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Steroidal glycoalkaloids (SGAs) are toxic specialized metabolites found in members of the Solanaceae, such as Solanum tuberosum (potato) and Solanum lycopersicum (tomato). The major potato SGAs are α-solanine and α-chaconine, which are biosynthesized from cholesterol. Previously, we have characterized two cytochrome P450 monooxygenases and a 2-oxoglutarate-dependent dioxygenase that function in hydroxylation at the C-22, C-26 and C-16α positions, but the aminotransferase responsible for the introduction of a nitrogen moiety into the steroidal skeleton remains uncharacterized. Here, we show that PGA4 encoding a putative γ-aminobutyrate aminotransferase is involved in SGA biosynthesis in potatoes. The PGA4 transcript was expressed at high levels in tuber sprouts, in which SGAs are abundant. Silencing the PGA4 gene decreased potato SGA levels and instead caused the accumulation of furostanol saponins. Analysis of the tomato PGA4 ortholog, GAME12, essentially provided the same results. Recombinant PGA4 protein exhibited catalysis of transamination at the C-26 position of 22-hydroxy-26-oxocholesterol using γ-aminobutyric acid as an amino donor. Solanum stipuloideum (PI 498120), a tuber-bearing wild potato species lacking SGA, was found to have a defective PGA4 gene expressing the truncated transcripts, and transformation of PI 498120 with functional PGA4 resulted in the complementation of SGA production. These findings indicate that PGA4 is a key enzyme for transamination in SGA biosynthesis. The disruption of PGA4 function by genome editing will be a viable approach for accumulating valuable steroidal saponins in SGA-free potatoes.

Summary Glycyrrhizin, a sweet triterpenoid saponin found in the roots and stolons of Glycyrrhiza species (licorice), is an important active ingredient in traditional herbal medicine. We previously identified two cytochrome P450 monooxygenases, CYP88D6 and CYP72A154, that produce an aglycone of glycyrrhizin, glycyrrhetinic acid, in Glycyrrhiza uralensis. The sugar moiety of glycyrrhizin, which is composed of two glucuronic acids, makes it sweet and reduces its side‐effects. Here, we report that UDP‐glycosyltransferase (UGT) 73P12 catalyzes the second glucuronosylation as the final step of glycyrrhizin biosynthesis in G. uralensis; the UGT73P12 produced glycyrrhizin by transferring a glucuronosyl moiety of UDP‐glucuronic acid to glycyrrhetinic acid 3‐O‐monoglucuronide. We also obtained a natural variant of UGT73P12 from a glycyrrhizin‐deficient (83‐555) strain of G. uralensis. The natural variant showed loss of specificity for UDP‐glucuronic acid and resulted in the production of an alternative saponin, glucoglycyrrhizin. These results are consistent with the chemical phenotype of the 83‐555 strain, and suggest the contribution of UGT73P12 to glycyrrhizin biosynthesis in planta. Furthermore, we identified Arg32 as the essential residue of UGT73P12 that provides high specificity for UDP‐glucuronic acid. These results strongly suggest the existence of an electrostatic interaction between the positively charged Arg32 and the negatively charged carboxy group of UDP‐glucuronic acid. The functional arginine residue and resultant specificity for UDP‐glucuronic acid are unique to UGT73P12 in the UGT73P subfamily. Our findings demonstrate the functional specialization of UGT73P12 for glycyrrhizin biosynthesis during divergent evolution, and provide mechanistic insights into UDP‐sugar selectivity for the rational engineering of sweet triterpenoid saponins., Significance Statement Glycyrrhizin, a licorice‐derived sweet triterpenoid diglycoside, is one of the most important phytoconstituents in the pharmaceutical and food industries. Here, we identified UGT73P12 as the enzyme catalyzing the sugar–sugar linkage to produce glycyrrhizin from its less glycosylated precursor in the presence of UDP‐glucuronic acid, and found that Arg32 of UGT73P12 is essential for the high specificity for UDP‐glucuronic acid, which was probably acquired during evolution to enable the functional specialization of UGT73P12 for glycyrrhizin biosynthesis.

