Cell culture and treatment conditions
P. tricornutum was screened, identified [39], and grown in sterile artificial seawater (ASW) containing f/2 medium [40] at 20oC ± 1oC. The cultures were illuminated by fluorescent white lamps at an intensity of 80 μmol/m2/s under a light/dark cycle of 12/12 h.
For different cell density treatments, the algal cells were cultured under white light-emitting diode light (80 μmol/m2/s) till the mid-exponential growth phase and harvested by centrifugation at 2000×g for 5 min. Then, the cells were inoculated into fresh medium and the cell densities were adjusted to low (1.6×106 cells/mL), medium (3.2×106 cells/mL), and high (6.4×106 cells/mL), cell density and cultured under light (80 μmol/m2/s), or dark conditions for 24 h. Subsequently, the cells were harvested by centrifugation at 6000×g for 1 min and quickly frozen with liquid nitrogen.
For different light treatments, the algal cells at various cell densities were cultured under blue light (BL, 460 nm, 80 μmol/m2/s) and white light (WL, 80 μmol/m2/s) for 24 h. Then, the cells were harvested by centrifugation at 6000×g for 1 min and quickly frozen with liquid nitrogen. Multi-cultivator MC 1000 (Photon Systems Instruments, Czech Republic) was used for eight different light quality treatments (at wavelengths of WL, 405 nm, 450 nm, 470 nm, 540 nm, 590 nm, 660 nm, and 730 nm, respectively, with the illumination of 80 μmol/m2/s).
For cell culture medium extraction treatment, the cells were harvested in a mid-exponential growth phase, and their density was adjusted to low (1.6×106 cells/mL), medium (3.2×106 cells/mL), and high (6.4×106 cells/mL) and cultured under light (80 μmol/m2/s) for 24 h. After that, the algae cells were collected by centrifugation, and the upper medium was transferred to a clean sterile Erlenmeyer flask for later use. Then, the cell pellet was harvested and ultrasonicated on ice for 3 min, and the broken cells with low, medium, and high-cell densities were centrifuged and then collected. Clear, remix with the low, medium, and high upper layer medium obtained in the previous step. Use the resulting mixed solution to culture new algae cells in the dark for 24 h.
For “Sandwich” device treatment, bottle A and bottle C were black-coated on the outside and a red filter was plated between bottle B and A&C. P. tricornutum cells in bottle A and bottle C were excited by applying direct light in the x-axis to emit chlorophyll fluorescence in the y-axis. By altering the cell density in bottles A and C, the intensity of the excited fluorescence could be changed.
To obtain iron-deplete medium, PVC bottles were cleaned with HNO3 overnight. Then, ASW was prepared by using ddH2O, microwave-sterilized, and passed through a column containing Chelex 100 beads (Bio-Rad Laboratories) to remove the iron contaminant. To obtain an iron-replete medium, 11.7 μM FeCl3 was added to ASW. Cell growth was monitored at OD730 nm using a UV-VIS spectrophotometer (UV-1800; Shimadzu, Kyoto, Japan).
Plasmid construction, nuclear transformation, and selection
The total RNA from P. tricornutum cells was isolated using RNA prep Pure Plant kit (polysaccharide and polyphenolic-rich) (Tiangen, Beijing, China) according to the manufacturer’s instructions. Subsequently, cDNA synthesis was performed using reverse transcription system (Takara, Beijing, China) according to the manufacturer’s instructions. The vectors for ISIP2a knockdown (Gene ID: 7200478) were generated by cloning the ISIP2a gene using primers (S-ISIP2a-F, S-ISIP2a-R; Additional file 1 Table S1) containing EcoRI and HindIII sites. The fragments were inserted into the multiple cloning sites of the pPha-T1 vector by EcoRI and HindIII restriction enzyme digestion for 15 min. The transgenic algal strain was constructed by biolistic transformation, and subsequent screening was performed as described earlier [41, 42]. The algal cells were transferred to a plate containing 100 μg/mL zeocin after they were allowed to recover for 24 h under low light. After 4 weeks, individual algal colonies were picked up and lysed for PCR analysis using T1yz-F and T1yz-R primers (Additional file 1: Table S1) to verify target gene integration. The positive colonies were cultured in a liquid medium containing 80 μg/mL zeocin and further verified using quantitative real-time PCR (qRT-PCR). To measure the growth curve of ISIP2a-Si strains, the cells were cultivated in iron-replete and iron-deplete ASW media, respectively. The iron-deplete ASW cultures were obtained using the trace metal clean method according to Kazamia et al. [12].
