptides were first separated into 80 fractions over 80 min using a gradient of 2%–60% acetonitrile (pH 9), after which the peptides were combined into 18 fractions and dried by vacuum centrifugation. The peptides were then subjected to a nanospray ionization source followed by tandem mass spectrometry with Q Exactive™ Plus (Thermo-Scientific) coupled online to ultra-performance liquid chromatography.
The tandem mass spectrometry data were processed using the Maxquant search engine (v.1.5.2.8). Tandem mass spectra were searched against the P. tricornutum UniProt database combined with a reverse decoy database. Trypsin/P was specified as a cleavage enzyme, allowing up to two missing cleavages. The mass tolerance for precursor ions was set at 20 ppm in the first search and 5 ppm in the main search. The mass error was set to 0.02 Da for the precursor ions and fragment ions. Carbamidomethylation on cysteine was specified as a fixed modification and oxidation on methionine was specified as a variable modification. FDR thresholds for protein, peptide, and modification site identification were specified at 1%. TMT-6-plex was selected as the quantification method. All other parameters in MaxQuant were set to default values.
The functional annotation of the differentially expressed proteins (DEPs) was annotated to the GO and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases. The DEP IDs were converted to UniProt IDs, and then the UniProt IDs were used to match the GO ID, and the corresponding information was retrieved from the UniProt-GOA database (http://www.ebi.ac.uk/GOA/) according to the GO ID. Un-annotated proteins were annotated by InterProScan using the protein sequence alignment method. All annotated proteins were then classified into three categories: biological process (BP), cellular component (CC), and molecular function (MF). The KEGG database (http://www.genome.jp/kegg/) was used to annotate the protein pathway.
Statistical analysis
Data are expressed as the mean value of four independent experiments (± standard deviation). Data were analyzed using analysis of variance with the SPSS 13.0 statistical software (IBM, Armonk, NY, USA). An independent-samples t-test was used at the α = 0.05 significance level to determine whether significant differences existed between the WT, 2015KO, and oeT strains.