Subcellular localization of DWARF5-YFP protein in Arabidopsis <i>dwarf5-2::DWARF5-YFP</i> plants.

<p>(A) Confocal images of leaves showing protein distribution in the ER and (D) to the periphery of the cell (yellow arrow). (B) Chlorophyll autofluorescence. (E) In red is shown the chlorophyll autofluorescence combined with the FM4-64 fluorescence localized to the PM. The overlay images show (C) the complete separation of red and yellow signal and (F) the co-localization of DWARF5-YFP and FM4-64 indicating the PM association of DWARF5. Scale bars = 25 µm.</p

Genetic complementation of <i>ste1-1</i>, <i>dwarf5-2</i> and <i>dim</i> sterol biosynthetic mutants expressing <i>STE1-YFP</i>, <i>DWARF5-YFP</i>, and <i>DIM-YFP</i> cDNAs, respectively.

<p>GC-FID chromatograms of steryl acetates are shown. (A) <i>dwarf5-2</i> mutant; (B) <i>dwarf5-2</i>/<i>DWARF5-YFP</i> partially complemented mutant; (C) <i>dwarf5-2</i>/<i>DWARF5-YFP</i> fully complemented mutant. (D) <i>dim</i> mutant; (E), <i>dim</i>/<i>DIM-YFP</i> partially complemented mutant; (F) <i>dim</i>/<i>DIM-YFP</i> fully complemented mutant. (G) <i>ste1-1</i> mutant; (H), <i>ste1-1</i>/<i>STE1-YFP</i> partially complemented mutant; (I) <i>ste1-1</i>/<i>STE1-YFP</i> fully complemented mutant. Sterol peaks identified by their retention time and confirmed by GC-MS (prominent mass fragments not shown here) are: 1, cholesterol; 2, Δ^5,7-cholesterol; 3, Δ⁷-cholesterol; 4, campesterol; 5, Δ⁷-campesterol; 6, Δ^5,7-campesterol; 7, Δ^5,7-stigmasterol; 8, Δ⁸-sitosterol; 9, Δ^5,7-sitosterol; 10, sitosterol; 11, isofucosterol; 12, Δ⁷-sitosterol; 13, 24-methylene cholesterol; 14, stigmasterol; 15, Δ⁷-avenasterol. Full complementation of <i>dwarf5-2</i>, <i>dim</i> and <i>ste1-1</i> results in the accumulation of sitosterol (10) instead of Δ^5,7-sitosterol (9), isofucosterol (11) and Δ⁷-sitosterol (12), respectively. The relevant peaks in each complementation are labelled in bold in the relevant panels.</p

Sterol composition of wild type and <i>dwarf5-2</i> (<i>d5</i>), <i>dim</i> (<i>d1</i>) and <i>ste1-1</i> (<i>s1</i>) Arabidopsis mutant plants.

<p>The values refer to the partially and fully complemented plants compared to the not complemented. The numbers in parentheses refers to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056429#pone-0056429-g001" target="_blank">Figure 1</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056429#pone-0056429-g002" target="_blank">Figure 2</a>. tr = trace amount; - = compound not detected. Accurate sterol nomenclature can be found at IUPAC <a href="http://www.iupac.org" target="_blank">http://www.iupac.org</a>.</p

Plant Sterol Metabolism. Δ⁷-Sterol-C₅-Desaturase (STE1/DWARF7), Δ^5,7-Sterol-Δ⁷-Reductase (DWARF5) and Δ²⁴-Sterol-Δ²⁴-Reductase (DIMINUTO/DWARF1) Show Multiple Subcellular Localizations in <em>Arabidopsis thaliana</em> (Heynh) L

<div><p>Sterols are crucial lipid components that regulate membrane permeability and fluidity and are the precursors of bioactive steroids. The plant sterols exist as three major forms, free sterols, steryl glycosides and steryl esters. The storage of steryl esters in lipid droplets has been shown to contribute to cellular sterol homeostasis. To further document cellular aspects of sterol biosynthesis in plants, we addressed the question of the subcellular localization of the enzymes implicated in the final steps of the post-squalene biosynthetic pathway. In order to create a clear localization map of steroidogenic enzymes in cells, the coding regions of Δ⁷-sterol-C₅-desaturase (STE1/DWARF7), Δ²⁴-sterol-Δ²⁴-reductase (DIMINUTO/DWARF1) and Δ^5,7-sterol-Δ⁷-reductase (DWARF5) were fused to the yellow fluorescent protein (YFP) and transformed into <em>Arabidopsis thaliana</em> mutant lines deficient in the corresponding enzymes. All fusion proteins were found to localize in the endoplasmic reticulum in functionally complemented plants. The results show that both Δ^5,7-sterol-Δ⁷-reductase and Δ²⁴-sterol-Δ²⁴-reductase are in addition localized to the plasma membrane, whereas Δ⁷-sterol-C₅-desaturase was clearly detected in lipid particles. These findings raise new challenging questions about the spatial and dynamic cellular organization of sterol biosynthesis in plants.</p> </div

Phenotype of <i>dwarf5-2</i>, <i>dim</i> and <i>ste1-1</i> mutants complemented with <i>DWARF5-YFP</i>, <i>DIM-YFP</i> and <i>STE1-YFP</i>, respectively.