Licorice (Glycyrrhiza) produces glycyrrhizin, a valuable triterpenoid saponin, which exhibits persistent sweetness and broad pharmacological activities. In the genus Glycyrrhiza, three species, Glycyrrhiza uralensis, Glycyrrhiza glabra and Glycyrrhiza inflata, produce glycyrrhizin as their main triterpenoid saponin, which has a ketone group at C-11. Other Glycyrrhiza species produce mainly oleanane-type saponins, which harbor homoannular or heteroannular diene structures that lack the C-11 ketone. Although the glycyrrhizin biosynthetic pathway has been fully elucidated, the pathway involving saponins with diene structures remains unclear. CYP88D6 from G. uralensis is a key enzyme in glycyrrhizin biosynthesis, catalyzing the sequential two-step oxidation of β-amyrin at position C-11 to produce 11-oxo-β-amyrin. In this study, we evaluated the functions of CYP88D6 homologs from the glycyrrhizin-producing species G. glabra and G. inflata and from the non-glycyrrhizin-producing species Glycyrrhiza pallidiflora and Glycyrrhiza macedonica, using yeast engineered to supply β-amyrin as a substrate. Yeast expressing CYP88D6 homologs from glycyrrhizin-producing species produced 11-oxo-β-amyrin. However, yeast expressing CYP88D6 homologs (such as CYP88D15) from the non-glycyrrhizin-producing Glycyrrhiza species accumulated oleana-9(11),12-dien-3β-ol and oleana-11,13(18)-dien-3β-ol; these diene compounds are non-enzymatic or yeast endogenous enzymatic dehydration derivatives of 11α-hydroxy-β-amyrin, a direct reaction product of CYP88D15. These results suggest that the activities of CYP88D6 homologs, particularly their ability to catalyze the second oxidation, could influence glycyrrhizin productivity and diversify the chemical structures of saponins in Glycyrrhiza plants. A synthetic biological approach to engineer CYP88D15 could enable the production of pharmacologically active saponins with diene structures, such as saikosaponins, whose biosynthetic pathways have yet to be fully characterized.

Aluminum tolerance of aluminum-sensitive rice was enhanced under darkness by multiple changes in membrane sterols: decreased stigmasterol, increased precursor partitioning for sterols biosynthesis, and increased expression of HMG genes., Aluminum-sensitive rice (Oryza sativa L.) cultivars showed increased Al tolerance under dark conditions, because less Al accumulated in the root tips (1 cm) under dark than under light conditions. Under dark conditions, the root tip concentration of total sterols, which generally reduce plasma membrane permeabilization, was higher in the most Al-sensitive japonica cultivar, Koshihikari (Ko), than in the most Al-tolerant cultivar, Rikuu-132 (R132), but the phospholipid content did not differ between the two. The Al treatment increased the proportion of stigmasterol (which has no ability to reduce membrane permeabilization) out of total sterols similarly in both cultivars under light conditions, but it decreased more in Ko under dark conditions. The carotenoid content in the root tip of Al-treated Ko was significantly lower under dark than under light conditions, indicating that isopentenyl diphosphate transport from the cytosol to plastids was decreased under dark conditions. HMG2 and HMG3 (encoding the key sterol biosynthetic enzyme 3-hydroxy-3-methylglutaryl CoA reductase) transcript levels in the root tips were enhanced under dark conditions. We suggest that the following mechanisms contribute to the increase in Al tolerance under dark conditions: inhibition of stigmasterol formation to retain membrane integrity; greater partitioning of isopentenyl diphosphate for sterol biosynthesis; and enhanced expression of HMGs to increase sterol biosynthesis.

