Plant Oxidosqualene Metabolism: Cycloartenol Synthase–Dependent Sterol Biosynthesis in <i>Nicotiana benthamiana</i>

<div><p>The plant sterol pathway exhibits a major biosynthetic difference as compared with that of metazoans. The committed sterol precursor is the pentacyclic cycloartenol (9β,19-cyclolanost-24-en-3β-ol) and not lanosterol (lanosta-8,24-dien-3β-ol), as it was shown in the late sixties. However, plant genome mining over the last years revealed the general presence of lanosterol synthases encoding sequences (<i>LAS1</i>) in the oxidosqualene cyclase repertoire, in addition to cycloartenol synthases (<i>CAS1</i>) and to non-steroidal triterpene synthases that contribute to the metabolic diversity of C₃₀H₅₀O compounds on earth. Furthermore, plant LAS1 proteins have been unambiguously identified by peptidic signatures and by their capacity to complement the yeast lanosterol synthase deficiency. A dual pathway for the synthesis of sterols through lanosterol and cycloartenol was reported in the model <i>Arabidopsis thaliana</i>, though the contribution of a lanosterol pathway to the production of 24-alkyl-Δ⁵-sterols was quite marginal (Ohyama et al. (2009) <i>PNAS</i> 106, 725). To investigate further the physiological relevance of <i>CAS1</i> and <i>LAS1</i> genes in plants, we have silenced their expression in <i>Nicotiana benthamiana</i>. We used virus induced gene silencing (VIGS) based on gene specific sequences from a <i>Nicotiana tabacum CAS1</i> or derived from the solgenomics initiative (<a href="http://solgenomics.net/" target="_blank">http://solgenomics.net/</a>) to challenge the respective roles of <i>CAS1</i> and <i>LAS1</i>. In this report, we show a CAS1-specific functional sterol pathway in engineered yeast, and a strict dependence on CAS1 of tobacco sterol biosynthesis.</p></div

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

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

Sterol profile determind by GC-MS of <i>erg7</i> expressing a tobacco cycloartenol synthase CAS1.

<p><b>A</b>, TIC of a total unsaponifiable extract of <i>erg7</i> transformed with a void vector. <b>B</b>, TIC of a total unsaponifiable extract of <i>erg7::NtCAS1</i>. <b>C</b>, 9β,19-cyclopropylsterol biosynthetic pathway in yeast. Compounds are: <b>1</b>, 2,3-oxidosqualene; <b>2</b>, ergosterol; <b>3</b>, cycloartenol; <b>4</b>, 31-norcycloartenol; <b>5</b>, 24-dehydropollinastanol; <b>6</b>, cycloeucalenol; <b>7</b>, 24-methylene pollinastanol. Compounds are identified according to their mass spectra and to those of authentic standards for 3, 6, and 7 purified from plant material as previously described <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0109156#pone.0109156-Lovato1" target="_blank">[25]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0109156#pone.0109156-Men1" target="_blank">[26]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0109156#pone.0109156-Wang2" target="_blank">[44]</a>. Peaks that are not numbered are not sterols.</p

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

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

Alignment of selected 2,3-oxidosqualene-cycloartenol cyclases (CAS1) and 2,3-oxidosqualene-lanosterol cyclases (LAS1) from <i>solanaceae</i>.

<p>At, <i>Arabidopsis thaliana</i>; Nt, <i>Nicotiana tabacum</i>; Nb, <i>Nicotiana benthamiana</i>; Sl, <i>Solanum lycopersicon</i>; Ca, <i>Capsicum annuum</i>. Dashes are for gaps that maximize the alignment made with GeneDoc <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0109156#pone.0109156-Nicholas1" target="_blank">[48]</a>. Conserved residues are highlighted in black or grey. The DCTAE motif is boxed (in green for CAS1; in red for LAS1). Important catalytic residues specifying cyclization of 2,3-oxidosqualene into cycloartenol or lanosterol are marked with arrowheads (Tyr 410, His 477 and Ile 481, <i>Arabidopsis thaliana</i> numbering). A terpene synthase signature DGSWyGsWAVcFtYG is underlined.</p

VIGS of CAS1 and LAS1 in <i>Nicotiana benthamiana</i>.

<p><b>A</b>, Morphological phenotype of <i>PVX</i> (left) and <i>PVX::CAS1</i> (right) plants 4 weeks after inoculation, the close-up shows bleaching of veins. <b>B</b>, Morphological phenotype of <i>PVX</i> (left) and <i>PVX::CAS1</i> (right) plants 5 weeks after inoculation, the close-up shows leaf wilting and necrosis. <b>C</b>, Relative gene expression in <i>PVX::CAS1</i> plants, <i>CAS1</i> is a measurement of the endogenous <i>NbCAS1</i> level, <i>PVX::CAS1</i> is a measurement of the viral <i>NtCAS1</i> transcript. <b>D</b>, Relative gene expression in <i>PVX::LAS1</i> plants, <i>LAS1</i> is a measurement of the endogenous <i>NbLAS1</i> level, <i>PVX::LAS1</i> is a measurement of the viral <i>NbLAS1</i> transcript. <b>E</b>, squalene epoxide amounts measured by GC-FID in silenced plants. <b>F</b>, sterol composition of <i>PVX</i> and <i>PVX::CAS1</i> plants. Structure of the compounds detected here are shown in <b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0109156#pone.0109156.s001" target="_blank">Fig. S1</a></b>. The pictures in <b>A</b> and <b>B</b> are representative of 4 independent experiments that included all 3 plants inoculated with each type of viral transcripts.</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

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