<p>(A, D, G) <i>dwarf5-2</i>, <i>dim</i> and <i>ste1-1</i> (sterol profiles given in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056429#pone-0056429-g002" target="_blank">Figure 2</a>). (B, E, H) <i>dwarf5-2, dim and ste1.1</i> partial complemented. (C, F, I) <i>dwarf5-2</i>, <i>dim</i> and <i>ste1-1</i>fully complemented. (J) Wild-type (sterol profile given in Supplemental <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056429#pone-0056429-g002" target="_blank">Figure 2</a>).</p

Subcellular localization of STE1-YFP protein in Arabidopsis <i>ste1-1::STE1-YFP</i> plants (panel A to D) and Nile Red staining of ste1-1 mutants (panel E to H).

<p>(A) STE1-YFP localization to structures resembling ER (white arrow) and LPs (yellow arrows). (B) and (F) chlorophyll autofluorescence. (C) and (G) LPs stained with Nile Red (yellow arrows). (D) Overlay image of (A), (B) and (C) showing the overlap of the Nile Red and YFP signal (yellow arrow) and the ER localization (white arrow) of STE1-YFP. (E) YFP signal absent in <i>ste1-1</i> mutant. (H) Overlay image of (E), (F) and (G) showing the distribution of LPs in cell of <i>ste1-1</i> plant. Scale bars = 10 µm.</p

Subcellular localization of DIM-YFP protein in Arabidopsis <i>dim::DIM-YFP</i> plants.

<p>(A) The DIM-YFP signal is localized to structures surrounding the nuclei resembling ER (white arrow) and to the cell periphery (yellow arrow). (B) In red is shown the chlorophyll autofluorescence combined with the FM4-64 fluorescence localized to the PM. (C) The overlay image shows the co-localization of DIM-YFP and FM4-64 suggesting the PM association of DIM. Scale bars = 50 µm.</p

Sterol composition of wild type (W303) and mutant (<i>erg3</i> and <i>erg4</i>) yeast strains expressing the corresponding YFP-fused proteins compared to the non-transformed strains.

<p>One representative analysis out of three independent experiments is shown. The letters in parentheses refers to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056429#pone-0056429-g001" target="_blank">Figure 1</a>.- = compound not detected.</p

Multiplexed Quantification of Plant Thylakoid Proteins on Western Blots Using Lanthanide-Labeled Antibodies and Laser Ablation Inductively Coupled Plasma Mass Spectrometry (LA-ICP-MS)

We have developed a novel calibration method that allows concurrent quantification of multiple proteins by laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) after Western blotting. Calibrants were made of nitrocellulose membranes doped with lanthanide standards. Excellent linearity was obtained in the interval from 0 to 24 ng lanthanide cm^–2. Cerium-labeled lysozyme was introduced as an internal reference protein, enabling correction for up to 50% difference in transfer efficiency during the blotting of membranes. The sensitivity of the LA-ICP-MS method was comparable to state-of-the-art chemiluminescence detection and was further improved by a factor of 20, using a polymer tag. Our method allowed reproducible and multiplexed quantification of five thylakoid proteins extracted from chloroplasts of the plant species Arabidopsis thaliana (relative standard deviation (RSD) of ≤ 5% in three independent analytical series). The method was capable of measuring the L subunit in photosystem I of an <i>Arabidopsis</i> mutant containing <5% of this particular protein, relative to the wild type. We conclude that the developed calibration method is highly suited for multiplexed and comparative protein studies, allowing for intermembrane comparisons with high sensitivity and reproducibility

Verification of product formation in living cells.

<p>A) Extracted ion chromatograms (<i>m/z</i> 152) from the LC-MS analysis of metabolites extracted from the growth medium of <i>Synechococcus</i> sp. PCC 7002 WT and PsaM-CYP79A1 cultures for detection of <i>in vivo</i> activity of the PsaM-CYP79A1 complex, compared to a <i>p</i>-hydroxyphenylacetaldoxime standard (C+). The two peaks are the E and Z isomers of the <i>p</i>-hydroxyphenylacetaldoxime. B) LC-MS extracted ion chromatograms as in A, from analysis of extracts of PsaM-CYP79A1 cyanobacteria or growth medium.</p