Steroidal glycoalkaloids (SGAs) are toxic specialized metabolites that are found in the Solanaceae. Potato (Solanum tuberosum) contains the SGAs α-solanine and α-chaconine, while tomato (Solanum lycopersicum) contains α-tomatine, all of which are biosynthesized from cholesterol. However, although two cytochrome P450 monooxygenases that catalyze the 22- and 26-hydroxylation of cholesterol have been identified, the 16-hydroxylase remains unknown. Feeding with deuterium-labeled cholesterol indicated that the 16α- and 16β-hydrogen atoms of cholesterol were eliminated to form α-solanine and α-chaconine in potato, while only the 16α-hydrogen atom was eliminated in α-tomatine biosynthesis, suggesting that a single oxidation at C-16 takes place during tomato SGA biosynthesis while a two-step oxidation occurs in potato. Here, we show that a 2-oxoglutarate-dependent dioxygenase, designated as 16DOX, is involved in SGA biosynthesis. We found that the transcript of potato 16DOX (St16DOX) was expressed at high levels in the tuber sprouts, where large amounts of SGAs are accumulated. Biochemical analysis of the recombinant St16DOX protein revealed that St16DOX catalyzes the 16α-hydroxylation of hydroxycholesterols and that (22S)-22,26-dihydroxycholesterol was the best substrate among the nine compounds tested. St16DOX-silenced potato plants contained significantly lower levels of SGAs, and a detailed metabolite analysis revealed that they accumulated the glycosides of (22S)-22,26-dihydroxycholesterol. Analysis of the tomato 16DOX (Sl16DOX) gene gave essentially the same results. These findings clearly indicate that 16DOX is a steroid 16α-hydroxylase that functions in the SGA biosynthetic pathway. Furthermore, St16DOX silencing did not affect potato tuber yield, indicating that 16DOX may be a suitable target for controlling toxic SGA levels in potato.

Several enzymes of the CYP716A subfamily have been reported to be involved in triterpenoid biosynthesis. Members of this subfamily oxidize various positions along the triterpenoid backbone and the majority of them catalyze a three-step oxidation at the C-28 position. Interestingly, C-28 oxidation is a common feature in oleanolic acid, ursolic acid, and betulinic acid, which are widely distributed in plants and exhibit important biological activities. In this work, three additional CYP716A enzymes isolated from olive, sugar beet, and coffee, were characterized as multifunctional C-28 oxidases. Semi-quantitative comparisons of in vivo catalytic activity were made against the previously characterized enzymes CYP716A12, CYP716A15, and CYP716A52v2. When heterologously expressed in yeast, the isolated enzymes differed in both catalytic activity and substrate specificity. This study indicates that the screening of enzymes from different plants could be a useful means of identifying enzymes with enhanced catalytic activity and desired substrate specificity. Furthermore, we show that "naturally-evolved" enzymes can be useful in the heterologous production of pharmacologically and industrially important triterpenoids.

α-Solanine and α-chaconine, steroidal glycoalkaloids (SGAs) found in potato (Solanum tuberosum), are among the best-known secondary metabolites in food crops. At low concentrations in potato tubers, SGAs are distasteful; however, at high concentrations, SGAs are harmful to humans and animals. Here, we show that POTATO GLYCOALKALOID BIOSYNTHESIS1 (PGA1) and PGA2, two genes that encode cytochrome P450 monooxygenases (CYP72A208 and CYP72A188), are involved in the SGA biosynthetic pathway, respectively. The knockdown plants of either PGA1 or PGA2 contained very little SGA, yet vegetative growth and tuber production were not affected. Analyzing metabolites that accumulated in the plants and produced by in vitro enzyme assays revealed that PGA1 and PGA2 catalyzed the 26- and 22-hydroxylation steps, respectively, in the SGA biosynthetic pathway. The PGA-knockdown plants had two unique phenotypic characteristics: The plants were sterile and tubers of these knockdown plants did not sprout during storage. Functional analyses of PGA1 and PGA2 have provided clues for controlling both potato glycoalkaloid biosynthesis and tuber sprouting, two traits that can significantly impact potato breeding and the industry.

