Culture conditions of N. oceanica
Nannochloropsis oceanica IMET1 was inoculated into the modified f/2 liquid medium, which was prepared with 35 g L−1 sea salt (Real Ocean, USA), 1 g L−1 NaNO3, 67 mg L−1 NaH2PO4*H2O, 3.65 mg L−1 FeCl3*6H2O, 4.37 mg L−1 Na2EDTA*2H2O, trace metal mix (0.0196 mg L−1 CuSO4*5H2O, 0.0126 mg L−1 NaMoO4*2H2O, 0.044 mg L−1 ZnSO4*7H2O, 0.01 mg L−1 CoCl2 and 0.36 mg L−1 MnCl2*4H2O) and vitamin mix (2.5 µg L−1 VB12, 2.5 µg L−1 biotin and 0.5 µg L−1 thiamine HCl) . The cells were first cultured in f/2 medium at 25 °C with 80 ± 5 μmol m−2 s−1 continuous irradiation in a 1-L column reactor (inner diameter 5 cm). The seed cultures were bubbled with 5% CO2. At the logarithmic phase (OD750 = 3.0), cells were harvested by centrifugation and then washed with fresh medium, before being used for the following experiments.
In total, six identical column reactors were employed for the wild-type N. oceanica culture. Each reactor contains 800 mL of fresh modified f/2 liquid medium, which was supplemented with 10 mM Tris–HCl buffer (pH = 8.2) in order to accurately control the pH during the culture. Equal numbers of the seed cells from six independent reactors were re-inoculated into each of the six new column reactors with fresh medium to an OD750 of 1.5, respectively. The light intensity was maintained at 80 ± 5 μmol m−2 s−1. The six algal cultures were first aerated with air enriched with 5% CO2 (“high-CO2” conditions, or HC) for 1 h. After the preadaption phase, three of the algal cultures proceeded under HC as the control condition, whereas the other three were switched to aeration with 0.01% CO2 (“very low-CO2” conditions, or VLC; the customized CO2 gas was provided by Dehai Gas Company, China) for CCM induction (Additional file 1: Figure S1; [17, 48]). After switching to the designated culture condition (e.g., VLC), cell aliquots were taken at 0, 3, 6, 12 and 24 h from each column by syringe for physiological measurement (including OD, inorganic carbon concentration, chlorophyll content, photosynthetic rate, etc.), transcriptomic profiling, proteomic profiling and metabolite analysis. Three biological replicates of algal cultures, corresponding to the collectively six column reactors, were established under each of the above VLC and HC conditions, respectively.
Tracking the photosynthetic activity of N. oceanica
Chlorophyll fluorescence parameters sensitively reflect the instantaneous photosynthetic state of microalgae and their acclimation to current environmental conditions . Fv/Fm (the variable/maximum fluorescence ratio), the maximum photochemical quantum yield of PSII reaction centers, represents the minimum fluorescence yield when PSII reaction centers are fully open and reflects the photosynthetic light energy conversion efficiency. On the other hand, Fv′/Fm′ represents the active PSII activity; therefore, Fv/Fm and Fv′/Fm′ are both measured here to depict photosynthetic performance and acclimation status . Fm is the maximum fluorescence yield when PSII reaction centers are completely closed; thus, it reflects the PSII electron transport capacity. Fv is the variable fluorescence (Fv = Fm − Fo), reflecting reduction in the PSII primary electron acceptor QA, thus indicating the photochemical activity of PSII reaction centers. Fo is the minimum fluorescence yield. (Damage to or irreversible loss of activity of PSII reaction centers will cause a decrease in the Fo value.) Fv/Fm and Fv′/Fm′ were calculated according to these two formulas Fv/Fm = (Fm − Fo)/Fm and Fv′/Fm′ = Fm′ − Ft/Fm′ . To measure these parameters, N. oceanica cultures were kept in the dark for 20 min and then exposed to a saturating light pulse (1000 mol m−2 s−1) for l s, while the chlorophyll fluorescence intensities were measured with a pulse amplitude-modulated (PAM) kinetics using IMAGING-PAM M-Series (Walz, Germany) following the manufacturer’s recommendations.