qRT-PCR
The expression levels of ISIP2a and ISIP1 genes were determined by qRT-PCR. The cDNA was obtained as described earlier and quantified by qRT-PCR using FastStart Essential DNA Green Master kit (Roche Diagnostics GmbH, Mannheim, Germany) and IQ5 multicolor real-time PCR detection system (Bio-Rad, Hercules, CA, USA) with Bio-Rad optical system software. The internal control was the 30S ribosomal protein subunit gene, and the primers used for qRT-PCR are shown in Additional file 1: Table S1.
Chlorophyll fluorescence parameters
The photosynthetic activities of the algal cells were measured during the exponential growth phase using the Dual-PAM-100 fluorometer (Walz, Effeltrich, Germany). The algal cells were dark-adapted for 10 min, and the intrinsic fluorescence (F0) was measured. Subsequently, maximum fluorescence yield (Fm) was detected when a saturation pulse was applied to calculate the Fv/Fm. Then, the light curve of Fluo and P700 was measured under stepwise illumination with increasing light intensities. The Fluo and P700 parameters were ascertained by applying a saturation pulse at the end of each light step, and the maximum fluorescence yield under illumination (Fm′) was observed. The effective PSII quantum yield [Y(II)] was calculated using the formula: Y(II) = (Fm–F)/Fm, where F is the real-time fluorescence averaged for 0.2 s. Non-photochemical quenching (NPQ) was calculated as (Fm–Fm′)/Fm′. The rETR(II) is a relative measure of the rate of electron transport (rate of charge separation at the PSII reaction centers), similar to rETR(I). For chlorophyll fluorescence measurement, the algal cells were illuminated with WL or BL, and plant lighting analyzer (PLA-20, EVERFINE Corporation, China) was used to measure fluorescence in the vertical direction.
Pigment extraction and analysis
The P. tricornutum cultures were centrifuged at 6000×g for 1 min, and the algal cells obtained as pellet were desiccated using a vacuum freeze dryer. Then, the cells were ground (JX-FSTPRP-24 grinder, Shanghai, China), and the pigments were extracted using a solvent containing acetone/methanol (1:1). After 12 h of extraction at −20°C, the extracts were centrifuged at 10,000×g for 10 min at 4°C, and the supernatants were collected and filtered through a 0.22-μm nylon filter. Then, the supernatants were analyzed by high-performance liquid chromatography (HPLC) using a ZORBAX reverse-phase C18 column (Agilent1200, USA) at a constant temperature of 50°C and total flow rate of 0.8 mL/min. The assay was set as a linear gradient from mobile phase I (H2O: MeOH: ACN, 15:30:50) to phase II (MeOH: ACN, 15:85) during the first 15 min, phase II to phase III (H2O: MeOH: ACN: EtOAc,15:15:35:35) between 15 and 17 min, and phase III to phase IV (MeOH: EtOAc, 30:70) over the final 13 min. The compounds were examined based on absorbance at 443 nm. The pigment standards for chlorophyll a (Chl a), diadinoxanthin (Ddx), and diatoxanthin (Dtx) were obtained from Sigma-Aldrich (St. Louis, MO, USA), and de-epoxidation state (DEPS) was calculated as follows: DEPS = Dtx/(Ddx+Dtx).
