Proteomic Analysis in Nitrogen-Deprived <i>Isochrysis galbana</i> during Lipid Accumulation

<div><p>The differentially co-expressed proteins in N-deprived and N-enriched <i>I. galbana</i> were comparatively analyzed by using two dimensional electrophoresis (2-DE) and matrix-assisted laser desorption/ionization-time-of-flight/time-of-flight-mass spectrometry (MALDI-TOF/TOF-MS) with the aim of better understanding lipid metabolism in this oleaginous microalga. Forty-five of the 900 protein spots showed dramatic changes in N-deprived <i>I. galbana</i> compared with the N-enriched cells. Of these, 36 protein spots were analyzed and 27 proteins were successfully identified. The identified proteins were classified into seven groups by their molecular functions, including the proteins related to energy production and transformation, substance metabolism, signal transduction, molecular chaperone, transcription and translation, immune defense and cytoskeleton. These altered proteins slowed cell growth and photosynthesis of <i>I. galbana</i> directly or indirectly, but at the same time increased lipid accumulation. Eight key enzymes involved in lipid metabolism via different pathways were identified as glyceraldehyde-3-phosphate dehydrogenase (GAPDH), phosphoglycerate kinase (PGK), enolase, aspartate aminotransferase (AST), fumarate hydratase (FH), citrate synthase (CS), O-acetyl-serine lyase (OAS-L) and ATP sulfurylase (ATPS). The results suggested that the glycolytic pathway and citrate transport system might be the main routes for lipid anabolism in N-deprived <i>I. galbana</i>, and that the tricarboxylic acid (TCA) cycle, glyoxylate cycle and sulfur assimilation system might be the major pathways involved in lipid catabolism. </p> </div

The hypothesized pathways for lipid metabolism in N-deprived <i>I</i>.

<div><p><b><i>galbana</i></b>: </p> <p>① the glycolytic pathway, and involved enzymes GAPDH, PGK and enolase. the citrate transport system, and the involved enzyme CS. the TCA cycle, and the involved enzymes CS and FH. the glyoxylate cycle, and the involved enzymes AST, CS and FH. the pathways of cysteine (sulfur assimilation), glutathione and ethylene biosynthesis, and the involved enzymes OAS-L, ATPS, GS and ACC synthase.</p></div

Global Inventory, Long-Range Transport and Environmental Distribution of Dicofol

The uncertainties on whether dicofol can be identified as a persistent organic pollutant (POP) in terms of its long-range transport (LRT) potential and global distribution, are always a controversial topic during international regulation deliberations. The lack of monitoring data in remote background regions necessitates a model-based evaluation approach for assessing the global distribution of dicofol. However, few model simulations are available at present, as there is no inventory available for global historical usage of dicofol that has sufficiently high spatial and temporal resolution. To describe the current status of global emission, we first developed an inventory of global dicofol usage for the period of 2000–2012 at 1° × 1° latitude/longitude resolution. We then assessed the LRT potential of dicofol by calculating its Arctic Contamination Potential using the Globo-POP model. In addition, we simulated the global mass distribution and the fate of dicofol in the environment using the BETR-Global model at 15° × 15° latitude/longitude resolution. Our estimated inventory established that over the period of 13 years, a total of 28.2 kilo tonnes (kt) of dicofol was applied and released into the environment. East and Southeast Asia, the Mediterranean Coast, and Northern and Central America were identified as hotspots of usage and release. Dicofol exhibited a higher Arctic Contamination Potential than several confirmed Arctic contaminants, and a larger current volume of consumption than most existing POPs. The results of our BETR-Global simulation suggest that (i) dicofol can indeed be transported northward, most likely driven by both atmospheric and oceanic advections from source regions at midlatitudes, and (ii) dicofol will be enriched in remote background regions. Continuous use of dicofol in source regions will result in exposure both locally and in remote regions, and the examination of the potential for adverse effects is therefore of paramount importance. Proactive restrictions at the international level may be warranted

The growth and lipid changes of <i>I</i>. <i>galbana</i> in L₁ and N-deprived L₁ (L₁-N) media.

