Cycloartenol Synthase Research Articles

Compounds of Betula Platyphylla Cell Suspension Cultures in Response to Fungal Elicitor

ABSTRACTTo determine which triterpenoid compounds respond to fungal elicitors, four oxidosqualene cyclase (OSC) genes involved in triterpenoid biosynthesis, namely, β-amyrin synthase (β-AS), lupeol synthase (LUS), cycloartenol synthase (CAS1 and CAS2), were investigated in cell suspension cultures of Betula platyphylla using real-time quantitative PCR. The results showed that the fungal elicitor significantly increased the mRNA levels of the four OSCs. However, the expression profile of the four studied OSCs in response to fungal elicitation was different; the CAS expression rate was about 50% in the four OSCs after 2 h to 8 h of fungal elicitation, LUS expression was about 70% after 8 h of fungal elicitation, and the LUS mRNA level and its product betulin reached their maxima (18-fold and 2.5-fold that of the control) at 24 h. This result suggests that the lupeol synthase pathway is the major source of triterpenoid production by B. platyphylla cell suspension cultures in response to fungal elicitors. In addition, the activity ofphenylalanine ammonia-lyase (PAL) and peroxidase (POD) in the fungal treated cell cultures increased, reaching their maxima after 6 h of fungal elicitation and then slowly declining thereafter. This result was in accordance with CAS gene expression and in contrast with that of LUS. Therefore, OSCs may have different roles and synergistic interactions in response to fungal elicitation.

Salt-dependent increase in triterpenoids is reversible upon transfer to fresh water in mangrove plants Kandelia candel and Bruguiera gymnorrhiza

This study examined the salinity dependence of triterpenoid content and triterpenoid synthase gene expression in mangrove plants, Kandelia candel and Bruguiera gymnorrhiza (Rhizophoraceae) after long-term exposure to salinity and subsequent re-adaptation. Seedlings of the two mangrove species grown in varying salt concentrations for 4 months were divided into two treatment groups and grown for another 4 months, one group continued under the respective saline condition and the other in fresh water for re-adaptation. The total content of triterpenoids increased with increasing salinity in roots and leaves of K. candel, but only in roots in B. gymnorrhiza. This increase was reversed to a variable extent, depending on the species and organ, after transfer to fresh water. In contrast, the total content of phytosterols showed no correlation with salinity throughout the experiment. The increase in total triterpenoids was accompanied by an up-regulation of several triterpenoid synthase genes: KcMS, a multifunctional triterpenoid synthase, in roots and leaves of K. candel and BgLUS, a lupeol synthase, and BgbAS, a β-amyrin synthase, in roots of B. gymnorrhiza. The expression of root KcCAS, a cycloartenol synthase, which is involved in phytosterol biosynthesis, was not modulated by the salinity conditions but decreased with increasing salinity in leaves, followed by the restoration to the initial level after transfer to fresh water. The concentrations of individual triterpenoids, but not of phytosterols, in the roots positively correlated with the salinity. These results reinforced the importance of triterpenoids in the adaptation of mangroves to withstand salt and/or water stress.

Effects of irrigation on fruit ripening behavior and metabolic changes in olive

Olive (Olea europaea, cv Leccino) fruits grown under different water regimes were analyzed by metabolomics and specific transcript accumulation analyses. The fruit from non-irrigated (rain-fed) and irrigated trees cultivated under field conditions, with a seasonal water amount equivalent to the calculated crop evapotranspiration (ETc) was compared in the last developmental phase and, in particular, at commercial harvest. Metabolomics (GC–MS) analysis identified several hundred metabolites in ripe mesocarp, 46 of which showed significantly different contents in the rain-fed and irrigated samples. Some compounds involved in primary metabolism (carbohydrates, amino acids, organic acids) and secondary metabolism (squalene, simple phenols) appeared to be more abundant when irrigation was performed. Higher levels of total polyphenol were observed in the rain-fed fruit, which at ripening showed an increase in anthocyanin concentration. These data indicate that ripening in olives is affected by irrigation. In addition, expression analyses of three key polyphenol biosynthetic genes (phenylalanine ammonia lyase (PAL), chalcone synthase (CHS), dihydroflavonol reductase (DFR)) and two genes involved in triterpenoid metabolism (β-amyrin synthase and cycloartenol synthase) were also performed. The analyzed genes showed different expression patterns throughout ripening, and the resulting PAL, DFR and β-amyrin synthase transcript accumulation was found to be affected by the different water regimes at specific stages of fruit development.

