Functional expression of Saccharomyces cerevisiae CYP51A1 encoding lanosterol-14-demethylase in tobacco results in bypass of endogenous sterol biosynthetic pathway and resistance to an obtusifoliol-14-demethylase herbicide inhibitor

Nicotiana tabacum protoplasts have been transformed by Agrobacterium tumefaciens containing a T-DNA in which the gene CYP51A1 encoding lanosterol-14-demethylase (LAN14DM) from Saccharomyces cerevisiae is under the control of a cauliflower mosaic virus (CaMV) 35S promoter. Two transformants strongly expressed the LAN14DM as shown by Northern and Western experiments. These transgenic calli were killed by LAB 170250F (LAB) (a phytotoxic fungicide inhibiting both plant obtusifoliol-14-demethylase (OBT14DM) and LAN14DM) but were resistant to γ-ketotriazole (γ-kt), a herbicide which has been shown to inhibit OBT14DM but not LAN14DM at a concentration that was lethal to control calli. However, these transgenic calli were killed by mixtures of γ-kt plus fungicide inhibitors of LAN14DM such as ketoconazole, itraconazole or flusilazole which alone were not effective. Further analysis of the transgenic calli grown in the presence of γ-kt showed that their Δ⁵-sterol content was close to that of untreated control calli obtained from protoplasts transformed with control plasmid; this is in agreement with evidence that the LAN14DM expressed from the transgene could bypass the blocked OBT14DM by using the plant substrate obtusifoliol. In contrast, control calli when treated with γ-kt, displayed a sterol content strongly enriched in 14α-methyl sterols and depressed in physiological Δ⁵-sterols. When the transgenic calli were cultured in mixtures of γ-kt and LAN14DM inhibitors sterol compositions enriched in 14α-methyl sterols were obtained, reflecting a strong inhibition of both ‘endogenous’ OBT14DM and ‘exogenous’ LAN14DM. Taken together these results show that in tobacco calli transformed with CYP51A1, resistance to a triazole herbicide arises from expression of a functional LAN14DM enzyme; its activity in transgenic tissues creates a bypass of the sterol biosynthetic pathway at the 14-demethylase level when this latter is blocked by an OBT14DM herbicide inhibitor.     

Metabolism of Δ0-, Δ5-, and Δ7-Sterols by Larvae of Heliothis zea

Heliothis zea was reared on artificial diets containing Δ⁵-sterols (cholesterol, campesterol, or sitosterol), Δ⁷-sterols (lathosterol, epifungisterol, or spinasterol), or Δ⁰-sterols (cholestanol, epicoprostanol, campestanol, or sitostanol) in order to determine how different dietary sterols affect the type of sterols present in the tissues of the late-sixth-instar larva. Although all of the dietary sterols (except epicoprostanol) supported the growth of the larvae, not all of the sterols were metabolized to the same end products. In each case, at least 80% of the sterols in the tissues of the larvae retained the same nucleus as that of the dietary sterol, indicating that H. zea carries out very little metabolism of ring B of Δ⁵-, Δ⁷-, and Δ⁰-sterols. The larvae dealkylated the Δ⁵-, Δ⁷-, and Δ⁰-alkylsterols to 24-desalkylsterols, but a greater percentage of the Δ⁵-alkylsterols were metabolized in this manner. The sterols present as free sterols in the larva were also present as esterifed sterols which accounted for 2–4% of the total sterols. Therefore, the sterol composition of the tissues of H. zea can be altered by varying the dietary sterols.     

Co-occurrence of Δ5- and Δ7 -sterols in the fungus Dictyuchus monosporus

The desmethyl sterol composition of the oomycete Dictyuchus monosporus is unusual in that it is a mixture of 56.9 % Δ⁵-sterols and 42.6 % Δ⁷-sterols. The Δ⁵-sterols are cholesterol, 24 methylenecholesterol and fucosterol; the Δ⁷-sterols are cholest-7-enol, ergosta-7,24(28)-dienol and stigmasta-7,E-24(28)-dienol. Stigmasta-7,E-24(28)-dienol, is identified for the first time from natural sources. In addition, traces of lanosterol are present.     

