Arabidopsis thaliana Squalene Epoxidase 1 Is Essential for Root and Seed Development
Squalene epoxidase converts squalene into oxidosqualene, the precursor of all known angiosperm cyclic triterpenoids, which include membrane sterols, brassinosteroid phytohormones, and non-steroidal triterpenoids. In this work, we have identified six putative Arabidopsis squalene epoxidase (SQE) enzymes and used heterologous expression in yeast to demonstrate that three of these enzymes, SQE1, SQE2, and SQE3, can epoxidize squalene. We isolated and characterized Arabidopsis sqe1 mutants and discovered severe developmental defects, including reduced root and hypocotyl elongation. Adult sqe1–3 and sqe1–4 plants have diminished stature and produce inviable seeds. The sqe1–3 mutant accumulates squalene, consistent with a block in the triterpenoid biosynthetic pathway. Therefore, SQE1 function is necessary for normal plant development, and the five SQE-like genes remaining in this mutant are not fully redundant with SQE1. Squalene epoxidase converts squalene into oxidosqualene, the precursor of all known angiosperm cyclic triterpenoids, which include membrane sterols, brassinosteroid phytohormones, and non-steroidal triterpenoids. In this work, we have identified six putative Arabidopsis squalene epoxidase (SQE) enzymes and used heterologous expression in yeast to demonstrate that three of these enzymes, SQE1, SQE2, and SQE3, can epoxidize squalene. We isolated and characterized Arabidopsis sqe1 mutants and discovered severe developmental defects, including reduced root and hypocotyl elongation. Adult sqe1–3 and sqe1–4 plants have diminished stature and produce inviable seeds. The sqe1–3 mutant accumulates squalene, consistent with a block in the triterpenoid biosynthetic pathway. Therefore, SQE1 function is necessary for normal plant development, and the five SQE-like genes remaining in this mutant are not fully redundant with SQE1. Plants are estimated to produce more than 500,000 secondary metabolites (1Hadacek F. CRC Crit. Rev. Plant Sci. 2002; 21: 273-322Crossref Scopus (174) Google Scholar). These compounds have many functions, including attracting pollinators, communicating with neighboring plants, and defending against pathogens and herbivores (2Swain T. Annu. Rev. Plant Physiol. 1977; 28: 479-501Crossref Google Scholar, 3Pichersky E. Gershenzon J. Curr. Opin. Plant Biol. 2002; 5: 237-243Crossref PubMed Scopus (818) Google Scholar). The importance of secondary metabolites is highlighted by the extensive resources that plants invest in producing these compounds. Although once thought to be metabolically simple, more than 170 secondary metabolites have been identified in Arabidopsis thaliana (reviewed in Ref. 4D'Auria J.C. Gershenzon J. Curr. Opin. Plant Biol. 2005; 8: 308-316Crossref PubMed Scopus (206) Google Scholar). Triterpenoids are the 30-carbon subset of terpenoids, the largest class of secondary metabolites. Triterpenoid biosynthesis is diagramed in Fig. 1. Isopentenyl diphosphate and dimethylallyl diphosphate are synthesized from mevalonate and oligomerized to farnesyl diphosphate by farnesyl diphosphate synthase (FPS). 5The abbreviations used are: FPS, farnesyl diphosphate synthase; BR, brassinosteroid; cDNA, complementary DNA; GC-MS, gas chromatography-mass spectrometry; PN, plant nutrient; RT, reverse transcription; SQE, squalene epoxidase; T-DNA, transfer-DNA; TUB, tubulin. 