Measurement of total inorganic carbon content in the medium
Total dissolved inorganic carbon content (TIC) in the medium was measured using a high-temperature TOC/TNb analyzer (LiquiTOC II, Elementar, Germany) coupled with automatic sampling instrument . To prepare for the measurement, 3 mL algal medium was diluted 10 times with the distilled Milli-Q water and transferred into a 30 mL brown glass reagent bottle. Another 3 mL medium filtered by pre-combusted GF/F filter (0.7 µm pore size, 25 mm) was diluted 10 times with distilled water and then transferred to a 30 mL brown glass reagent bottle. Both samples were acidified with 100 µL nitric acid and then stored at 20 °C for the measurement.
Measurement of lipid, carbohydrate and protein
To quantify the amount of carbohydrate, protein and lipid, N. oceanica cells were harvested after 24 h cultivation by centrifugation (at 4000 rpm for 5 min) under VLC and HC. For dry algal powder, cells were lyophilized for 2 days. Extraction and assaying of lipid, carbohydrate and protein in the microalgal biomass were performed based on our published protocols .
Metabolite analysis by GC–mass spectrometry
A 10 mg (DW) sample of the microalgal biomass, which had been frozen in liquid nitrogen and stored at − 80 °C, was extracted for metabolite analysis according to Lisec et al. with slight modifications . Lyophilized algal culture was carefully weighted to about 5.00 mg and transferred to a microcentrifuge tube. 500 μL of 100% (v/v) methanol supplemented with 2 μg of ribitol for sample normalization was added to the algae powder. Metabolite extraction was performed by 15 min of shaking in a thermomixer (1200 rpm) at 70 °C. Cell debris was centrifuged at 16,000×g for 5 min, and 100 μL of the supernatant solution was dried in a vacuum evaporator for 3 h. Dried samples were derivatized by the addition of 20 μL of a 20 mg/mL solution of methoxylamine hydrochloride (Sigma-Aldrich, USA) in pyridine (30 °C for 90 min). 30 μL of N-methyl-N-(trimethylsilyl) trifluoroacetamide (MSTFA) was then added and shaken for a further 30 min at 37 °C. Totally 10 μL of an alkane standard mix containing 50 ng each of C12, C15, C19, C22, C28, C32 and C36 in chloroform was added for retention index determination with high accuracy [58, 59]. Samples were randomized, and 1 μL of derivatized sample was injected splitless into an Agilent 6890 GC fitted with an Agilent 5975 MSD. Helium was used as the carrier gas at a constant flow of 1 ml/min. Inlet temperature was set at 300 °C. Oven temperature was initially set at 70 °C for 1 min, ramped at 1 °C/min until 76 °C and then ramped at 6 °C/min until 325 °C, with a final hold of 10 min. A Varian Factor 4 capillary column (VF-5 ms, 30 m × 0.25 mm, 0.25 μm plus 10 m EZ-Guard) was used. The MSD transfer line heater was kept at 300 °C. MS quadrupole temperature was kept at 150 °C and source temperature at 230 °C. Mass detection range was set from 40 to 600 atomic mass units. Spectral data files were processed with AMDIS (version 2.65) for metabolite identification. Metabolites were identified by retention index and spectral comparison with pre-run standards or by searching the NIST library. All identified metabolites were entered into MSD ChemStation (version E.02.00.493), and a quantitation database was created using specific target ions and qualifier ions unique to each metabolite. All spectra were manually reviewed. Normalization was performed to the internal standard ribitol and to the tissue weight. Student’s t test was used to compare the two datasets (VLC and HC, n = 6) at the same time point. If the test gave a P value ≤ 0.05, the difference between VLC and HC was interpreted as being significant.