<p>A, cell numbers; B, chlorophyll; C, Fv/Fm ; D, P.I. ; E, total lipid; F, relative content of fatty acid. </p

The workflow outlining the proteomic analysis of <i>I</i>. <i>galbana</i> in N-deprived L₁ and L₁ media.

<p>To analyze the growth, lipid accumulation and protein variation of <i>I</i>. <i>galbana</i>, three replicates per group were performed in this experiment, and three samples were took from each replicate. In total, nine samples per group were analyzed. But in the proteomic analysis, the protein of every group was extracted from the mixture of nine samples.</p

Schematic diagram of SDO sub-segment expression.

<p>Del-1: no structural amino acid site; Del-2: two metal I binding site residues, two metal II binding site residues and no GSH binding site; Del-3: complete metal I binding site, three metal II binding site residues and one GSH binding site; Del-4: complete metal I binding site and metal II binding site as well as three GSH binding sites; complete ORF: complete metal I binding sites and metal II binding sites as well as GSH binding sites. The numbers indicated the expressed sub-segment amino acid length.</p

Characteristics of the five sub-segment-expressed recombinant SDO proteins.

<p>A. SDO specific activities (mean±SE, n = 3) of five recombinant SDO proteins. B. GSH affinities (mean±SE, n = 3) of the recombinant SDO proteins. Groups containing the same letters on the bar indicate no significant difference while different letters on the bar indicate a significant difference (p<0.05).</p

<i>De Novo</i> Transcriptome Analysis of an Aerial Microalga <i>Trentepohlia jolithus:</i> Pathway Description and Gene Discovery for Carbon Fixation and Carotenoid Biosynthesis

<div><p>Background</p><p>Algae in the order Trentepohliales have a broad geographic distribution and are generally characterized by the presence of abundant β-carotene. The many monographs published to date have mainly focused on their morphology, taxonomy, phylogeny, distribution and reproduction; molecular studies of this order are still rare. High-throughput RNA sequencing (RNA-Seq) technology provides a powerful and efficient method for transcript analysis and gene discovery in <i>Trentepohlia jolithus</i>.</p><p>Methods/Principal Findings</p><p>Illumina HiSeq 2000 sequencing generated 55,007,830 Illumina PE raw reads, which were assembled into 41,328 assembled unigenes. Based on NR annotation, 53.28% of the unigenes (22,018) could be assigned to gene ontology classes with 54 subcategories and 161,451 functional terms. A total of 26,217 (63.44%) assembled unigenes were mapped to 128 KEGG pathways. Furthermore, a set of 5,798 SSRs in 5,206 unigenes and 131,478 putative SNPs were identified. Moreover, the fact that all of the C4 photosynthesis genes exist in <i>T. jolithus</i> suggests a complex carbon acquisition and fixation system. Similarities and differences between <i>T. jolithus</i> and other algae in carotenoid biosynthesis are also described in depth.</p><p>Conclusions/Significance</p><p>This is the first broad transcriptome survey for <i>T. jolithus</i>, increasing the amount of molecular data available for the class Ulvophyceae. As well as providing resources for functional genomics studies, the functional genes and putative pathways identified here will contribute to a better understanding of carbon fixation and fatty acid and carotenoid biosynthesis in <i>T. jolithus</i>.</p></div

Putative pathway of carbon fixation in <i>T. jolithus</i>, generated by KEGG.

<p>The numbers within the small boxes are enzyme codes. The boxes with a red border are enzymes identified in this study. The boxes with a black border are enzymes not identified in this study.</p

Rocks covered with red <i>T. jolithus</i>.

<p>(A) Red-Stone-Valley in winter. (B) Reddish stones along rivers. (C) Microscopic structure of dried <i>T. jolithus</i>. (D) Microscopic structure of rehydrated <i>T. jolithus</i> after a few drops of water was added to the dried material.</p