Cloning and bioinformatics analysis of cycloartenol synthase (HcCAS1) gene in Huperzia carinata

To clone and sequence the open reading frame of cycloartenol synthase (CAS) from Huperzia carinata. After searching the transcriptome dataset of H. carinata, one unique sequence containing oxide squalene cyclases domain was discovered. The primers were designed according to the cDNA sequence of CAS from the dataset. And then, the open reading frame of CAS was cloned by RT-PCR strategy with the template of mixed RNA extracted from root, stem and leaf of H. carinata. The bioinformatic analysis of this gene and its corresponding protein was performed. One unique sequence of CAS, named as HcCAS1 (GenBank accession number JN790125) , was cloned from H. carinata. The open reading frame of HcCAS1 consists of 2 474 bp, encoding one polypeptide with 757 amino acids. This study cloned and analyzed CAS from H. carinata for the first time. The result will provide a foundation for exploring the mechanism of sterol biosynthesis in Huperziaceae plants.

Molecular cloning and characterization of a cDNA encoding cycloartenol synthase from Fritillaria thunbergii Miq.

Fritillaria thunbergii Miq., known as the bulbous plants of the genus fritillaria , produces a large amount of sterols. Homology based polymerase chain reactions (PCRs) with degenerate primers designed from the conserved sequences among the known cycloartenol synthase (CAS) resulted in cloning of a CAS from the young leaves of F. thunbergii Miq . A putative cycloartenol synthase cDNA ( FtCAS ) consists of a 2271 bp open reading frame, which encodes for 756 amino acids. The deduced amino acid sequence shows 82% homology to cycloartenol synthases from Dioscorea zingiberensis . Heterologous expression of the FtCAS in the methylotrophic yeast, Pichia pastoris , resulted in production of cycloartenol. Our results indicate that FtCA S encode cycloartenol synthase.

Biochemical and molecular analysis of Ginsenoside biosynthesis in Panax ginseng during flower and berry development

Panax ginseng produces a large number of ginsenosides throughout the parts of the ginseng plant. Several genes in the ginsenoside biosynthesis pathway were cloned, including those encoding farnesyl diphosphate synthase, squalene synthase, squalene epoxidase, dammarenediol-II synthase, lanosterol synthase, β- amyrin synthase, and cycloartenol synthase. The expression levels of these seven genes were examined in the roots and different developmental stages of the flowers and berries of 4-year-old ginseng plants using quantitative real-time polymerase chain reaction. The levels of ten ginsenosides (Rb1, Rb2, Rb3, Rc, Rd, Re, Rf, Rg1, Rg2, and Rh1) were determined by high performance liquid chromatography analysis. Gene expression varied during flower and berry development. Total saponin levels were highest in the early berry stage and correlated well with those in the ginseng root. Therefore, berries should be harvested at the early berry stage to ensure optimal ginsenoside levels for pharmaceutical interest.

Molecular cloning and characterization of a cDNA encoding cycloartenol synthase from Fritillaria thunbergii Miq.

Fritillaria thunbergii Miq., known as the bulbous plants of the genus fritillaria , produces a large amount of sterols. Homology based polymerase chain reactions (PCRs) with degenerate primers designed from the conserved sequences among the known cycloartenol synthase (CAS) resulted in cloning of a CAS from the young leaves of F. thunbergii Miq. A putative cycloartenol synthase cDNA (FtCAS) consists of a 2271 bp open reading frame, which encodes for 756 amino acids. The deduced amino acid sequence shows 82% homology to cycloartenol synthases from Dioscorea zingiberensis. Heterologous expression of the FtCAS in the methylotrophic yeast, Pichia pastoris , resulted in production of cycloartenol. Our results indicate that FtCAS encode cycloartenol synthase. Key words : Fritillaria thunbergii Miq., cycloartenol synthase, cDNA cloning, 2,3-oxidosqualene cyclase.