5The abbreviations used are: FPS, farnesyl diphosphate synthase; BR, brassinosteroid; cDNA, complementary DNA; GC-MS, gas chromatography-mass spectrometry; PN, plant nutrient; RT, reverse transcription; SQE, squalene epoxidase; T-DNA, transfer-DNA; TUB, tubulin. Farnesyl diphosphate is dimerized to squalene by squalene synthase. Squalene epoxidase (SQE)-mediated oxidation then produces oxidosqualene, which triterpene synthases cyclize to >80 triterpene skeletons (5Xu R. Fazio G.C. Matsuda S.P.T. Phytochemistry. 2004; 65: 261-291Crossref PubMed Scopus (435) Google Scholar, 6Phillips D.R. Rasbery J.M. Bartel B. Matsuda S.P.T. Curr. Opin. Plant Biol. 2006; 9: 305-314Crossref PubMed Scopus (292) Google Scholar). Further metabolism of these compounds produces membrane sterols, brassinosteroid phytohormones, saponins, other defense compounds, cuticular waxes, and numerous triterpenoids that have not been functionally characterized. The yeasts and mammals that have been investigated each encode a single squalene epoxidase. In contrast, several plants have multiple genes predicted to encode squalene epoxidases, a diversity suggesting that this step may be subject to additional or unique regulation in plants. Two Medicago truncatula SQE enzymes have been biochemically characterized (7Suzuki H. Achnine L. Xu R. Matsuda S.P.T. Dixon R.A. Plant J. 2002; 32: 1033-1048Crossref PubMed Scopus (235) Google Scholar). The Brassica napus (8Schafer U.A. Reed D.W. Hunter D.G. Yao K. Weninger A.M. Tsang E.W. Reaney M.J. MacKenzie S.L. Covello P.S. Plant Mol. Biol. 1999; 39: 721-728Crossref PubMed Scopus (22) Google Scholar), Populus trichocarpa, and Oryza sativa genomes each have multiple predicted SQE enzymes. Despite the likely importance of SQE to plant growth and development, no plant mutants with defects in these enzymes have been reported. In this work, we heterologously expressed the six Arabidopsis putative SQE enzymes in Saccharomyces cerevisiae lacking squalene epoxidase to determine which have squalene epoxidase activity. We isolated Arabidopsis sqe1 loss-of-function mutants and found that these mutants display severe developmental defects and accumulate squalene. Our results demonstrate that SQE1 is particularly important for oxidosqualene production in the roots and reproductive tissues of Arabidopsis. Chemicals—Bis(trimethylsilyl)trifluoroacetamide, pyridine, and potassium hydroxide were from Aldrich Chemical Company (Milwaukee, WI). Lovastatin (mevinolin), squalene, epibrassinolide, hemin, ergosterol, raffinose, TRI Reagent, and cholesterol ethyl ether were from Sigma-Aldrich (St. Louis, MO). Methanol, methyl t-butyl ether, dichloromethane, and hexane (OmniSolv grade) were from EM Science (Gibbstown, NJ). Ammonium glufosinate (Basta) was from Sigma-Aldrich and Crescent Chemical (Augsburg, Germany). Sitosterol was obtained from wild-type (Col-0, Columbia) Arabidopsis plant extracts by high pressure liquid chromatography purification and was identified by NMR and GC-MS. Oxidosqualene was synthesized according to previously published procedures (9Nadeau R.G. Hanzlik R.P. Methods Enzymol. 1968; 15: 346-351Crossref Scopus (63) Google Scholar). Yeast Expression—SQE1 (At1g58440), SQE3 (At4g37760), SQE5 (At5g24150), and SQE6 (At5g24160) were PCR-amplified from an Arabidopsis cDNA library in the pSPORT vector (10LeClere S. Bartel B. Plant Mol. Biol. 2001; 46: 695-703Crossref PubMed Scopus (70) Google Scholar). The SQE2 (At2g22830) cDNA was reverse-transcribed and PCR-amplified from Arabidopsis mRNA using a RETRO-script kit (Ambion). A full-length SQE4 (At5g24140) cDNA derived from a λ-PRL2 cDNA library (11Newman T. de Bruijn F.J. Green P. Keegstra K. Kende H. McIntosh L. Ohlrogge J. Raikhel N. Somerville S. Thomashow M. Retzel E. Somerville C. Plant Physiol. 