Effect of Light on Sterol Changes in Medicago sativa

Most vascular plants contain Δ⁵-sterols as the predominant type; however, a few species such as Medicago sativa, have mainly Δ⁷-sterols. The Δ⁷-sterols of alfalfa are isomers of the common Δ⁵-sterols and are generally assumed to be their immediate precursors. Light had a significant influence on the sterol status of M. sativa. High light intensity and a long day favored the accumulation of dihydrospinasterol; a short day and low light intensity, particularly darkness, favored spinasterol accumulation. These data for Δ⁷-sterol plants agree with those reported for Δ⁵-sterol plants; light favors the accumulation of the monounsaturated 29 carbon sterols and darkness favors the accumulation of the diunsaturated sterols. Proposed is a mechanism to explain the effect of light on the accumulation of Δ⁷- and Δ⁵-sterols.     

Utilization of Δ5,7 - and Δ8-sterols by larvae of Heliothis zea

Larvae from two populations of Heliothis zea were reared on artificial diets containing various sterols, which supported suboptimal growth, and their tissue sterols were characterized in order to determine how these dietary sterols are utilized by this insect. The sterols studied included Δ^5,7-sterols (7-dehydrocholesterol or ergosterol), Δ⁸-sterols (lanosterol and/or 24-dihydrolanosterol), and a Δ⁵-sterol (4,4-dimethylcholesterol). Although larvae did not develop on 4,4-dimethylcholesterol, those fed primarily Δ⁸-4,4,14-trimethylsterols developed to the third instar. When the latter sterols were spared with cholesterol, the larvae reached the sixth instar and contained 4,4,14-trimethylsterols as well as cholesterol in their tissues. When larvae were fed 7-dehydrocholesterol, <1% of the larvae from one population developed to the sixth instar and these larvae contained 7-dehydrocholesterol as their principal sterol. The other larvae successfully completed their larval stage when they were transferred from the diet containing 7-dehydrocholesterol (or no sterol) to a diet containing cholesterol within at least 9 days. The sterol composition of larvae transferred from a diet containing cholesterol to a diet containing 7-dehydrocholesterol, after they had reached 60% of their final weight, was 54% cholesterol and 46% 7-dehydrocholesterol. The major sterol isolated from the tissues of the larvae fed ergosterol was also 7-dehydrocholesterol. Therefore, although the larva of H. zea can dealkylate and saturate the side chain of the Δ^5,7,22-24β-methylsterol, it carries out little metabolism of the B ring of the nucleus. These studies demonstrate that, when Δ^5,7- or Δ⁸-sterols are the principal sterols in the diet of H. zea, they are absorbed and incorporated into its tissues, although they slow the rate of growth and may prevent complete development of the larva.     

Investigations on the Δ23-, Δ24(28)- and Δ25-Sterols of zea mays

The sterols of Zea mays shoots were isolated and characterized by TLC, HPLC, GC/MS and ¹H NMR techniques. In all, 22 4-demethyl sterols were identified and they included trace amounts of the Δ²³-, Δ²⁴- and Δ²⁵-sterols, 24-methylcholesta-5,E-23-dien-3β-ol, 24-methylcholesta-5,Z-23-dien-3β-ol, 24-methylcholesta-5,25-dien-3β-ol, 24-ethylcholesta-5,25-dien-3β-ol and 24-ethylcholesta-5,24-dien-3β-ol. In the 4,4-dimethyl sterol fraction, cycloartenol and 24-methylenecycloartanol were the major sterol components but small amounts of the Δ²³-compound, cyclosadol, and the Δ²⁵-compound, cyclolaudenol, were recognized. These various Δ²³- and Δ²⁵-sterols may have some importance in alternative biosynthetic routes to the major sterols, particularly the 24β-methylcholest-5-en-3β-ol component of the C₂₈-sterols. Radioactivity from both [2-¹⁴C]MVA and [methyl-¹⁴C]methionine was incorporated by Z. mays shoots into the sterol mixture. Although 24-methylene and 24-ethylidene sterols were relatively highly labelled, the various Δ²³- and Δ²⁵-sterols contained much lower levels of radioactivity, which is possibly indicative of their participation in alternative sterol biosynthetic routes. (24R)-24-Ethylcholest-5-en-3β-ol (sitosterol) had a significantly higher specific activity than the 24-methylcholest-5-en-3β-ol indicating that the former is synthesized at a faster rate.     

