Metabolomics- and Proteomics-Assisted Genome Annotation and Analysis of the Draft Metabolic Network of Chlamydomonas reinhardtii

Abstract

We present an integrated analysis of the molecular repertoire of Chlamydomonas reinhardtii under reference conditions. Bioinformatics annotation methods combined with GCxGC/MS-based metabolomics and LC/MS-based shotgun proteomics profiling technologies have been applied to characterize abundant proteins and metabolites, resulting in the detection of 1069 proteins and 159 metabolites. Of the measured proteins, 204 currently do not have EST sequence support; thus a significant portion of the proteomicsdetected proteins provide evidence for the validity of in silico gene models. Furthermore, the generated peptide data lend support to the validity of a number of proteins currently in the proposed model stage. By integrating genomic annotation information with experimentally identified metabolites and proteins, we constructed a draft metabolic network for Chlamydomonas. Computational metabolic modeling allowed an identification of missing enzymatic links. Some experimentally detected metabolites are not producible by the currently known and annotated enzyme set, thus suggesting entry points for further targeted gene discovery or biochemical pathway research. All data sets are made available as supplementary material as well as web-accessible databases and within the functional context via the Chlamydomonas-adapted MapMan annotation platform. Information of identified peptides is also available directly via the JGIChlamydomonas genomic resource database (http:/ /genome.jgi-psf.org/Chlre3/Chlre3.home.html).

Nitrogen-Sparing Mechanisms in Chlamydomonas Affect the Transcriptome, the Proteome, and Photosynthetic Metabolism

“…Visual inspection classified these into increased versus decreased (up versus down) abundance relative to the 0-h time point, with 306 proteins in the former and 329 in the latter, each of the two groups consisting of three different temporal patterns (Supplemental Figure 4). Proteins were mapped into functional categories according to the C. reinhardtii MapMan ontology May et al, 2008) and enrichment within the up-or downregulated groups was assessed (Supplemental Data Set 4). (Figure 2A).…”

System Response of Metabolic Networks in Chlamydomonas reinhardtii to Total Available Ammonium

“…nome-scale reconstruction of the Chlamydomonas reinhardtii metabolic network consists of 263 pathways and 1419 reactions (25), catalyzed by 713 enzymes that have been annotated experimentally or computationally to a DNA sequence of C. reinhardtii. In order to perform comprehensive analysis of primary metabolite responses to nitrogen depletion, we have used mass spectrometry-based profiling of proteins and metabolites in these cultures in a semiquantitative way.…”

“…Hence, the outcome of cellular regulation should encompass metabolite measurements to truly depict a systemic view of stress responses, called metabolomics. Metabolomics uses a range of platforms for surveying combinations of chemical compounds in biological systems (23)(24)(25)(26). Although measurements of proteins, especially enzymes, concomitant with their substrates, products and allosteric modifiers appear to be a logical choice of tools to study metabolic response to nutritional stress, there have been only a few investigations on metabolomics and proteomics data integration to enable a system level understanding of cellular metabolism.…”

“…C. reinhardtii is a microalga that has importance as model for algal biofuels (11). Availability of a sequenced genome (27), molecular biology protocols (28,29), a proteomic database (25), and metabolomics protocols (30,31) benefit the exploitation of Chlamydomonas to investigate global metabolic changes in response to environmental or genetic alterations (32,33).…”

Systems-Level Analysis of Nitrogen Starvation-Induced Modifications of Carbon Metabolism in a Chlamydomonas reinhardtii Starchless Mutant

“…reinhardtii, a unicellular chlorophyte alga containing a pyrenoid within its single chloroplast representing the site of starch accumulation and ribulose-1,5-bis-phosphate carboxylase/ oxygenase function, is an excellent reference system for addressing this issue (Harris, 2001). A draft genome sequence (http://www.phytozome.net/search.php for the version 5 assembly), resources for transcriptomics, proteomics, metabolomics (Bölling and Fiehn, 2005;May et al, 2008;Atteia et al, 2009;Rolland et al, 2009;Mühlhaus et al, 2011;Urzica et al, 2012;Wang et al, 2012), and well-characterized mutants in starch metabolism are available (Ball et al, 1991;Mouille et al, 1996;Wattebled et al, 2002Wattebled et al, , 2003. In addition, the growth medium and nutrient requirements are well defined, and there is considerable ongoing effort to discover and understand the operation of carbon metabolism pathways under different trophic situations, including anaerobiosis, respiratory, and phototrophic conditions (Kropat et al, 2011;Brueggeman et al, 2012;Catalanotti et al, 2012;Fang et al, 2012;Johnson and Alric, 2012).…”