1994; 106: 1241-1255Crossref PubMed Scopus (558) Google Scholar) was obtained from the Arabidopsis Biological Resource Center (clone 129F12T7). The Arabidopsis cDNAs and the yeast ERG1 coding sequence were subcloned into the galactose-inducible yeast expression vector pRS426Gal (12Hart E.A. Hua L. Darr L.B. Wilson W.K. Pang J. Matsuda S.P.T. J. Am. Chem. Soc. 1999; 121: 9887-9888Crossref Scopus (94) Google Scholar) cut with SalI and NotI or SacII and NotI (for SQE3). Inserts were sequenced to ensure that cDNAs were free of PCR-derived errors. S. cerevisiae strains were RXY6 (MATa erg7::HIS3 erg1::Kan-MX4 hem1::TRP1 ura3-52 trp1-Δ63 leu2–3,112 his3-Δ200 ade2 Gal+) (13Fazio G.C. Xu R. Matsuda S.P.T. J. Am. Chem. Soc. 2004; 126: 5678-5679Crossref PubMed Scopus (72) Google Scholar) and RXY6.2, an oxidosqualene auxotroph created by transforming RXY6 with pSM61.21, a LEU2-integrating plasmid carrying ScERG7 driven from the GAL1 promoter (14Corey E.J. Matsuda S.P.T. Baker C.H. Ting A.Y. Cheng H. Biochem. Biophys. Res. Commun. 1996; 219: 327-331Crossref PubMed Scopus (69) Google Scholar). The hem1 lesion in this strain allows the import of sterol under aerobic conditions (15Gollub E.G. Liu K. Dayan J. Adlersberg M. Sprinson D.B. J. Biol. Chem. 1977; 252: 2846-2854Abstract Full Text PDF PubMed Google Scholar). Transformants were selected for uracil prototrophy at 30 °C on synthetic medium lacking uracil (16Ausubel F.M. Brent R. Kingston R.E. Moore D.D. Seidman J.G. Smith J.A. Struhl K. Current Protocols in Molecular Biology. Wiley-Interscience, New York1999: 13.0.1-13.13.7Google Scholar) and with hemin, ergosterol, and and with were for prototrophy by growth on synthetic medium lacking uracil and with raffinose, hemin, and RXY6 were for oxidosqualene by in synthetic medium lacking uracil and with raffinose, hemin, and were by in of and by with Squalene and oxidosqualene in the extracts were by chromatography in ether and with from (MATa erg7::HIS3 hem1::TRP1 leu2–3,112 his3-Δ200 ade2 Gal+) (14Corey E.J. Matsuda S.P.T. Baker C.H. Ting A.Y. Cheng H. Biochem. Biophys. Res. Commun. 1996; 219: 327-331Crossref PubMed Scopus (69) Google Scholar) and from with or were for oxidosqualene production using GC-MS. were by for at 30 °C in of synthetic medium lacking uracil (16Ausubel F.M. Brent R. Kingston R.E. Moore D.D. Seidman J.G. Smith J.A. Struhl K. Current Protocols in Molecular Biology. Wiley-Interscience, New York1999: 13.0.1-13.13.7Google Scholar) and with raffinose, hemin, ergosterol, and which was in the medium were with and in of and at °C for The were to to and were three with of The hexane extracts were under a and in of to which was using the °C Plant and sqe1–3 were isolated from the J.M. H. P. J. P. R. C. C. E. H. L. N. H. M. N. E. M. C. R. P. L. M. T. E. PubMed Scopus Google Scholar) in the sqe1–4 was isolated from the R.A. Sci. S. PubMed Scopus Google Scholar, P. T. S. C. H. R.A. 9: PubMed Scopus Google Scholar) in the plants were identified by from a or with to the the wild-type or to a including the of the the mutant were by the the and the The mutant was identified by with a and which a The SQE1 was identified by with and which a The mutant was identified by with and which a The SQE1 was identified by with and which a The sqe1–3 mutant was identified by with and which a and the SQE1 was identified by with and which a sqe1–4 mutants were identified using an and which a and the SQE1 was identified by with and which a Plant were in a with for with and on plant medium Somerville C. Mol. Scopus Google Scholar) with to and with under at °C were to and under at were from medium to liquid were the liquid by roots a in a of the of the hypocotyl from an were in