Sterols and related metabolites from five species of sponges

Free sterol fractions were isolated from the marine sponges Phyllospongia madagascarensis, Scalarispongia sp., Oceanapia sp., Monanchora clathrata and studied by GLC, GLC–MS, and spectroscopy NMR. P. madagascarensis and Scalarispongia sp. contained common Δ⁵-sterols; cholesterol was shown to be a main sterol of both the sponges. Oceanapia sp. contained stanols and minor Δ⁵-sterols with 24R-24,25-methylene-5α-cholestan-3β-ol as a main constituent. Many free sterols from M. clathrata were Δ⁷-series compounds, and latosterol was a main sterol. Δ⁴-3-Ketosteroids and Δ⁵-sterol esters were found in the Antarctic sponge Haliclona sp., but free sterols were practically absent except for trace amount of cholesterol. A chemotaxonomic application of sterols in relation to the genera Phyllospongia, Oceanapia and the family Crambeidae is provided. The known cases of the absence of sterols in sponges and probable reasons of the phenomenon are discussed.     

Cloning, sequencing, and disruption of the gene encoding sterol C-14 reductase in Saccharomyces cerevisiae.

A sterol C-14 reductase (erg24-1) mutant of Saccharomyces cerevisiae was selected in a fen1, fen2, suppressor background on the basis of nystatin resistance and ignosterol (ergosta-8,14-dienol) production. The erg24-1 allele segregated genetically as a single, recessive gene. The wild-type ERG24 gene was cloned by complementation onto a 12-kb fragment from a yeast genomic library, and subsequently subcloned onto a 2.4-kb fragment. This was sequenced and found to contain an open reading frame of 1,314 bp, predicting a polypeptide of 438 amino acids (M(r) 50,612). A 1,088-bp internal region of the ERG24 gene was excised, replaced with a LEU2 gene, and integrated into the chromosome of the parental strain, FP13D (fen1, fen2) by gene replacement. The ERG24 null mutant produced ergosta-8,14-dienol as the major sterol, indicating that the delta 8-7 isomerase, delta 5-desaturase and the delta 22-desaturase were inactive on sterols with the C14 = 15 double bond.     

Isolation of the ERG2 Gene,Encoding Δ8 → Δ7 Sterol Isomerase,from the Maize Smut Pathogen Ustilago maydis

Bailey, A. M., Burden, R. S., James, C. S., Keon, J. P. R., Croxen, R., Bard, M., and Hargreaves, J. A. 1993. Isolation of the ERG2 gene, encoding Δ⁸ → Δ⁷ sterol isomerase, from the maise smut pathogen Ustilago maydis. Experimental Mycology 18, 87-92. The ERG2 gene encoding Δ⁸ → Δ⁷ sterol isomerase has been isolated from the fungal plant pathogen Ustilago maydis. This was accomplished by screening an U. maydis genomic library with a fragment of the Saccharomyces cerevisiae ERG2 gene. The identity of the U. maydis ERG2 gene was confirmed by complementation of an U. maydis Erg2 mutant and by comparing the deduced amino acid sequence encoded by the U. maydis ERG2 gene with that of the S. cerevisiae ERG2 gene product.     

Differential effects of fenpropimorph and fenhexamid, two sterol biosynthesis inhibitor fungicides, on arbuscular mycorrhizal development and sterol metabolism in carrot roots

