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Plant oxidosqualene metabolism: cycloartenol synthase-dependent sterol biosynthesis in Nicotiana benthamiana.

Gas-Pascual E, Berna A, Bach TJ, Schaller H - PLoS ONE (2014)

Bottom Line: The plant sterol pathway exhibits a major biosynthetic difference as compared with that of metazoans.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 Arabidopsis thaliana, though the contribution of a lanosterol pathway to the production of 24-alkyl-Δ(5)-sterols was quite marginal (Ohyama et al. (2009) PNAS 106, 725).

View Article: PubMed Central - PubMed

Affiliation: Institut de Biologie Moléculaire des Plantes du CNRS & Université de Strasbourg, Institut de Botanique, Strasbourg, France.

ABSTRACT
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 (LAS1) in the oxidosqualene cyclase repertoire, in addition to cycloartenol synthases (CAS1) 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 Arabidopsis thaliana, though the contribution of a lanosterol pathway to the production of 24-alkyl-Δ(5)-sterols was quite marginal (Ohyama et al. (2009) PNAS 106, 725). To investigate further the physiological relevance of CAS1 and LAS1 genes in plants, we have silenced their expression in Nicotiana benthamiana. We used virus induced gene silencing (VIGS) based on gene specific sequences from a Nicotiana tabacum CAS1 or derived from the solgenomics initiative (http://solgenomics.net/) to challenge the respective roles of CAS1 and LAS1. In this report, we show a CAS1-specific functional sterol pathway in engineered yeast, and a strict dependence on CAS1 of tobacco sterol biosynthesis.

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Alignment of selected 2,3-oxidosqualene-cycloartenol cyclases (CAS1) and 2,3-oxidosqualene-lanosterol cyclases (LAS1) from solanaceae.At, Arabidopsis thaliana; Nt, Nicotiana tabacum; Nb, Nicotiana benthamiana; Sl, Solanum lycopersicon; Ca, Capsicum annuum. Dashes are for gaps that maximize the alignment made with GeneDoc [48]. 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, Arabidopsis thaliana numbering). A terpene synthase signature DGSWyGsWAVcFtYG is underlined.
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pone-0109156-g003: Alignment of selected 2,3-oxidosqualene-cycloartenol cyclases (CAS1) and 2,3-oxidosqualene-lanosterol cyclases (LAS1) from solanaceae.At, Arabidopsis thaliana; Nt, Nicotiana tabacum; Nb, Nicotiana benthamiana; Sl, Solanum lycopersicon; Ca, Capsicum annuum. Dashes are for gaps that maximize the alignment made with GeneDoc [48]. 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, Arabidopsis thaliana numbering). A terpene synthase signature DGSWyGsWAVcFtYG is underlined.

Mentions: We cloned by RT-PCR a cDNA fragment from N.benthamiana leaf RNA, that corresponded to an expressed NbLAS1 (Table S3), confirmed by qRT-PCR analysis. The alignement of amino acid sequences of CAS1 and LAS1 from Nicotiana tabacum, N.benthamiana, Solanum lycopersicon and Capsicum annuum (Fig. 3) shows features that support the identification of CAS1 and LAS1 sequences in these solanaceae. In fact, these two enzymes have been studied extensively. Molecular evolution and site-directed mutagenesis experiments revealed important aminoacid residues and conserved motifs that govern the cyclization of OSC into cycloartenol or into lanosterol. All CAS1 enzymes have a strict requirement for His477 and I481 (Arabidopsis numbering) in the vicinity of the conserved DCTAE motif implicated in substrate protonation, whereas all LAS1 proteins have a strict requirement for a V481 (Fig. 3) [31], [32]. A thorough analysis of the cyclization reactions, which required the heterologous expression of the plant CAS1 or LAS1 enzymes in the yeast erg7 (lanosterol-deficient) mutant led to a current understanding of the catalytic differences in both reactions [33].


