Systems Biology Analysis of Chlorella vulgaris Figure 4. Gene Ontology enrichment analysis. Functional distribution of the complete annotated C. vulgaris transcriptome (grey bars) and the 2,949 transcripts corresponding to proteins identified in sub-proteomic soluble fraction via MS/MS analysis (black bars). Distribution is represented as percent of total transcripts in respective fractions. doi:10.1371/journal.pone.0025851.g004 group of malonyl-CoA is transferred to acyl carrier protein (ACP) forming malonyl-ACP in a reaction catalyzed by MAT. The subsequent series of four condensation reactions is then catalyzed by KAS, KAR, HD, and enoyl-ACP reductase (ENR). These condensation reactions ultimately lengthen precursor acyl-ACP chains by two carbons per cycle. Termination of elongation is catalyzed by an acyl-ACP thioesterase (FAT), leading to free fatty acid release and export to the cytosol, or via direct transfer of the acyl group to glycerol-3-phosphate and/or monoacylglycerol-3- phosphate in the TAG biosynthetic pathway (discussed below). For extensive review of plant and microalgal fatty acid biosynthesis, refer to Ohlrogge and Browse, 1995 and Hu et al., 2008. Microalgal TAG biosynthesis is proposed to occur via sequential transfer of fatty acids from CoA to glycerol-3-phosphate (G3P) via the direct glycerol pathway [1,47] (summarized in Figure 6). Fatty acid transfer to position one of G3P results in the formation of lyso-phosphatidic acid (LPA), in a reaction catalyzed by GPAT. Subsequent acyl transfer to position two of LPA leads to formation of phosphatidic acid (PA), in a reaction catalyzed by LPAAT. PA can also be formed via phosphorylation of diacylglycerol (DAG) in a reaction catalyzed by DAG kinase (DAGK) [48]. The penultimate step of TAG biosynthesis is catalyzed by PAP, resulting in dephosphorylation of PA and formation of DAG. DGAT ultimately catalyzes the final and committed step of TAG biosynthesis, in which a third acyl chain is transferred to position 3 of G3P, forming a neutral triacylglyceride [1,49]. We examined changes in spectral counts (the total number of MS/MS spectra identifying a protein) for the components of the fatty acid and triacylglyceride biosynthetic pathways under nitrogen-replete and nitrogen-deplete conditions. Normalized spectral abundance factor (NSAF) values were utilized to calculate spectral count fold-changes, as described by Zybailov et al. [39]. Figure 6 summarizes the spectral count fold-change for the components of fatty acid and TAG biosynthetic pathways under nitrogen-deplete conditions with respect to nitrogen-replete conditions (the ratio of N-deplete to N-replete). ACCase abundance was upregulated approximately 2-fold under nitro- gen-deplete conditions, while AMPK abundance was downregu- PLoS ONE | www.plosone.org 8 October 2011 | Volume 6 | Issue 10 | e25851