Asymmetric somatic hybridization between Bupleurum scorzonerifolium Willd. and Taxus chinensis var. mairei

In an attempt to transfer the genetic components needed for taxol synthesis into a more tractable plant, protoplasts of Taxus chinensis var. mairei were UV irradiated for various times prior to their fusion with protoplasts of Bupleurum scorzonerifolium. Of the 60 presumptive hybrid calli obtained, selections of the 9 most rapidly growing were recognized as hybrid clones based on a combination of their callus morphology, esterase profile, chromosome number, and RAPD genotype. The RAPD data showed that 82.4-96.8% of the hybrid genome was inherited from the recipient (B. scorzonerifolium) and 4.6-13.9% from the donor (T. chinensis), with 1.6-6.9% of the RAPD fragments being not present in either parent. None of the hybrid clones expressed a detectable quantity of taxol, but four produced more of the triterpenoid oleanolic acid than did calli of the recipient. With the use of semi-quantitative reverse transcriptase PCR, it was possible to show variation in the expression of several triterpenoid biosynthetic pathway related genes between B. scorzonerifolium and the hybrids. Increasing the concentration of oleanolic acid requires that the expression levels of the genes encoding IPP isomerase, squalene synthase, squalene epoxidase and β-amyrin synthase are increased, while at the same time, those of the genes encoding the branching enzymes cycloartenol synthase and lupeol synthase need to be reduced.

Investigation of the potential for triterpene synthesis in rice through genome mining and metabolic engineering.

The first committed step in sterol biosynthesis in plants involves the cyclization of 2,3-oxidosqualene by the oxidosqualene cyclase (OSC) enzyme cycloartenol synthase. 2,3-Oxidosqualene is also a precursor for triterpene synthesis. Antimicrobial triterpenes are common in dicots, but seldom found in monocots, with the notable exception of oat. Here, through genome mining and metabolic engineering, we investigate the potential for triterpene synthesis in rice. The first two steps in the oat triterpene pathway are catalysed by a divergent OSC (AsbAS1) and a cytochrome P450 (CYP51). The genes for these enzymes form part of a metabolic gene cluster. To investigate the origins of triterpene synthesis in monocots, we analysed systematically the OSC and CYP51 gene families in rice. We also engineered rice for elevated triterpene content. We discovered a total of 12 OSC and 12 CYP51 genes in rice and uncovered key events in the evolution of triterpene synthesis. We further showed that the expression of AsbAS1 in rice leads to the accumulation of the simple triterpene, β-amyrin. These findings provide new insights into the evolution of triterpene synthesis in monocots and open up opportunities for metabolic engineering for disease resistance in rice and other cereals.

A Paris Polyphylla Var. Yunnanensis Gene Encoding Cycloartenol Synthase Isolated and Introduced Into Tomato by Agrobacterium-Mediated Transformation

A Paris Polyphylla Var. Yunnanensis Gene Encoding Cycloartenol Synthase Isolated and Introduced Into Tomato by Agrobacterium-Mediated Transformation

Gene regulation patterns in triterpene biosynthetic pathway driven by overexpression of squalene synthase and methyl jasmonate elicitation in Bupleurum falcatum

The root of Bupleurum falcatum L. (Apiaceae) has long been one of the most important traditional herbal medicines in Asian countries. A group of triterpene saponins (saikosaponins) are the major constituents of this plant. Squalene synthase (SS) may play a regulatory role in directing triterpene intermediates and sterol pathways. Here, we investigated the regulatory role of the squalene synthase (BfSS1) gene in the biosynthesis of phytosterol and triterpene in B. falcatum. BfSS1 mRNA accumulated ubiquitously in plant organs and markedly increased in roots after treatment with methyl jasmonate (MeJA), ABA and ethephon. Transgenic B. falcatum constructs overexpressing BfSS1 in the sense and antisense orientations were assembled using the Agrobacterium-mediated method. Transgenic roots overexpressing BfSS1 in the sense orientation resulted in enhanced production of both phytosterol and saikosaponins. Overexpression of the BfSS1 gene in the sense orientation increased the mRNA accumulation of downstream genes such as squalene epoxidase and cycloartenol synthase but unexpectedly decreased the mRNA levels of β-amyrin synthase (β-AS), a triterpene synthase mRNA. MeJA treatment of wild-type roots strongly stimulated β-AS mRNA accumulation and saikosaponin production but suppressed phytosterol production. MeJA treatment of transgenic roots overexpressing BfSS1 in the sense orientation failed to stimulate β-AS mRNA accumulation but still enhanced saikosaponin and phytosterol production. These results indicate that overexpression of BfSS1 in B. falcatum regulates more powerfully the downstream genes than elicitor (MeJA) treatment in triterpene and phytosterol biosynthesis.