Plant oxidosqualene metabolism: cycloartenol synthase-dependent sterol biosynthesis in Nicotiana benthamiana.

Gas-Pascual E, Berna A, Bach TJ, Schaller H - PLoS ONE (2014)

Alignment of selected 2,3-oxidosqualene-cycloartenol cyclases (CAS1) and 2,3-oxidosqualene-lanosterol cyclases (LAS1) from solanaceae.At, Arabidopsis thaliana; Nt, Nicotiana tabacum; Nb, Nicotiana benthamiana; Sl, Solanum lycopersicon; Ca, Capsicum annuum. Dashes are for gaps that maximize the alignment made with GeneDoc [48]. 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, Arabidopsis thaliana numbering). A terpene synthase signature DGSWyGsWAVcFtYG is underlined.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4208727&req=5

pone-0109156-g003: Alignment of selected 2,3-oxidosqualene-cycloartenol cyclases (CAS1) and 2,3-oxidosqualene-lanosterol cyclases (LAS1) from solanaceae.At, Arabidopsis thaliana; Nt, Nicotiana tabacum; Nb, Nicotiana benthamiana; Sl, Solanum lycopersicon; Ca, Capsicum annuum. Dashes are for gaps that maximize the alignment made with GeneDoc [48]. 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, Arabidopsis thaliana numbering). A terpene synthase signature DGSWyGsWAVcFtYG is underlined.
Mentions: We cloned by RT-PCR a cDNA fragment from N.benthamiana leaf RNA, that corresponded to an expressed NbLAS1 (Table S3), confirmed by qRT-PCR analysis. The alignement of amino acid sequences of CAS1 and LAS1 from Nicotiana tabacum, N.benthamiana, Solanum lycopersicon and Capsicum annuum (Fig. 3) shows features that support the identification of CAS1 and LAS1 sequences in these solanaceae. In fact, these two enzymes have been studied extensively. Molecular evolution and site-directed mutagenesis experiments revealed important aminoacid residues and conserved motifs that govern the cyclization of OSC into cycloartenol or into lanosterol. All CAS1 enzymes have a strict requirement for His477 and I481 (Arabidopsis numbering) in the vicinity of the conserved DCTAE motif implicated in substrate protonation, whereas all LAS1 proteins have a strict requirement for a V481 (Fig. 3) [31], [32]. A thorough analysis of the cyclization reactions, which required the heterologous expression of the plant CAS1 or LAS1 enzymes in the yeast erg7 (lanosterol-deficient) mutant led to a current understanding of the catalytic differences in both reactions [33].

Bottom Line: The plant sterol pathway exhibits a major biosynthetic difference as compared with that of metazoans.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 Arabidopsis thaliana, though the contribution of a lanosterol pathway to the production of 24-alkyl-Δ(5)-sterols was quite marginal (Ohyama et al. (2009) PNAS 106, 725).

View Article: PubMed Central - PubMed

Affiliation: Institut de Biologie Moléculaire des Plantes du CNRS & Université de Strasbourg, Institut de Botanique, Strasbourg, France.

ABSTRACT
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 (LAS1) in the oxidosqualene cyclase repertoire, in addition to cycloartenol synthases (CAS1) 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 Arabidopsis thaliana, though the contribution of a lanosterol pathway to the production of 24-alkyl-Δ(5)-sterols was quite marginal (Ohyama et al. (2009) PNAS 106, 725). To investigate further the physiological relevance of CAS1 and LAS1 genes in plants, we have silenced their expression in Nicotiana benthamiana. We used virus induced gene silencing (VIGS) based on gene specific sequences from a Nicotiana tabacum CAS1 or derived from the solgenomics initiative (http://solgenomics.net/) to challenge the respective roles of CAS1 and LAS1. In this report, we show a CAS1-specific functional sterol pathway in engineered yeast, and a strict dependence on CAS1 of tobacco sterol biosynthesis.

Show MeSH
Related in: MedlinePlus