Transcriptome sampling, sequencing and analysis
For transcriptomic analyses, the cells were harvested by centrifugation for 5 min at 2500g and then were immediately quenched with liquid N2 and stored in − 80 °C freezer. Total algal RNA was extracted using Trizol reagents (Invitrogen, USA). The concentration and purity of the RNA were determined spectrophotometrically (NanoDrop-1000, Thermo Scientific, USA).
For mRNA-Seq, the poly (A)-containing mRNA molecules were purified using Sera-Mag Magnetic Oligo (dT) Beads (Thermo Scientific, USA) and were fragmented into 200- to 300-bp fragments by incubation in RNA Fragmentation Reagent (Ambion, USA) according to the manufacturer’s instructions. The fragmented mRNA was then purified from the fragmentation buffer using Agencourt® RNA Clean beads (Beckman Coulter, USA). The purified, fragmented mRNA was converted into double-stranded cDNA using the SuperScript Double-Stranded cDNA Synthesis Kit (Invitrogen, USA) by priming with random hexamers. Strand nonspecific transcriptome libraries were prepared using the NEBNext® mRNA Library Prep Reagent Set (New England Biolabs, USA) and sequenced for 2 × 90 bp runs (paired-end, PE) using Illumina HiSeq 2000.
To ensure quality, the raw data (2 × 90 bp PE reads) were modified as follows: First, adapter pollutions in reads were deleted, and then, because the sequence qualities of Illumina reads degrade quickly toward the 3′ end, all reads were trimmed from the 3′ end until the 3′-end–most position with Phred equivalent score was 20 or greater. The raw data were deposited in NCBI GEO with the reference series number GSE55861. These filtered Illumina reads were aligned to our previously published N. oceanica IMET1 genome  with TopHat (version 2.0.4, allowing no more than two segment mismatches) . Reads mapped to more than one location were excluded. Thirdly, the short read mapping results from TopHat were used for the differential gene expression analysis with Cufflinks (version 2.0.4), as was described .
For each of the mRNA-Seq datasets under each experimental condition, gene expression was measured as the numbers of aligned reads to annotated genes by Cufflinks (version 2.0.4) and normalized to FPKM values (fragments per kilobase of exon model per million mapped fragments). Genes were considered to be significantly differentially expressed if either of the conditions was met: (i) Their expression values showed at least twofold change with a false discovery rate (FDR)-corrected p value ≤ 0.05 (provided by Cuffdiff from the Cufflinks package) between control and stressed conditions, and moreover, their FPKM values at either condition were ≥ 10. (ii) Their expression values showed 1.5- to less than twofold change with a FDR-adjusted p-value ≤ 0.05 between control and stressed conditions for at least two time points, and moreover, their FPKM values at either of the conditions were ≥ 10.
The 2933 differentially expressed genes were grouped into 16 clusters based on their temporal expression patterns by the k-means clustering using the Multiple Experiment Viewer 4.8 (MeV4.8; http://www.tm4.org/mev/) with the Euclidean distance . The optimal number of clusters was identified and investigated by performing a figure of merit (FOM) analysis within MeV4.8 . FOM analysis showed that the value was stabilized after a partitioning into 12–18 clusters using k-means algorithm. Therefore, the transcripts were split into 16 clusters, each of which exhibits a particular pattern of temporal dynamics.
Validation of transcript abundance using Real-time qPCR
To further test the validity of the mRNA-Seq results, RNA extracted from the same cultures for mRNA-Seq was subjected to the PrimeScript® RT reagent Kit with gDNA Eraser (Takara, Japan) for cDNA synthesis. Also, qRT-PCR was performed by standard methods (Roche, Switzerland) as previously described . Ct values were determined for triplicate independent technical experiments performed on triplicate biological cultures (n = 3). Relative fold differences were calculated based on the ΔCt method using the actin amplification product as an internal standard. Primer pairs used for qRT-PCR analyses are listed in Additional file 5: Table S3. Sizes of amplification products were 100 to 300 bp. The correlation coefficient between the qPCR results and the mRNA-Seq results for the 12 genes tested was 0.94 (R2; Additional file 6: Figure S3).