the for to the at °C for five additional were and were to medium for an additional in the of was used to determine the or of each root from an were on medium at °C for were and of was used to determine the of each and and and from and sqe1–3 were for was isolated using TRI according to the with reverse used a and of the cDNA was using the sqe1–3 the of the and SQE1 of the and SQE2, and SQE3, and and SQE4 and and and and sqe1–3 SQE1, and SQE5 cDNAs were from the using SalI and NotI and subcloned into the (10LeClere S. Bartel B. Plant Mol. Biol. 2001; 46: 695-703Crossref PubMed Scopus (70) Google Scholar) vector cut with and were into C. J. Mol. Scopus Google Scholar), which was used to plants using the Plant J. PubMed Google Scholar). Transformants were selected on medium with mutants were identified in the or using and for the were selected by the of glufosinate in the and were using a Plant and tissues from sqe1–3 and on medium were for these plants were selected from the of a by the root at were selected from plants, and for and were selected from plants. from or and wild-type plants was used for plant and root for and root for and root for and root for and root for and root for was with for were under a with and extracts in were of and of root for of and of root for In extracts were of and of root for of and of root for In extracts were of and of root for of and of root for of the extracts of of was for of cholesterol ethyl ether to Sitosterol was in this for an to for purification for squalene the of the extracts in of of was to in for for These were at °C under for The were then to to and with methyl t-butyl ether for for and with the was of the extracts were using of of with of cholesterol ethyl ether to purification was on from the squalene was and more was The remaining of the extracts were in of and of with were with of and methyl t-butyl ether in and methyl t-butyl ether, and all were to and squalene, and and ether were by of with of at by for for and were on an to an with a 30 were at °C in was at were with at and a °C a of Two were In the the was at °C for at to by an at to and then at °C for at for squalene, at for cholesterol ethyl ether, and at for The used an of °C at for squalene, at for cholesterol ethyl ether, and at for were identified by and with of of squalene and were by to the cholesterol ethyl ether, on Arabidopsis Squalene putative squalene epoxidase genes were identified in the Arabidopsis using a E.W. J. Mol. Biol. PubMed Scopus Google Scholar) with the characterized Medicago SQE enzymes (7Suzuki H. Achnine L. Xu R. Matsuda S.P.T. Dixon R.A. Plant J. 2002; 32: 1033-1048Crossref PubMed Scopus (235) Google Scholar). We identified additional plant using a with the Arabidopsis SQE1 sequence and used these to a putative Arabidopsis epoxidase enzymes SQE2, and are to characterized Medicago SQE enzymes. In contrast, the other three putative Arabidopsis and are to characterized Medicago are more to Brassica putative SQE enzymes (8Schafer U.A. Reed D.W. Hunter D.G. Yao K. Weninger A.M. Tsang E.W. Reaney M.J. MacKenzie S.L. Covello P.S. Plant Mol. Biol. 1999; 39: 721-728Crossref PubMed Scopus (22) Google Scholar). SQE1, SQE2, and SQE3 are to the characterized SQE enzymes from yeast and and SQE6 are to these enzymes sequence of characterized SQE enzymes. The Arabidopsis SQE were with other characterized SQE using and the in at six are in are in the of in sqe1–3 and The a predicted J. R. T. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). P. J. T. Biochem. Biophys. PubMed Scopus Google Scholar), and T. L. T. H. Biochem. Biophys. Res. Commun. PubMed Scopus Google Scholar) that are important for R. ERG1 activity. that the of R. ERG1 for squalene oxidosqualene T. L. T. H. Biochem. Biophys. Res. Commun. PubMed Scopus Google determine of the Arabidopsis SQE have squalene epoxidase we expressed each Arabidopsis cDNA in a S. cerevisiae which the ERG1 squalene epoxidase and is a sterol auxotroph F. F. Mol. 1977; PubMed Scopus (70) Google Scholar), and the growth of the yeast on medium lacking We found that the Arabidopsis SQE1, SQE2, and SQE3 cDNAs the yeast mutant to a the yeast ERG1 A and suggesting that these cDNAs encode squalene In contrast, Arabidopsis and SQE6 to the mutant A and consistent with the that these putative SQE enzymes are by the we not have to the SQE we not the that and SQE6 not accumulate expressed in the of the SQE enzymes, we expressed the Arabidopsis in an which the ERG1 squalene epoxidase and the synthase E.J. Matsuda S.P.T. Bartel B. Sci. S. 1994; PubMed Scopus Google Scholar), the that oxidosqualene heterologous SQE chromatography of extracts from these strains that oxidosqualene to accumulate to in yeast or SQE6 to in yeast SQE1, SQE2, or SQE3 not that yeast SQE1, SQE2, or SQE3 all produce oxidosqualene and Fig. from the of these genes to the yeast mutant the yeast and demonstrate that SQE1, SQE2, and SQE3 encode squalene not the of and In to we found that Arabidopsis SQE1 and SQE3 of and and Fig. expressed in the yeast ERG1 to and In contrast, SQE2 expression in yeast oxidosqualene, was not These that the Arabidopsis SQE enzymes may have the expression of the Arabidopsis SQE we from the expression P. M. L. Plant Physiol. 2004; PubMed Scopus Google Scholar). SQE1 and SQE3 in plant SQE2 and SQE4 to be more expressed SQE5 to be expressed in plant and SQE6 is expressed in and We these using of SQE in several tissues from wild-type plants of to the in of the SQE genes with squalene epoxidase SQE2, and by the of loss-of-function mutants from SQE1, we isolated three mutants from the J.M. H. P. J. P. R. C. C. E. H. L. N. H. M. N. E. M. C. R. P. L. M. T. E. PubMed Scopus Google Scholar) and from the R.A. Sci. S. PubMed Scopus Google Scholar, P. T. S. C. H. R.A. 9: PubMed Scopus Google Scholar). The in and and of the SQE1 coding sqe1–3 is in SQE1 and the in sqe1–4 is in SQE1 with that SQE1 mRNA was not in sqe1–3 mutant the is in the mRNA of the was Although we not defects in the or not which of the SQE1 coding sqe1–3 and sqe1–4 mutants were plants that a of to determine sqe1–3 defects are we from a plant on medium for and then the plants to and a for the of development, were from of the production of several was the stature of the sqe1–3 plants In to the normal of sqe1–3 tissues in of sqe1–3 in the were than wild-type is with mutants with reduced brassinosteroid T. S. S. S. J. J. Plant Physiol. 1999; PubMed Scopus Google Scholar, J. P. Plant PubMed Google Scholar). determine the sqe1–3 hypocotyl defects be to reduced we a on medium with We found that the hypocotyl of was by from S. J. H. S. T. T. H. T. J. Plant 9: PubMed Scopus Google Scholar). sqe1–3 not with that the brassinosteroid is not the of sqe1–3 elongation. In to defects, sqe1–3 plants which we by plants or on sqe1–3 mutants roots and and the which a the sqe1–3 root was not by not The root may to the stature of sqe1–3 plants in sqe1–3 plants were to wild-type sqe1–3 plants root that in and sqe1–3 plants were in or of sqe1–3 plants sqe1–3 were than and inviable and to sqe1–4 plants severe growth defects, including and and not suggesting that the defects were to sqe1 and not to in the of plants that the sqe1–3 lesion was determine the defects in the sqe1–3 mutant from SQE1 than