Cloning and Characterization of Oxidosqualene Cyclases from Kalanchoe daigremontiana: ENZYMES CATALYZING UP TO 10 REARRANGEMENT STEPS YIELDING FRIEDELIN AND OTHER TRITERPENOIDS

The first committed step in triterpenoid biosynthesis is the cyclization of oxidosqualene to polycyclic alcohols or ketones C(30)H(50)O. It is catalyzed by single oxidosqualene cyclase (OSC) enzymes that can carry out varying numbers of carbocation rearrangements and, thus, generate triterpenoids with diverse carbon skeletons. OSCs from diverse plant species have been cloned and characterized, the large majority of them catalyzing relatively few rearrangement steps. It was recently predicted that special OSCs must exist that can form friedelin, the pentacyclic triterpenoid whose formation involves the maximum possible number of rearrangement steps. The goal of the present study, therefore, was to clone a friedelin synthase from Kalanchoe daigremontiana, a plant species known to accumulate this triterpenoid in its leaf surface waxes. Five OSC cDNAs were isolated, encoding proteins with 761-779 amino acids and sharing between 57.4 and 94.3% nucleotide sequence identity. Heterologous expression in yeast and GC-MS analyses showed that one of the OSCs generated the steroid cycloartenol together with minor side products, whereas the other four enzymes produced mixtures of pentacyclic triterpenoids dominated by lupeol (93%), taraxerol (60%), glutinol (66%), and friedelin (71%), respectively. The cycloartenol synthase was found expressed in all leaf tissues, whereas the lupeol, taraxerol, glutinol, and friedelin synthases were expressed only in the epidermis layers lining the upper and lower surfaces of the leaf blade. It is concluded that the function of these enzymes is to form respective triterpenoid aglycones destined to coat the leaf exterior, probably as defense compounds against pathogens or herbivores.

Upregulation of phytosterol and triterpene biosynthesis in Centella asiatica hairy roots overexpressed ginseng farnesyl diphosphate synthase

Farnesyl diphosphate synthase (FPS) plays an essential role in organ development in plants. However, FPS has not previously been identified as a key regulatory enzyme in triterpene biosynthesis. To elucidate the functions of FPS in triterpene biosynthesis, C. asiatica was transformed with a construct harboring Panax ginseng FPS (PgFPS)-encoding cDNA coupled to the cauliflower mosaic virus 35S promoter. Higher levels of CaDDS (C. asiatica dammarenediol synthase) and CaCYS (C. asiatica cycloartenol synthase) mRNA were detected in all hairy root lines overexpressing when compared with the controls. However, no differences were detected in any expression of the CaSQS (C. asiatica squalene synthase) gene. In particular, the upregulation of CaDDS transcripts suggests that FPS may result in alterations in triterpene biosynthesis capacity. Squalene contents in the T17, T24, and T27 lines were increased to 1.1-, 1.3- and 1.5-fold those in the controls, respectively. The total sterol contents in the T24 line were approximately three times higher than those of the controls. Therefore, these results indicated that FPS performs a regulatory function in phytosterol biosynthesis. To evaluate the contribution of FPS to triterpene biosynthesis, we applied methyl jasmonate as an elicitor of hairy roots expressing PgFPS. The results of HPLC analysis revealed that the content of madecassoside and asiaticoside in the T24 line was transiently increased by 1.15-fold after 14 days of MJ treatment. This result may indicate that FPS performs a role not only in phytosterol regulation, but also in triterpene biosynthesis.

