A Comprehensive GC–MS Sub-Microscale Assay for Fatty Acids and its Applications

Fatty acid analysis is essential to a broad range of applications including those associated with the nascent algal biofuel and algal bioproduct industries. Current fatty acid profiling methods require lengthy, sequential extraction and transesterification steps necessitating significant quantities of analyte. We report the development of a rapid, microscale, single-step, in situ protocol for GC–MS lipid analysis that requires only 250 μg dry mass per sample. We furthermore demonstrate the broad applications of this technique by profiling the fatty acids of several algal species, small aquatic organisms, insects and terrestrial plant material. When combined with fluorescent techniques utilizing the BODIPY dye family and flow cytometry, this micro-assay serves as a powerful tool for analyzing fatty acids in laboratory and field collected samples, for high-throughput screening, and for crop assessment. Additionally, the high sensitivity of the technique allows for population analyses across a wide variety of taxa

Ligand Effects on the Oxidative Addition of Halogens to (dpp-nacnac^R)Rh(phdi)

The treatment of (dpp-nacnac^R)­Rh­(phdi) {(dpp-nacnac^R)⁻ = CH­[C­(R)­(N-^<i>i</i>Pr₂C₆H₃)]₂^–; R = CH₃, CF₃; phdi = 9,10-phenanthrenediimine} with X₂ oxidants afforded octahedral rhodium­(III) products in the case of X = Cl and Br. The octahedral complexes exhibit well-behaved cyclic voltammograms in which a two-electron reduction is observed to regenerate the initial rhodium­(I) complex. When treated with I₂, (dpp-nacnac^CH3)­Rh­(phdi) produced a square pyramidal η¹-I₂ complex, which was characterized by NMR and UV–vis spectroscopies, mass spectrometry, and X-ray crystallography. The more electron poor complex (dpp-nacnac^CF3)­Rh­(phdi) reacted with I₂ to give a mixture of two products that were identified by ¹H NMR spectroscopy as a square pyramidal η¹-I₂ complex and an octahedral diiodide complex. Reaction of the square pyramidal (dpp-nacnac^CH3)­Rh­(I₂)­(phdi) with HBF₄ resulted in protonation of the (dpp-nacnac^CH3)⁻ backbone to provide an octahedral rhodium­(III) diiodide species. These reactions highlight the impact that changes in the electron-withdrawing nature of the supporting ligands can have on the reactivity at the metal center

Ligand Effects on the Oxidative Addition of Halogens to (dpp-nacnac^R)Rh(phdi)

The treatment of (dpp-nacnac^R)­Rh­(phdi) {(dpp-nacnac^R)⁻ = CH­[C­(R)­(N-^<i>i</i>Pr₂C₆H₃)]₂^–; R = CH₃, CF₃; phdi = 9,10-phenanthrenediimine} with X₂ oxidants afforded octahedral rhodium­(III) products in the case of X = Cl and Br. The octahedral complexes exhibit well-behaved cyclic voltammograms in which a two-electron reduction is observed to regenerate the initial rhodium­(I) complex. When treated with I₂, (dpp-nacnac^CH3)­Rh­(phdi) produced a square pyramidal η¹-I₂ complex, which was characterized by NMR and UV–vis spectroscopies, mass spectrometry, and X-ray crystallography. The more electron poor complex (dpp-nacnac^CF3)­Rh­(phdi) reacted with I₂ to give a mixture of two products that were identified by ¹H NMR spectroscopy as a square pyramidal η¹-I₂ complex and an octahedral diiodide complex. Reaction of the square pyramidal (dpp-nacnac^CH3)­Rh­(I₂)­(phdi) with HBF₄ resulted in protonation of the (dpp-nacnac^CH3)⁻ backbone to provide an octahedral rhodium­(III) diiodide species. These reactions highlight the impact that changes in the electron-withdrawing nature of the supporting ligands can have on the reactivity at the metal center

Genome Sequence and Transcriptome Analyses of <i>Chrysochromulina tobin</i>: Metabolic Tools for Enhanced Algal Fitness in the Prominent Order Prymnesiales (Haptophyceae)

<div><p>Haptophytes are recognized as seminal players in aquatic ecosystem function. These algae are important in global carbon sequestration, form destructive harmful blooms, and given their rich fatty acid content, serve as a highly nutritive food source to a broad range of eco-cohorts. Haptophyte dominance in both fresh and marine waters is supported by the mixotrophic nature of many taxa. Despite their importance the nuclear genome sequence of only one haptophyte, <i>Emiliania huxleyi</i> (Isochrysidales), is available. Here we report the draft genome sequence of <i>Chrysochromulina tobin</i> (Prymnesiales), and transcriptome data collected at seven time points over a 24-hour light/dark cycle. The nuclear genome of <i>C</i>. <i>tobin</i> is small (59 Mb), compact (∼40% of the genome is protein coding) and encodes approximately 16,777 genes. Genes important to fatty acid synthesis, modification, and catabolism show distinct patterns of expression when monitored over the circadian photoperiod. The <i>C</i>. <i>tobin</i> genome harbors the first hybrid polyketide synthase/non-ribosomal peptide synthase gene complex reported for an algal species, and encodes potential anti-microbial peptides and proteins involved in multidrug and toxic compound extrusion. A new haptophyte xanthorhodopsin was also identified, together with two “red” RuBisCO activases that are shared across many algal lineages. The <i>Chrysochromulina tobin</i> genome sequence provides new information on the evolutionary history, ecology and economic importance of haptophytes.</p></div

Phylogenetic placement of haptophyte, dinoflagellate, and cryptophyte xanthorhodopsins.

<p>Bayesian phylogeny inferred from a 231 amino acid alignment of rhodopsins, with posterior probabilities and maximum-likelihood bootstrap support values shown at key nodes. Clades of xanthorhodopsins, novel cryptophyte-specific rhodopsins, sensory type I, II, and III rhodopsins, bacteriorhodopsins, xenorhodopsins, halorhodopsins and the proteorhodopsin outgroup are indicated.</p

<i>Chrysochromulina</i> MATE domain detail.

<p><i>Chrysochromulina</i> MATE domain detail.</p

Identification of fatty acid synthesis genes of <i>Chrysochromulina tobin</i> and corresponding RNA transcript FPKM over a 12 hr light: 12 hr dark photoperiod.

<p>The genes identified as fatty acid synthesis genes and the corresponding chloroplast and endoplasmic reticulum pathway schematic (from [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005469#pgen.1005469.ref037" target="_blank">37</a>]).</p

<i>Chrysochromulina tobin</i> cell structure.

<p>(A) Scanning electron micrograph of <i>C</i>. <i>tobin</i>. Two flagella are visible (marked F) along with the prominent coiled haptonema (white arrow). Scale bar represents 2.5 microns. (B) Electron micrograph of whole cell: Lipid body (LB); Mitochondrion (M); Chloroplast (C). Scale bar represents 500 nanometers.</p

<i>Chrysochromulina tobin</i> displays photoperiod controlled cell division and lipid metabolism.

<p>(A) Cell division is observed to be highest during the light to dark transition. (B) Change in lipid body size is correlated to photoperiod when detected by BODIPY 505/515 dye incorporation (green); chloroplast auto-fluorescence (red).</p