Sterols composition of transformed carrot roots incubated in presence of increasing concentrations of fenpropimorph (0.02; 0.2; 2 mg l⁻¹) and fenhexamid (0.02; 0.2; 2; 20 mg l⁻¹), colonized or not by Glomus intraradices was determined. In mycorrhizal roots treated with fenpropimorph, normal Δ⁵-sterols were replaced by unusual compounds such as 9β,19-cyclopropylsterols (24-methylpollinastanol), Δ^8,14-sterols (ergosta-8,14-dienol, stigmasta-8,14-dienol), Δ⁸-sterols (Δ⁸ sitosterol) and Δ⁷-sterols (ergosta-7,22-dienol). After application of fenpropimorph, a drastic reduction of the mycorrhizal root growth, root colonization and extraradical fungal development was observed. Application of fenhexamid did not modify sterol profiles and the total colonization of roots. But the arbuscule frequency of the fungal partner was significantly affected.Comparison of the effects caused by the tested fungicides indicates that the usual phytosterols may be involved in symbiosis development. Indeed, observed modifications of root sterols composition could explain the high fenpropimorph toxicity to the AM symbiosis. However, the absence of sterolic modifications in the roots treated with fenhexamid could account for its more limited impact on mycorrhization.     

"Dinoflagellate Sterols" in marine diatoms

Sterol compositions for three diatom species, recently shown to contain sterols with side chains typically found in dinoflagellates, were determined by HPLC and ¹H NMR spectroscopic analyses. The centric diatom Triceratium dubium (= Biddulphia sp., CCMP 147) contained the highest percentage of 23-methylated sterols (37.2% (24R)-23-methylergosta-5,22-dienol), whereas the pennate diatom Delphineis sp. (CCMP 1095) contained the cyclopropyl sterol gorgosterol, as well as the 27-norsterol occelasterol. The sterol composition of Ditylum brightwellii (CCMP 358) was the most complex, containing Δ⁰- and Δ⁷-sterols, in addition to the predominant Δ⁵-sterols. A pair of previously unknown sterols, stigmasta-5,24,28-trienol and stigmasta-24,28-dienol, were detected in D. brightwellii and their structures were determined by NMR spectroscopic analysis and by synthesis of the former sterol from saringosterol. Also detected in D. brightwellii was the previously unknown 23-methylcholesta-7,22-dienol.     

Neue sterine in Helianthus annuus

Six Δ⁷-sterols were isolated from sunflower seed oil by preparative TLC. On the basis of physica and chemical data five of the Δ⁷-sterols were identified: Δ⁷-stigmastenol, Δ⁷-campestenol, Δ^7,24(28)-stigma-stadienol, Δ^7,24(25)-stigmastadienol, and Δ^{7,9(11),24(28)}-stigmastatrienol. The last two sterols have not previously been detected in nature.     

Metabolic engineering of soybean affords improved phytosterol seed traits

Different combinations of three rate‐limiting enzymes in phytosterol biosynthesis, the Arabidopsis thaliana hydroxyl methylglutaryl CoA1 (HMGR1) catalytic subunit linked to either constitutive or seed‐specific β‐conglycinin promoter, and the Glycine max sterol methyltransferase1 (SMT1) and sterol methyltransferase2‐2 (SMT2‐2) genes, under the control of seed‐specific Glycinin‐1 and Beta‐phaseolin promoters, respectively, were engineered in soybean plants. Mature seeds of transgenic plants displayed modest increases in total sterol content, which points towards a tight control of phytosterol biosynthesis. However, in contrast to wild‐type seeds that accumulated about 35% of the total sterol in the form of intermediates, in the engineered seeds driven by a seed‐specific promoter, metabolic flux was directed to Δ⁵‐24‐alkyl sterol formation (99% of total sterol). The engineered effect of end‐product sterol (sitosterol, campesterol, and stigmasterol) over‐production in soybean seeds resulted in an approximately 30% increase in overall sitosterol synthesis, a desirable trait for oilseeds and human health. In contradistinction, increased accumulation of cycloartenol and 24(28)‐methylencylartanol (55% of the total sterol) was detected in plants harbouring the constitutive t‐HMGR1 gene, consistent with the previous studies. Our results support the possibility that metabolic flux of the phytosterol family pathway is differentially regulated in leaves and seeds.     

Sterine in Trichosanthes kirilowii

Four Δ⁵-sterols and six Δ⁷-sterols were isolated from the seed oil of Trichosanthes kirilowii and identified as campesterol, sitosterol, stigmasterol, Δ⁷-campesterol, Δ⁷-stigmasterol, Δ^7,22-stigmastadienol, 24-ethylcholesta-5,25-diene-3β-ol, 24-ethylcholesta-7,24(25)-diene-3β-ol, 24-ethylcholesta-7,25-diene-3β-ol, and 24-ethylcholesta-7,22,25-trine-3β-ol.     

