PAGE. Entire lanes were excised from the gel and cut into 40 segments. Gel segments were reduced, alkylated, and tryptically digested robotically, using a ProGest protein digestion station (DigiLab, Inc.) to provide peptide-containing liquid fractions suitable for LC/MS/MS analysis on a Waters NanoAcquity HPLC system interfaced to a ThermoFisher LTQ Orbitrap Velos mass spectrometer. Peptides were loaded on a trapping column and eluted over a 75-mm analytical column at 350 nL/min; both columns were packed with Jupiter Proteo resin (Phenomenex). The mass spectrometer was operated in data-dependent mode, with MS performed in the Orbitrap at 60,000 FWHM resolution and MS/MS performed in the LTQ. The fifteen most abundant ions were selected for MS/MS. For all proteomic analyses, three biological replicates were examined. In-house Awk and Python scripts were used to convert the annotated transcriptome into a format suitable for input to the proteomic Mascot program [37]. Mascot was used to perform in silico six-frame translations of the annotated transcriptome, and the product ion data were searched against the resultant database. Product ion data were also searched against concatenated forward and reverse Chlorophyta databases (using all available sequenced microalgae). Databases were appended with commonly observed background proteins (cRAP) to prevent false assignment of peptides derived from those proteins. Mascot DAT output files were parsed into the Scaffold program (Proteome Software) for validation and filtering to assess false discovery rates (FDR), which allowed only statistically significant protein identifications. Scaffold parameters were set to a minimum of 2 peptides per protein with minimum probabilities of 95% at the protein level and 50% (Prophet scores) at the corresponding peptide level in order to ensure ,1% FDR. ANOVA statistical analysis and principal component analysis was applied using ArrayTrack [38] in order to identify differential significance between nutrient-replete and depleted samples, as well as between biological replicates. Only those positive protein identifications for which p-values less than or equal to 0.05 were obtained were considered statistically significant for the data presented. Data normalization was applied based upon the total number of spectral counts under nitrogen- deplete conditions as described by Zybailov et al. [39]. Results Growth and lipid accumulation of C. vulgaris under nitrogen stress C. vulgaris UTEX395 was cultured photoautotrophically in small-scale, Roux-bottle photobioreactors under nitrogen-replete and nitrogen-deplete conditions. Cells were harvested from cultures in nitrogen-replete media, followed by buffer exchange to nitrogen-free media, and final harvesting after growth in nitrogen-deplete media in order to obtain mRNA and protein fractions for transcriptomic and proteomic analysis (Figure 1a, circled data points). These harvest points were selected to maximize the differential in lipid accumulation based upon culture sampling throughout the growth curve. Growth under nitrogen- replete conditions led to both faster growth rates as well as higher cell densities compared to nitrogen-depleted cultures, indicative of nitrogen-limitation in the latter. Nitrogen-deplete cultures contin- ued to grow at rates similar to nitrogen-replete cultures for approximately 24 hours before their growth rates declined. It is an interesting observation that cells continue to grow post-nitrogen deprivation. This is likely due to the utilization of internal nitrogen stores, and potentially through the mobilization of nitrogen contained in cell wall chitin (discussed further below; Gerken and Knoshaug, unpublished results). With the decrease in growth Systems Biology Analysis of Chlorella vulgaris rate, the appearance of the culture changed, turning from dark green to yellow (Figure 1a, inset). We selected harvest points for transcriptomic and proteomic analysis based upon total fatty acid content, as opposed to the expression levels of specific transcripts or proteins, in order to maximize the differential in protein expression specifically with respect to oil accumulation. Lipid accumulation was quantitatively examined via transesterification of glycerolipids into FAME. We chose this method of lipid analysis because in our view, all fatty acids are potential feedstocks for biofuel production and because the conversion of fatty acids and recovery and quantitation of FAMEs provide a much more accurate measure of lipid content than standard gravimetric analyses such as Bligh-Dyer [40]. Under nutrient replete conditions, C. vulgaris accumulated 10%62% fatty acids on a dry cell weight basis (dcw) (Figure 1b). When incubated for ,5 days in the presence of nitrogen free media, C. vulgaris accumulated 60%66% fatty acids (dcw) (Figure 1b). A major shift in the profile of fatty acids occurs under nitrogen deprivation when compared to nutrient replete conditions. Most notably there is a 9- fold increase in C18:1n9 and a 4-fold decrease of C18:3 (Figure 1c). This result is similar to that observed by Stephenson et al. [41] where C. vulgaris cultures grown during nitrogen deprivation accumulated a significant amount of C18:1, while the amount of more highly unsaturated fatty acids (C18:2, C18:3, C16:2) decreased. Conversely, the fatty acid profile observed for C. vulgaris under nitrogen deprivation dramatically differs from that observed for C. reinhardtii under nitrogen stress where palmitic and linoleic acid (C16:0 and C18:2n9,12), as well as oleic acid (C18:1), significantly increase [42]. We also examined qualitatively the accumulation of neutral lipids under nitrogen replete and nitrogen-deplete conditions by fluorescence microscopy, using the neutral lipid dye BODIPY 493/503 (Figure 1d). Under nutrient replete conditions, represen- tative C. vulgaris cells were found to accumulate a few small, discrete green fluorescent lipid droplets. However, under nitrogen deprivation, most C. vulgaris cells accumulated large lipid droplets that appear to encompass the bulk of the intercellular space. No notable increase in cell size was observed. This is in contrast to lipid accumulation observed in C. reinhardtii, in which there is an increase in the number rather than an increase in size of small lipid droplets [11,13]. De novo Transcriptome Assembly and Annotation In order to sequence the C. vulgaris transcriptome, a comple- mentary DNA (cDNA) library was prepared from total mRNA, and sequenced-by-synthesis using Illumina technology. Illumina Pipeline analysis produced 27 million, 55-base reads (Figure 2). We were unable to use the C. variabilis NC64A and Coccomyxa C- 169 genome sequences as scaffolds for assembly because the sequence homology was less than 5% for the short reads (http:// genome.jgi-psf.org/Coc_C169_1/Coc_C169_1.home.html, data not shown). Therefore, Velvet and Oases program suites were utilized for de novo transcriptome assembly. The resultant assembly yielded 29,237 transcripts, with an average transcript length of 970 nt (Figure 2). Unique transcripts were aligned against all NCBI Chlorophyta non-redundant sequences, including whole genome sequences. 7,067 transcripts had hits with significant Expect values (E-values) less than 1e-03, suggesting ,77% of assembled C. vulgaris transcripts are unique amongst available microalgae sequences. As noted by Rismani-Yazdi et al. [17], this identification rate is consistent with previously reported values for de novo assembled eukaryotic transcriptomes. Of the corresponding E-values, 315 (4.4%) were scored 0.0, which indicates that the corresponding hits PLoS ONE | www.plosone.org 4 October 2011 | Volume 6 | Issue 10 | e25851