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

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

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

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

Phosphoproteome Exploration Reveals a Reformatting of Cellular Processes in Response to Low Sterol Biosynthetic Capacity in <i>Arabidopsis</i>

Sterols are membrane-bound isoprenoid lipids that are required for cell viability and growth. In plants, it is generally assumed that 3-hydroxy-3-methylglutaryl-CoA-reductase (HMGR) is a key element of their biosynthesis, but the molecular regulation of that pathway is largely unknown. In an attempt to identify regulators of the biosynthetic flux from acyl-CoA toward phytosterols, we compared the membrane phosphoproteome of wild-type <i>Arabidopsis thaliana</i> and of a mutant being deficient in HMGR1. We performed a N-terminal labeling of microsomal peptides with a trimethoxyphenyl phosphonium (TMPP) derivative, followed by a quantitative assessment of phosphopeptides with a spectral counting method. TMPP derivatization of peptides resulted in an improved LC–MS/MS detection due to increased hydrophobicity in chromatography and ionization efficiency in electrospray. The phosphoproteome coverage was 40% higher with this methodology. We further found that 31 proteins were in a different phosphorylation state in the <i>hmgr1–1</i> mutant as compared with the wild-type. One-third of these proteins were identified based on novel phosphopeptides. This approach revealed that phosphorylation changes in the <i>Arabidopsis</i> membrane proteome targets major cellular processes such as transports, calcium homeostasis, photomorphogenesis, and carbohydrate synthesis. A reformatting of these processes appears to be a response of a genetically reduced sterol biosynthesis