reverse-transcribed using random hexamer primers and an Omniscript reverse transcription kit (QIAgen). Target genes (and β-actin as a housekeeping marker) were then subjected to quantitative PCR amplification in 96-well optical reaction plates in 20 μL mixtures per well, using a SYBR Green Kit (Takara Bio) following the manufacturer’s instructions and a 7300 Sequence Detection System (Applied Biosystems/Life Technologies). The threshold cycle (Ct) for each well was measured, and the mRNA levels of the target genes in N-deprived cells, relative to those of controls, were quantified after normalization to β-actin.
RNA-Seq data processing and metabolic pathway analysis
The draft P. tricornutum reference genome was obtained from http://genome.jgi-psf.org/Phatr2/Phatr2.download.ftp.html webcite. The sequence is composed of “finished chromosomes” (Phatr2) and “unmapped sequences” (Phatr2_bd), which were annotated separately, and both were included in this study for comprehensive analysis. We aligned RNA short reads (11751810 in total) with the reference genome using SOAP 2.21 and default parameters. All original and mapped data are available at http://guanlab.ccmb.med.umich.edu/data/Yang_N_deprivation/ webcite. This resulted in 10786429 mapped reads (91.79%). Any gene with one or no reads was discarded as non-confident, and with this constraint we aligned 12312844 reads obtained from samples of N-deprived cultures, with 11326275 (91.99%) mapped to the genome. RPKM (Reads Per Kilobase per Million mapped reads) values were estimated and log2-transformed  for each gene defined in the Phatr2 database. This allowed us to estimate the fold-change in their transcript levels under nitrogen deprivation. We downloaded the KEGG pathway database (a unique set of genes that can be confidently associated with each protein in the KEGG pathway repository) released on Jun 30, 2011. We compiled all KEGG-annotated genes for P. tricornutum, and all those of other species meeting an e-value cutoff constraint of 1e-5 identified by BLAST analysis, 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 