Regulation of ginsenoside and phytosterol biosynthesis by RNA interferences of squalene epoxidase gene in Panax ginseng

Squalene epoxidase catalyzes the first oxygenation step in phytosterol and triterpenoid saponin biosynthesis and is suggested to represent one of the rate-limiting enzymes in this pathway. Here, we investigated the roles of two squalene epoxidase genes ( PgSQE1 and PgSQE2) in triterpene and phytosterol biosynthesis in Panax ginseng. PgSQE1 and PgSQE2 encoded deduced proteins of 537 and 545 amino acids, respectively. Amino acid sequences deduced from PgSQE1 and PgSQE2 share 83% homology, but the N-terminal regions (first 60 amino acids) are highly different. PgSQE1 mRNA abundantly accumulated in all organs. PgSQE2 was only weakly expressed and preferentially in petioles and flower buds. Methyl jasmonate (MeJA) treatment enhanced the accumulation of PgSQE1 mRNA in roots, but rather suppressed expression of PgSQE2. Precursor (squalene) treatment coordinately upregulated the expression of both PgSQE1 and PgSQE2. In situ hybridization analysis established that both PgSQE1 and PgSQE2 mRNAs accumulated preferentially in vascular bundle tissue and resin ducts of petioles. RNA interference of PgSQE1 in transgenic P. ginseng completely suppressed PgSQE1 transcription. Concomitantly, the interference of PgSQE1 resulted in reduction of ginsenoside production. Interestingly, silencing of PgSQE1 in RNAi roots strongly upregulated PgSQE2 and PNX (cycloartenol synthase) and resulted in enhanced phytosterol accumulation. These results indicate that expression of PgSQE1 and PgSQE2 were regulated in a different manner, and that PgSQE1 will regulate ginsenoside biosynthesis, but not that of phytosterols in P. ginseng.

인삼 모상근의 생장과 Ginsenoside 생합성에 미치는 석결명의 영향

인삼 모상근의 생장과 ginsenosides의 함량을 증가시키기 위하여 생장조절제가 첨가되지 않은 1/2 MS 배지에 석결명의 농도와 처리시기를 달리하여 인삼 모상근 KGHR-8 세포주를 30일간 배양하였다. 실험 결과, 고체배양에서 10 mg/L 석결명을 첨가하였을 때 생장량이 가장 높으며, 300 mg/L 이상 처리구는 대조군에 비해 생장이 낮았다. 액체배양시 10, 15 mg/L 석결명을 첨가하였을 때 생장량이 증가하였고 ginsenoside 함량은 10, 15 mg/L 처리구에서 각각 8.8%, 11.8% 증가를 보였다. 석결명의 최적 처리시점을 구명하고자 첨가시점을 달리하여 인삼 모상근의 생장량을 조사한 결과, 대조군보다 14일 후 10 mg/L 석결명을 접종한 처리구에서 생체중량 22.9%, 건조중량 20.7%으로 가장 높은 인삼 모상근의 생장 증가를 보였다. 또한, 광조사 효과를 조사하기 위해 암실과 광조사 처리구로 나누어 30일간 배양한 결과, 대조군과 비교하여 광조사 처리구에서는 18% 정도 생장량이 증가하였고 암실 처리구에서 거의 차이가 없었다. RT-PCR 결과에서는 10, 20 mg/L 석결명 처리구가 대조군에 비해 사포닌 생합성 관련 유전자인 squalene synthase, squalene epoxidase, dammarenediol synthase, cycloartenol synthase 및 <TEX>$\beta$</TEX>-amyrin synthase의 전사량이 증가하였다. Bioreactor (2L)를 이용하여 인삼 모상근을 배양한 결과 처리구가 대조군에 비해 5% 증가를 보였으며, ginsenosides 함량도 2.63 g으로 대조군에 비해 증가하였다. 따라서 석결명은 이차 대사산물인 사포닌의 생산에 영향을 미치는 elicitor로써 역할을 하는 것으로 판단된다. In order to investigate the effects of elicitors on the growth and ginsenoside biosynthesis of ginseng hairy roots, we treated Panax ginseng hairy root with various concentrations of Haliotidis concha according to different time course. Haliotidis concha supplement increased the biomass and ginsenoside accumulation at 10 mg/L concentration. The growth rate of hairy root under a lighter concentration was greater than hairy root treated with a denser concentration. The highest content and productivity of ginsenosides appeared at 2 weeks after the treatment of 10 mg/L Haliotidis concha. The gene expression of squalene synthase, squalene epoxidase, dammarenediol synthase, cycloartenol synthase, <TEX>$\beta$</TEX>-amyrin synthase in hairy roots of ginseng were examined by RT-PCR. The Haliotidis concha treatment resulted in the obvious accumulation of the mRNA of triterpene biosynthesis in Panax ginseng hairy root as compared with the control. In this study, Haliotidis concha acts as a kind of elicitor for the production of ginsenosides.

