lysis, retaining the best hits. We then hand-curated these two sets to remove inconsistencies and incorrect annotations, which resulted in 3648 genes with a KEGG id in total, and transformed the expression fold-change to a color gradient, where red represents up-regulation and green down-regulation. The subcellular localization of the enzymes were based on the prediction in the GenBank database, the prediction softwares online including WoLF PSORT (http://wolfpsort.org/ webcite) and SignalP (http://www.cbs.dtu.dk/services/SignalP/ webcite), besides relevant references.
Measurement of photosynthesis activity
Chlorophyll fluorescence parameters sensitively reflect the instantaneous photosynthetic state of diatoms and their acclimation to current environmental conditions. Fv/Fm (the variable/maximum fluorescence ratio) indicates the maximum photochemical quantum yield of PSII reaction centers, reflecting the photosynthetic light energy conversion efficiency. Thus, it is a widely used index of photosynthetic performance and acclimation status . Fo is the minimum fluorescence yield when PSII reaction centers are fully open. Damage to or irreversible loss of activity of PSII reaction centers will cause a decrease in the Fo value. 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 of the PSII primary electron acceptor QA, thus indicating the photochemical activity of PSII reaction centers. To measure these parameters, P. tricornutum cultures were kept in the dark for 20 min, then exposed to a saturating light pulse (3000 mol · m-2 · s-1) for l sec while the chlorophyll fluorescence intensities were measured with a Handy-PEA chlorophyll fluorimeter (Hansatech Instruments Ltd) following the manufacturer’s recommendations.
Ultrastructural analysis by transmission electron microscopy
Samples were fixed in 2% v/v glutaraldehyde, 2% w/v paraformaldehyde in 100 mM sodium cacodylate buffer-NaOH, pH 7.4, for 2.5 h at 4°C and rinsed three times (20 min per rinse) with 130 mM sucrose, 10 mM 2-mercaptoethanol in 100 mM sodium cacodylate buffer-NaOH, pH 7.4. They were then post-fixed with 1% (w/v) osmium tetroxide in 100 mM sodium cacodylate buffer-NaOH, pH 7.4, rinsed three times (5 min per rinse) with ultrapure (Milli-Q) water and dehydrated through a graded series of acetone (20%, 50%, 70%, 90%, 100% v/v). After infiltration through a graded acetone/Epon/Spurr’s epoxy resin series, samples were embedded in 100% w/v Spurr’s epoxy resin and polymerized at 60°C for 24 h. Ultrathin sections were prepared using a Diatome diamond knife on an 8800 Ultratome III (LKB Instruments) and stained with uranyl acetate and lead citrate. The stained sections (about 20 per treatment) were examined under a JEM-1200EX transmission electron microscope (JEOL), and images were recorded on 4489 film (Eastman-Kodak).
RNA-seq: RNA-sequencing; MGDG: Monogalactosyldiacylglycerol; TCA: Tricarboxylic cycle; TAG: Triacylglycerol; PUFA: Polyunsaturated fatty acid; SFA: Saturated fatty acid; KEGG: Kyoto encyclopedia of genes and genomes; LHC: Light harvesting complex; PSII: Photosystem II; ROS: Reactive oxygen species; PEPC: Phosphoenolpyruvate carboxylase; OAA: Oxaloacetate; CCM: Carbon concentrating mechanism; GS: Glutamine synthetase; PEP: Phosphoenolpyruvate; ME: Malic enzyme; ACCase: Adetyl-CoA carboxylase; FAS: Fatty acid synthase; FTIR: Fourier transform infrared spectrometer; FBP: Fructose-1,6-bisphosphatase; DGAT: Diacylglycerol acyltransferase; PDAT: Phospholipid:diacylglycerol acyltransferase; PC: Phosphatidylcholine; RPKM: Reads per kilobase per million mapped reads; FA: Fatty acid; DW: Dry weight.
The authors declare that there are no competing interests.
HL and YG designed the research; ZY, YN, YM, JX, MZ, WY, JL, YG and HL performed research; YG, ZY and HL contributed analytic and computational tools; ZY, YN, WY, JL, SL, YG and HL analyzed data; ZY, YG and HL wrote the paper. All authors read and app