Intracellular iron quotas
To determine the intracellular iron concentrations, the P. tricornutum cells were washed thrice with a 10-mM EDTA, centrifuged, and freeze-dried using a vacuum freeze dryer. Subsequently, the cells were subjected to acid digestion to destroy the organic matter content. In brief, the cells were mixed with 5 mL of concentrated HNO3 and digested for 2 h at 150°C until the solution turned clear. Then, the solution was made up to 10 mL with ultrapure water. The iron content was determined in 2% HNO3 over a concentration range of 0.25–0.75 mg/L using calibration curves produced by an inductively coupled plasma optical emission spectrometer (ICP-OES, Optima 8000, PerkinElmer, USA).
Mathematical model for chlorophyll fluorescence calculation
In order to calculate the chlorophyll fluorescence photon flux (CFPF) received at the single site of each cell, we build a mathematical model based on the radius of plastid r and attenuation coefficient of light underwater Kd. Under laboratory conditions with incident blue light at 80 μmol/m2/s, we detected that the chlorophyll fluorescence photon flux density (CFPFD) in 1 mL cells was 2.418 × 10−2 μmol potons · m−2 · s−1 under the cell density N = 1.6 × 106 cells · mL−1. So, we can convert the CFPFD to the CFPF emitted by per cell u = 9.07 × 10−12μmol potons · s−1. And then, we can calculate the CFPFD received per cell using the formula:
$$ \mathrm{CFPFD}=\mu \varOmega /\left(\frac{S_{plastid}}{2}\right)=u\frac{\pi {r}^2}{4\pi {R}^2}/\left(\frac{S_{plastid}}{2}\right) $$
Furthermore, considering the huge biomass and large-scale during diatom blooms, we build another mathematical model to calculate the CFPFD under stages with different cell densities (S1, S2 and S3) during blooms in actual oceans (Fig. b). In case of evenly distributed cells in real oceans, the groups of cells at the same distant R contribute equally to the cell at the center (Fig. a). If we further treat these cells as point light source, the CFPF emitted per unit cell u decays with spatial angle Ω and distance, and then, in an infinite space, the integral can be written as follows:
$$ \mathrm{CFPF}={\int}_0^{\infty}\left(4\pi {R}^2\cdotp dR\right)\cdotp N\cdotp u\cdotp \frac{\pi {r}^2}{4\pi {R}^2}\cdotp {e}^{-{K}_d\cdotp R}=\frac{Nu\pi {r}^2}{K_d} $$
where the radius of plastid r = 3 μm and attenuation coefficient of light underwater Kd = 0.57/m [43].
In situ data analysis derived from Tara oceans
Correlations between normalized metatranscriptome (MetaT) expression of ISIPs and chlorophyll content among various Tara stations were conducted by GraphPad Prism 8.0.2. The ISIPs in each Tara station were expressed as a percentage of the total value of ISIP1, ISIP2a, ISIP2b, and ISIP3 and normalized by the total diatom unigene expression, respectively, which were derived from the research by Caputi et al. [9], and also accessible at ENA under the accession number PRJEB6609. The observed dissolved iron concentrations and chlorophyll content in each Tara station are available at PANGAEA [44].
Cis-acting element prediction
To predict the cis-acting elements in P. tricornutum, the 2000-bp upstream region of iron uptake-related genes in the P. tricornutum genome was extracted. Then, the upstream region sequences were submitted to the PlantCare (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/) to predict cis-acting elements. The cis-acting elements related to light response were visualized using TBtools [45].
Statistical analysis
All the results obtained in this study are expressed as mean values (n = 3). The data were first analyzed by one-way ANOVA and then subjected to post hoc analysis using Tukey’s test at an α = 0.05 significance level. All analyses were performed using SPSS 18.0 (SPSS Inc., Chicago, Il, USA). Details of statistical tests performed are in Additional File 2.