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

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.

Figure 1. Prominent 2,3-oxidosqualene cyclization products.

1, β-amyrin (olean-12-en-3β-ol); 2, cycloartenol (9β,19-cyclolanost-24-en-3β-ol); 3, lanosterol (lanosta-8,24-dien-3β-ol). βAMS, β-amyrin synthase; CAS1, cycloartenol synthase; LAS1, lanosterol synthase.

Figure 2. Phylogenetic tree illustrating the distribution of 2,3-oxidosqualene cyclases in plants, algae, and protists.

The distance between each sequence was calculated using the program CLUSTAL W. The phylogenetic tree was drawn using Phylodendron online tool (http://iubio.bio.indiana.edu/treeapp/treeprint-form.html). Scale represents 0.1 amino acid substitutions per site. Protein clusters are enhanced by colors as follows: red and orange, β-amyrin and promiscuous β-amyrin synthases, dark red, lupeol synthases; blue, plant lanosterol synthases; green, plant cycloartenol synthases; brown, cycloartenol synthases from algae or a planctomycete, and yellow, cycloartenol synthases from protists. Abbreviations and accession numbers are given in Table S1. All proteins displayed in the phylogenetic tree have either been functionally characterized or genome annotated (except CAS1 from Salpingoeca sp, Allium macrosternom, Avena strigosa, Avena ventricosa and Luffa cylindrica, which have been annotated based on sequence homology and/or expression evidence).

Figure 3. 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 . 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.

Figure 4. VIGS of CAS1 and LAS1 in Nicotiana benthamiana.

A, Morphological phenotype of PVX (left) and PVX::CAS1 (right) plants 4 weeks after inoculation, the close-up shows bleaching of veins. B, Morphological phenotype of PVX (left) and PVX::CAS1 (right) plants 5 weeks after inoculation, the close-up shows leaf wilting and necrosis. C, Relative gene expression in PVX::CAS1 plants, CAS1 is a measurement of the endogenous NbCAS1 level, PVX::CAS1 is a measurement of the viral NtCAS1 transcript. D, Relative gene expression in PVX::LAS1 plants, LAS1 is a measurement of the endogenous NbLAS1 level, PVX::LAS1 is a measurement of the viral NbLAS1 transcript. E, squalene epoxide amounts measured by GC-FID in silenced plants. F, sterol composition of PVX and PVX::CAS1 plants. Structure of the compounds detected here are shown in Fig. S1. The pictures in A and B are representative of 4 independent experiments that included all 3 plants inoculated with each type of viral transcripts.

Figure 5. Sterol profile determind by GC-MS of erg7 expressing a tobacco cycloartenol synthase CAS1.

A, TIC of a total unsaponifiable extract of erg7 transformed with a void vector. B, TIC of a total unsaponifiable extract of erg7::NtCAS1. C, 9β,19-cyclopropylsterol biosynthetic pathway in yeast. Compounds are: 1, 2,3-oxidosqualene; 2, ergosterol; 3, cycloartenol; 4, 31-norcycloartenol; 5, 24-dehydropollinastanol; 6, cycloeucalenol; 7, 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 , , . Peaks that are not numbered are not sterols.

Figure 6. Yeast spotting assay with ERG7 deficient gil77 or gil77::CAS1 strains.

Growth of gil77 yeast strain transformed with the pYeDP60 vector or pYeDP60-NtCAS1 was tested on SGal medium supplemented or not with 20 µg/ml of ergosterol. After 5 days at 30°C, plates supplemented with ergosterol showed fully-grown colonies, whereas inductive conditions (SGal without ergosterol) allowed only limited growth of yeast expressing NtCAS, indicating however that NtCAS1 expression partially overcomes ERG7 deficiency.