AtNHX1基因对荞麦的遗传转化及抗盐再生植株的获得

通过农杆菌介导法将拟南芥液泡膜Na⁺/H⁺反向转运蛋白基因AtNHX1转入荞麦中,在2.0mg/L 6-BA、0.1mg/L IAA、1mg/L KT、50mg/L卡那霉素和500mg/L头孢霉素的MS培养基上进行选择培养,从来源于864块外植体的36块抗性愈伤组织中共获得426棵再生植株（转化频率为4.17%）。经PCR、Southern印迹分析、RT-PCR和Northern检测,初步证实AtNHX1基因已整合至荞麦基因组中。用200mmol/L的盐水对转基因植株和对照植株进行胁迫处理6周,转基因植株能够生存,而对照植株死亡。用不同浓度的NaCl溶液处理转基因植株和对照植株,发现Na⁺及脯氨酸含量在转基因植株中的积累水平显著高于对照植株,而K⁺的含量在转基因植株中的积累水平低于对照植株。次生代谢产物黄酮类化合物芦丁在转基因植株根、茎和叶片中的含量也比对照植株明显要高。这些结果表明利用基因工程手段提高作物的耐盐性是可行的。     

Increased sterol biosynthesis in tobacco calli resistant to a triazole herbicide which inhibits demethylation of 14α-methyl sterols

The γ-keto triazole derivative 4,4-dimethyl-1-(2-methoxyphenyl)-1-(1,2,4-triazol-1-yl)-1-penten-3-one is toxic to Nicotiana tabacum L. cv. Xanthi plants or cell cultures. Analysis of the sterol composition of treated wild-type plant material demonstrates that this herbicide is an inhibitor of the C-14α-methyl demethylation process in sterol biosynthesis. Selection experiments, consisting of screening large populations of microcalli derived from UV-mutagenized tobacco protoplasts for resistance to a lethal dose (1 mg · 1^?1) of the γ-keto triazole, have resulted in the recovery of two groups of resistant calli. In the first group, selected calli show a sterol composition in the absence or presence of the inhibitor very similar to that of wild-type sensitive calli, whereas in the second group the main feature of the selected calli is a new sterol profile. These calli present an overproduction of sterols with a concomitant esterification of overproduced metaolites, just as it was demonstrated for calli previously selected in our laboratory for resistance to LAB 170250F, a triazole fungicide (Maillot-Vernier et al., 1991, Mol. Gen. Genet. 231, 33–40).     

Kinetics of Δ5,7-sterol accumulation during growth ofSaccharomyces cerevisiae

Saccharomyces cerevisiae accumulates Δ^5,7-sterols up to 4 mg per g biomass. The differential rate of sterol synthesis continually increases during growth, its value only being decreased at sterol levels higher than 30 mg per g biomass. The specific rate of sterol synthesis reaches a broad maximum during the growth phase. The gradual sterol accumulation pattern is dominant in cultures growing both on fermentable and nonfermentable carbon sources and is modulated by glucose repression. Limited feeding with sucrose has a significantly greater negative impact on sterol accumulation than feeding with ethanol as a carbon source.     

Accumulation of Δ8,14-sterols in suspension cultures of bramble cells cultured with an azasterol antimycotic agent (A25822B)

Bramble suspension cultures normally contain Δ⁵-sterols (sitosterol, campesterol and isofucosterol). When the cells were grown in a medium supplemented with 15-aza-24-methylene-d-homocholesta-8,14-dien- 3β-ol, Δ⁵-sterols disappeared almost completely whereas Δ^8,14-sterols accumulated strongly. Five Δ^8,14-sterols, including two new compounds, (24R)-24-ethyl-5α-cholesta-8,14-dien-3β-ol and 4α-methyl-5α-stigmasta-8,14, Z-24(28)-trien-3β-ol, were identified. The 15-azasterol probably inhibited the reduction of the Δ¹⁴-bond. Cell lines growing permanently in an azasterol-supplemented medium were obtained.