Upregulation of ginsenoside and gene expression related to triterpene biosynthesis in ginseng hairy root cultures elicited by methyl jasmonate

In this study, methyl jasmonate (MJ)-induced changes of triterpene saponins in ginseng (Panax ginseng C.A. Meyer) hairy roots and expression profiling of relevant responsive genes were analyzed. The transcription of PgSS (squalene synthase), PgSE (squalene epoxidase), and PNA (dammarenediol synthase-II) genes in hairy root cultures elicited by MJ treatment increased as compared with the controls, whereas that of PNX (cycloartenol synthase) decreased slightly. In order to select candidate genes encoding for cytochrome P450-dependent hydroxylase or glucosyltransferase associated with triterpene biosynthesis, RT-PCR analysis was conducted following MJ elicitation. No differences were observed in any expression among the five genes associated with the cytochrome P450 family, when compared to that of control. For candidates of the glucosyltransferase gene,expression of EST IDs PG07020C06, PG07025D04, and PG07029G02 was upregulated. In an effort to assess the effects of MJ elicitation on the biosynthesis of triterpene saponin, protopanaxadiol saponin (Rb group) and protopanaxatriol saponin (Rg group) contents in hairy roots were evaluated by HPLC analysis. With 7 days of MJ elicitation, levels of all ginseonsides of the two-groups increased much higher than that observed in the control. In particular, protopanaxadiol-type saponin contents increased by 5.5–9.7 times that of the control, whereas protopanaxatriol-type saponin contents were increased by 1.85–3.82-fold. In the case of Rg1 ginsenoside after MJ elicitation, the content was affected negatively in ginseng hairy root cultures.

Antisense Suppression of Cycloartenol Synthase Results in Elevated Ginsenoside Levels in Panax ginseng Hairy Roots

In Panax ginseng, both ginsenosides and phytosterols are derived from the precursor 2,3-oxidosqualene. The enzymes cycloartenol synthase (CS) and dammarenediol synthase (DS) are responsible for ginsenoside and phytosterol biosynthesis, respectively. Here, we report on enhanced ginsenoside levels detected in P. ginseng hairy roots, following Agrobacterium rhizogenes transformation, by antisense suppression of the CS gene. Several antisense hairy root transgenic lines were recovered exhibiting markedly reduced levels of the CS transcript and corresponding CS enzymatic activity relative to control. Phytosterol levels in tested antisense transgenic lines were ∼50% lower than those of control, whereas 2,3-oxidosqualene contents in tested antisense lines were higher than those in control lines during the early period of root growth; however, these levels were comparable in both groups at a later stage of root growth. Ginsenoside levels were comparable in both groups during the early stage of root growth; however, the final total ginsenoside contents in antisense lines were about 50–100% higher than those found in control roots. Interestingly, the DS enzyme activity of antisense-CS lines was higher than that of controls. It is proposed that in P. ginseng, the regulation of CS can control the metabolic flux from 2,3-oxidosqualene to both phytosterol and ginsenoside.

Dual biosynthetic pathways to phytosterol via cycloartenol and lanosterol in Arabidopsis

