transcriptome as a search database. ACAT peptides identified via mass spectrometry using the C. vulgaris transcriptome were aligned against all available Chlorophyta ACAT sequences. Peptide sequence alignment shows just a single amino acid differential between C. vulgaris and Chlorophyta peptides in two instances, both corresponding to a species of same genus, Chlorella variabilis, demonstrating both the limitation of using orthologous databases for peptide identification and the advantage of using a de novo assembled transcriptome as a search database (Figure 5). Improved identification capability using the transcriptome as a search database was further underscored on a more global pathway mapping scale, examining the fatty acid and TAG biosynthetic pathways. Orthologous searching identified only three enzyme components of the fatty acid biosynthetic pathway, and none of the TAG enzymatic components. Conversely, utilization of the C. vulgaris transcriptome as a search database allowed us to identify all enzymatic components of the fatty acid and TAG biosynthetic pathways. It is clear from these data that using the de novo assembled transcriptome dramatically improves proteomic identification capabilities. These results might not have been expected if the assembly or annotation of the C. vulgaris transcriptome was weak, or if the Chlorophyta database provided higher sequence identity. This is an important observation because it most notably confirms our hypothesis, and at the same time provides a measure of quality of our de novo assembly and annotation. Finally, utilization of the de novo assembled C.vulgaris transcrip- tome allowed for identification and differentiation of critically important protein isoforms. Though no protein isoforms were identified for the TAG biosynthetic pathway, homomeric and heteromeric ACCase isoforms, as well as multiple KAS isoforms, were identified during the annotation stage. Isoform differentiation can have a dramatic impact upon strain engineering strategies. For example, it has been suggested that overexpression of cytosolic homomeric ACCase, coupled with plastidial sub-cellular localiza- tion, as opposed to overexpression of the more complex, multi- subunit heteromeric plastidial isoform, may be a simpler and more efficient means to increase fatty acid content in oleaginous organisms [50]. Targeted strain improvement efforts and complete pathway analyses will thus be greatly facilitated by the isoform identification and maximal identification coverage a de novo assembled transcriptome search database affords. The gene ontology analysis was encouraging because it indicated that all classes of proteins were equally represented in the proteome in comparison to the transcriptome, including proteins that would be expected to reside mainly in the insoluble fraction. But it is also a warning that the majority of transcribed genes were not found in the proteome. It is likely that this failure to identify most transcribed gene products is due to some combination of quantitative limits to the GeLC-MS methodology and to post-transcriptional regulation. The distinction between these two possibilities can have a major impact on the quantitative proteomic analysis, especially as it would be applied to hypothesis- driven strain improvement programs and will need to be evaluated on a gene-by-gene basis for pathways of interest. Future work involving quantitative transcriptomic analysis, insoluble proteomic analysis, and more focused searches for specific missing proteins will help shed light on this missing piece of the puzzle. Changes in Fatty Acid and TAG Biosynthetic Pathways in a High-Lipid State As observed previously for C. cryptica [51], our analysis also found upregulation of ACCase under lipid-accumulating condi- tions. More importantly, though, our current work also established Systems Biology Analysis of Chlorella vulgaris that the majority of the other fatty acid biosynthetic pathway components are upregulated under nitrogen-depletion and concurrent lipid accumulation. Interestingly, AMPK was down- regulated under high-lipid producing conditions. AMPK is proposed to serve as a fatty acid beta-oxidation ‘‘metabolic master switch,’’ acting as a direct ACCase inhibitor (and indirect carnitine palmitoyltransferase (CPT-1) activator in higher eukaryotes) [52]. This lends potential insight into the regulation of fatty acid synthesis through rate-limiting ACCase activity and concurrent increase in beta-oxidation. Overexpression of the ACCase gene in both C. cryptica and N. saprofila failed to significantly increase lipid accumulation [53]. A number of mechanisms have been proposed to explain this observation, including post-transcriptional regula- tion and feedback inhibition. It is possible AMPK also played a critical role in driving the equilibrium between acetyl-CoA and malonyl-CoA in the reverse direction, ultimately slowing the rate of fatty acid biosynthesis and increasing the rates of fatty acid beta- oxidation. The activity of AMPK under nitrogen-replete and nitrogen-deplete conditions warrants further investigation. The possibility must be considered that our decision to harvest cultures at the onset of stationary phase yielded cells that were past their peak in abundance of the enzymatic components of the fatty acid biosynthetic pathway (even though many significant increases were observed). Resultant late stage down-regulation of synthesis may explain the decrease observed for KAR under nitrogen- limited conditions. As synthesis of fatty acid biosynthetic machinery is shut down, protein turnover is likely to occur, as cells may be under a state of high catabolic activity in order to recycle internal nitrogen stores. However, protein abundance is not directly reflective of protein expression rates or activity. Thus, it is possible that KAR enzymatic rates remain unchanged despite decreased abundance. It is also unclear whether the increased abundance of fatty acid biosynthetic components (and decrease in KAR abundance) is due to altered rates of mRNA expression and translation, or mRNA and protein turnover. A more complete integrated systems biology analysis, incorporating transcriptomic, proteomic, and metabolomic data will be necessary to fully elucidate potential flux bottlenecks in the fatty acid pathway. Our results demonstrate that TAG biosynthetic machinery abundance is upregulated significantly higher than the fatty acid synthesis machinery in a high-lipid accumulation state. This massive upregulation can again likely be attributed to the late- stage harvest of nitrogen-deplete cells. At this stage, cells have neared stationary phase, having exhausted internal nitrogen stores. Photosynthetic energy and carbon fixation can continue (albeit with presumably altered efficiency indicated by the reduction in pigmentation in the starved, chlorotic cells, Figure 1a). The most effective diversion of the fixed carbon and reducing equivalents generated by photosynthesis is their conversion to TAGs (as reflected in fluorescence imaging of neutral lipids, Figure 1d). As observed in C. reinhardtii lipidomics analysis, acyltransferases were amongst the most abundant lipid droplet-associated proteins observed [11]. As such, with the intracellular space largely encompassed by neutral lipids, it is to be expected that we would observe significant abundance of TAG-related acyltransferases. The dramatic differential between fatty acid biosynthetic and TAG biosynthetic components may imply TAG biosynthesis may also play a significant role in the rate-limiting production of neutral lipids, suggesting future studies aimed at strain improve- ment might be focused upon overexpression of TAG biosynthetic components in addition to fatty acid biosynthetic components. Notably, none of the TAG biosynthetic machinery was identified under nitrogen-replete conditions, suggesting TAG biosynthesis is either largely in an ‘‘off state’’ or at very low levels PLoS ONE | www.plosone.org 11 October 2011 | Volume 6 | Issue 10 | e25851