Dimunito/Dwarf1 (DWF1) is an oxidoreductase enzyme that is responsible for the conversion of C28- and C29-Δ24(28)-olefinic sterols to 24-methyl- and 24-ethylcholesterols. Generally, the reaction proceeds in two steps via the Δ24(25) intermediate. In this study, we characterized the ArDWF1 gene from an expression sequence tag library of Ajuga reptans var. atropurpurea hairy roots. The gene was functionally expressed in the yeast T21 strain. The in vivo and in vitro study of the transformed yeast indicated that ArDWF1 catalyzes the conversion of 24-methylenecholesterol to campesterol. A labeling study followed by GC-MS analysis suggested that the reaction proceeded with retention of the C-25 hydrogen. The 25-H retention was established by the incubation of the enzyme with (23,23,25-2H3,28-13C)-24-methylenecholesterol, followed by 13C NMR analysis of the resulting campesterol. Thus, it has been concluded that ArDWF1 directly reduces 24-methylenecholesterol to produce campesterol without passing through a Δ24(25) intermediate. This is the first characterization of such a unique DWF1 enzyme. For comparison purposes, Oryza sativa DWF1 (OsDWF1) was similarly expressed in yeast. An in vivo assay of OsDWF1 supported the generally accepted two-step mechanism because the C-25 hydrogen of 24-methylenecholesterol was eliminated during its conversion to 24-methylcholesterol. As expected, the 24-methylcholesterol produced by OsDWF1 was a mixture of campesterol and dihydrobrassicasterol. Furthermore, the 24-methylcholesterol contained in the Ajuga hairy roots was determined to be solely campesterol through its analysis using chiral GC-MS. Therefore, ArDWF1 has another unique property in that only campesterol is formed by the direct reduction catalyzed by the enzyme.

Triterpene saponins are widely distributed in higher plants [1,2] and belong to a class of natural products that includes various bioactive compounds found in medicinal plants [3]. The family Leguminosae, which includes plants such as licorice (Glycyrrhiza uralensis) [4], soybean (Glycine max) [5] and alfalfa (Medicago sativa) [6], are well-known triterpene saponin-producing plants. The roots and stolons of licorice accumulate glycyrrhizin as a major bioactive triterpene saponin, while the hypocotyls of soybean accumulate soyasaponin I. Glycyrrhizin and soyasaponin I are synthesized from a common biosynthetic intermediate, the triterpene aglycone b-amyrin, derived from the initial cyclization of 2,3-oxidosqualene. Licorice b-amyrin 11-hydroxylase (CYP88D6) [7] and b-amyrin 30-hydroxylase (CYP72A154) [8] catalyze the oxidation of b-amyrin at positions C-11 (two-step oxidation) and C-30 (three-step oxidation), respectively, to produce glycyrrhetinic acid, the triterpene aglycone of glycyrrhizin. In contrast, soybean b-amyrin 22-hydroxylase [unpublished] and b-amyrin 24-hydroxylase (CYP93E1) [9] catalyze the oxidation of b-amyrin at positions C-22 (one-step oxidation) and C-24 (one-step oxidation), respectively, to produce soyasapogenol B (22-hydroxy, 24-hydroxyb-amyrin), the triterpene aglycone of soyasaponin I (Figure 12.1). Construction of vector plasmids for multi-gene transformation is an essential technique for pathway engineering of secondary metabolites. The construction of complex plasmids for expressing multiple genes is considered to be a timeconsuming process, but we were able to reduce the difficulty by connecting the gene cassettes in tandem at the restriction sites of homing endonucleases [10], which recognize 18 to 39 specific bases, and using a polymerase chain reaction (PCR) technique based on primer overlap extension [11]. The terminator regulates the level of expression by controlling transcriptional termination and 30-endprocessing of mRNA. Arabidopsis heat shock protein18.2