The differences between the biosynthesis of sterols in higher plants and yeast/mammals are believed to originate at the cyclization step of oxidosqualene, which is cyclized to cycloartenol in higher plants and lanosterol in yeast/mammals. Recently, lanosterol synthase genes were identified from dicotyledonous plant species including Arabidopsis, suggesting that higher plants possess dual biosynthetic pathways to phytosterols via lanosterol, and through cycloartenol. To identify the biosynthetic pathway to phytosterol via lanosterol, and to reveal the contributions to phytosterol biosynthesis via each cycloartenol and lanosterol, we performed feeding experiments by using [6-(13)C(2)H(3)]mevalonate with Arabidopsis seedlings. Applying (13)C-{(1)H}{(2)H} nuclear magnetic resonance (NMR) techniques, the elucidation of deuterium on C-19 behavior of phytosterol provided evidence that small amounts of phytosterol were biosynthesized via lanosterol. The levels of phytosterol increased on overexpression of LAS1, and phytosterols derived from lanosterol were not observed in a LAS1-knockout plant. This is direct evidence to indicate that the biosynthetic pathway for phytosterol via lanosterol exists in plant cells. We designate the biosynthetic pathway to phytosterols via lanosterol "the lanosterol pathway." LAS1 expression is reported to be induced by the application of jasmonate and is thought to have evolved from an ancestral cycloartenol synthase to a triterpenoid synthase, such as beta-amyrin synthase and lupeol synthase. Considering this background, the lanosterol pathway may contribute to the biosynthesis of not only phytosterols, but also steroids as secondary metabolites.

Albinism and cell viability in cycloartenol synthase deficient Arabidopsis

Phenotypes of Arabidopsis thaliana that carry mutations in CYCLOARTENOL SYNTHASE 1 (CAS1) which is required in sterol biosynthesis have been described. Knockout mutant alleles are responsible of a male-specific transmission defect. Plants carrying a weak mutant allele cas1-1 accumulate 2,3-oxidosqualene, the substrate of CAS1, in all analyzed organs. Mutant cas1-1 plants develop albino inflorescence shoots that contain low amount of carotenoids and chlorophylls. The extent of this albinism, which affects Arabidopsis stems late in development, may be modulated by the light/dark regime. The fact that chloroplast differentiation and pigment accumulation in inflorescence shoots are associated with a low CAS1 expression could suggest the involvement of 2,3-oxidosqualene in a yet unknown regulatory mechanism linking the sterol biosynthetic segment, located in the cytoplasm, and the chlorophyll and carotenoid biosynthetic segments, located in the plastids, in the highly complex terpenoid network. CAS1 loss of function in a mosaic analysis of seedlings further demonstrated that leaf albinism associated with an accumulation of 2,3-oxidosqualene is a novel phenotype for plant sterol deficient mutant.Addendum to: Babiychuk E, Bouvier-Navé P, Compagnon V, Suzuki M, Muranaka T, Van Montagu M, Kushnir S, Schaller H. Allelic mutant series reveal distinct functions for Arabidopsis cycloartenol synthase 1 in cell viability and plastid biogenesis. Proc Natl Acad Sci USA 2008; 105:3163-8.

Allelic mutant series reveal distinct functions for Arabidopsis cycloartenol synthase 1 in cell viability and plastid biogenesis

Sterols have multiple functions in all eukaryotes. In plants, sterol biosynthesis is initiated by the enzymatic conversion of 2,3-oxidosqualene to cycloartenol. This reaction is catalyzed by cycloartenol synthase 1 (CAS1), which belongs to a family of 13 2,3-oxidosqualene cyclases in Arabidopsis thaliana. To understand the full scope of sterol biological functions in plants, we characterized allelic series of cas1 mutations. Plants carrying the weak mutant allele cas1-1 were viable but developed albino inflorescence shoots because of photooxidation of plastids in stems that contained low amounts of carotenoids and chlorophylls. Consistent with the CAS1 catalyzed reaction, mutant tissues accumulated 2,3-oxidosqualene. This triterpenoid precursor did not increase at the expense of the pathway end products. Two strong mutations, cas1-2 and cas1-3, were not transmissible through the male gametes, suggesting a role for CAS1 in male gametophyte function. To validate these findings, we analyzed a conditional CRE/loxP recombination-dependent cas1-2 mutant allele. The albino phenotype of growing leaf tissues was a typical defect observed shortly after the CRE/loxP-induced onset of CAS1 loss of function. In the induced cas1-2 seedlings, terminal phenotypes included arrest of meristematic activity, followed by necrotic death. Mutant tissues accumulated 2,3-oxidosqualene and contained low amounts of sterols. The vital role of sterols in membrane functioning most probably explains the requirement of CAS1 for plant cell viability. The observed impact of cas1 mutations on a chloroplastic function implies a previously unrecognized role of sterols or triterpenoid metabolites in plastid biogenesis.