an that was not by we the plants with a the wild-type SQE1 cDNA from the In to plants, we were to from the that were and wild-type The the root and hypocotyl of plants and not These results that the sqe1–3 from SQE1 We other SQE enzymes for to the sqe1–3 SQE1, expression of SQE4 or SQE5 from the promoter not the sqe1–3 defects not consistent with the of the SQE enzymes to the yeast mutant Chemical of sqe1–3 determine sqe1–3 were by triterpenoid we used to squalene and in three of sqe1–3 and wild-type plants. We found that squalene were at in sqe1–3 roots and tissues The of the SQE squalene in the sqe1–3 mutant that SQE1 squalene in plants, heterologously expressed in yeast The membrane sterol is the triterpenoid in Arabidopsis S. Phytochemistry. Scopus Google Scholar), and we that of SQE1 we not in sqe1–3 tissues to we that we more compounds of in and to compounds than to be reduced in sqe1–3 with wild-type tissues The in sqe1–3 roots a of root in the that the sqe1–3 are by of derived from from SQE1 several Arabidopsis sterol biosynthetic mutants with severe developmental defects have been identified M. N. H. K. H. K. S. T. S. S. T. Plant J. 2004; PubMed Scopus Google Scholar, J.C. S. M. H. S. M. S. J. Google Scholar, K. C. C. J. J. Google Scholar, K. C. C. J. Plant J. 2002; PubMed Scopus Google Scholar, M. J. M. J. K. R. K. Plant 2002; PubMed Scopus Google Scholar), plant mutants have not been we have that of the six putative Arabidopsis SQE SQE1, is for normal plant to previously identified sterol biosynthetic the sqe1–3 and sqe1–4 mutants have multiple developmental The sqe1–3 developmental defects other triterpenoid biosynthetic to the mutant M. N. H. K. H. K. S. T. S. S. T. Plant J. 2004; PubMed Scopus Google Scholar) in the sqe1–3 and The which are in a sterol produce roots M. J. M. J. K. R. K. Plant 2002; PubMed Scopus Google Scholar, F.M. S. T. B. Plant 1999; PubMed Scopus Google Scholar, F.M. S. S. S. T. Plant 2002; PubMed Scopus Google Scholar, H. Plant PubMed Scopus Google Scholar, K. Google Scholar), and the in a sterol severe and root defects J.C. S. M. H. S. M. S. J. Google Scholar, K. C. C. J. J. Google Scholar, M. J. M. J. K. R. K. Plant 2002; PubMed Scopus Google Scholar). mutants with and defects have been identified in several plants T. T. S. 2005; PubMed Scopus Google Scholar). Although sqe1–3 plants in the this was not that of sqe1–3 mutants severe defects in root producing roots and and not sqe1–3 defects and not suggesting the sqe1–3 defects are not to a of In to other triterpenoid biosynthetic which display defects in J. P. Plant PubMed Google Scholar, M. N. H. K. H. K. S. T. S. S. T. Plant J. 2004; PubMed Scopus Google Scholar, K. C. C. J. J. Google Scholar, K. C. C. J. Plant J. 2002; PubMed Scopus Google Scholar), sqe1–3 growth defects were in roots and and and severe in sqe1–3 plants were the mutant suggesting that growth conditions may in for the root of the In to the importance of SQE1 for results demonstrate an for SQE1 in Although on a on plants were inviable and that SQE1 to Chemical of sqe1–3 plants squalene with wild-type plants a sqe1–3 in the Arabidopsis oxidosqualene was not were to the remaining SQE enzymes in sqe1–3 can oxidosqualene for normal at the is an A for this is that a subset of in a on SQE1 and these accumulate squalene, is the squalene this subset of a in this may be by in the Although not to triterpenoids in be to the of the SQE enzymes in The sqe1 that these are the of the We not SQE1 mRNA by in sqe1–3 mRNA of the sqe1–3 was suggesting that a sqe1–3 the accumulate in sqe1–3 plants. The in sqe1–3 is the in ERG1 that and several that are important for T. L. T. H. Biochem. Biophys. Res. Commun. PubMed Scopus Google Scholar, P. J. T. Biochem. Biophys. PubMed Scopus Google Scholar), suggesting sqe1–3 be of Arabidopsis SQE in Arabidopsis of six SQE the of these genes have redundant or unique The sqe1–3 and sqe1–4 developmental defects and sqe1–3 squalene demonstrate that of the five remaining SQE genes fully for SQE1 function in the heterologous expression in we found that three of the six Arabidopsis SQE-like enzymes, SQE1, SQE2, and SQE3, have SQE and can epoxidize squalene to Further SQE1 and SQE3 can not oxidosqualene, and and Fig. SQE2 to produce oxidosqualene single can or the of ERG1 to oxidosqualene to T. L. T. H. Biochem. Biophys. Res. Commun. PubMed Scopus Google Scholar). Arabidopsis oxidosqualene can not oxidosqualene, H. M.J. Wilson W.K. S. Matsuda S.P.T. J. Am. Chem. Soc. 2005; PubMed Scopus Google Scholar), suggesting that may have in plants. In to multiple SQE genes may expression of In is expressed in plant expression is to N. Plant Mol. Biol. PubMed Scopus Google Scholar). P. M. L. Plant Physiol. 2004; PubMed Scopus Google Scholar) and that SQE1 and SQE3 have expression SQE4 and SQE6 expression may be more In to SQE enzymes may be to or in and to a subset of oxidosqualene Arabidopsis SQE2 and putative squalene epoxidase have predicted and Arabidopsis is to N. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar), suggesting the that triterpenoid biosynthesis is with In to and developmental the SQE genes may be to and SQE may be in with other triterpenoid biosynthetic enzymes to in to or In SQE is a SQE is (7Suzuki H. Achnine L. Xu R. Matsuda S.P.T. Dixon R.A. Plant J. 2002; 32: 1033-1048Crossref PubMed Scopus (235) Google Scholar). In SQE1 and SQE3 to be SQE2 and SQE4 to be expressed at may be under Plant to the diversity of plant in plant are at numerous by and in diversity of Although are to a single plants this diversity by producing triterpene skeletons (5Xu R. Fazio G.C. Matsuda S.P.T. Phytochemistry. 2004; 65: 261-291Crossref PubMed Scopus (435) Google Scholar). the characterized Arabidopsis oxidosqualene all can oxidosqualene a several of these enzymes are oxidosqualene to a of triterpene skeletons with D.R. Rasbery J.M. Bartel B. Matsuda S.P.T. Curr. Opin. Plant Biol. 2006; 9: 305-314Crossref PubMed Scopus (292) Google Scholar). In to the diversity by the oxidosqualene the of six SQE in Arabidopsis that squalene may an additional in triterpenoid The three that to function in yeast A and are and may have and SQE6 are more to Brassica SQE enzymes than to Arabidopsis SQE1, SQE2, SQE3, and putative SQE enzymes from other plants expression of SQE4 and SQE5 to the Arabidopsis sqe1–3 mutant not or the yeast mutant A and consistent with the that SQE4 and SQE5 the may have triterpenoids not an oxidosqualene and may be from squalene M. H. Scopus Google Scholar, H. J. Scopus Google Scholar). be to the and of of the of the that and of genes in function in plant defense Plant Physiol. 2005; PubMed Scopus Google Scholar, R. Plant 2004; PubMed Scopus Google Scholar). SQE in the plant may a for and Triterpenoid biosynthetic mutants have severe defects, the importance of an triterpenoid biosynthetic for plant We have that three of the six Arabidopsis SQE are squalene and that SQE1 is for plant sqe1 mutants have defects and to produce seeds. In sqe1 mutants accumulate squalene, suggesting that triterpenoid biosynthesis is the Arabidopsis SQE that to the Arabidopsis sqe1 mutant to the yeast consistent with the that a subset of Arabidopsis SQE enzymes may have unique Further and into the of the of SQE enzymes in plants.