Triterpenoid saponins are major components of secondary metabolites in soybean seeds and are divided into two groups: group A saponins, and 2,3-dihydro-2,5-dihydroxy-6-methyl-4H-pyran-4-one (DDMP) saponins. The aglycone moiety of group A saponins consists of soyasapogenol A (SA), which is an oxidized β-amyrin product, and the aglycone moiety of the DDMP saponins consists of soyasapogenol B (SB). Group A saponins produce a bitter and astringent aftertaste in soy products, whereas DDMP saponins have known health benefits for humans. We completed map-based cloning and characterization of the gene Sg-5, which is responsible for SA biosynthesis. The naturally occurring sg-5 mutant lacks group A saponins and has a loss-of-function mutation (L164*) in Glyma15g39090, which encodes the cytochrome P450 enzyme, CYP72A69. An enzyme assay indicated the hydroxylase activity of recombinant CYP72A69 against SB, which also suggested the production of SA. Additionally, induced Glyma15g39090 mutants (R44* or S348P) lacked group A saponins similar to the sg-5 mutant, indicating that Glyma15g39090 corresponds to Sg-5. Endogenous levels of DDMP saponins were higher in the sg-5 mutant than in the wild-type lines due to the loss of the enzyme activity that converts SB to SA. Interestingly, the genomes of palaeopolyploid soybean and the closely related common bean carry multiple Sg-5 paralogs in a genomic region syntenic to the soybean Sg-5 region. However, SA did not accumulate in common bean samples, suggesting that Sg-5 activity evolved after gene duplication event(s). Our results demonstrate that metabolic switching of undesirable saponins with beneficial saponins can be achieved in soybean by disabling Sg-5.

Steroidal glycoalkaloids (SGAs) are cholesterol-derived specialized metabolites produced in species of the Solanaceae. Here, we report that a group of jasmonate-responsive transcription factors of the ETHYLENE RESPONSE FACTOR (ERF) family (JREs) are close homologs of alkaloid regulators in Cathranthus roseus and tobacco, and regulate production of SGAs in tomato. In transgenic tomato, overexpression and dominant suppression of JRE genes caused drastic changes in SGA accumulation and in the expression of genes for metabolic enzymes involved in the multistep pathway leading to SGA biosynthesis, including the upstream mevalonate pathway. Transactivation and DNA-protein binding assays demonstrate that JRE4 activates the transcription of SGA biosynthetic genes by binding to GCC box-like elements in their promoters. These JRE-binding elements occur at significantly higher frequencies in proximal promoter regions of the genes regulated by JRE genes, supporting the conclusion that JREs mediate transcriptional co-ordination of a series of metabolic genes involved in SGA biosynthesis.

Chemical investigation of the glandular trichome exudate of Verbascum blattaria f. erubescens (Scrophulariaceae) led to the isolation of four new glycerides, 1-O-acetyl-2-O-[(R)-3-hydroxytetradecanoyl]-sn-glycerol, 1-O-acetyl-2-O-[(R)-3-hydroxyhexadecanoyl]-sn-glycerol, 2-O-[(R)-3-hydroxytetradecanoyl]glycerol and 2-O-[(R)-3-hydroxyhexadecanoyl]glycerol. Their structures, including the configurations at the stereogenic centres, were elucidated from spectral evidence and chemical transformations.

Plants produce structurally diverse triterpenoids, which are important for their life and survival. Most triterpenoids and sterols share a common biosynthetic intermediate, 2,3-oxidosqualene (OS), which is cyclized by 2,3-oxidosqualene cyclase (OSC). To investigate the role of an OSC, marneral synthase 1 (MRN1), in planta, we characterized a Arabidopsis mrn1 knock-out mutant displaying round-shaped leaves, late flowering, and delayed embryogenesis. Reduced growth of mrn1 was caused by inhibition of cell expansion and elongation. Marnerol, a reduced form of marneral, was detected in Arabidopsis overexpressing MRN1, but not in the wild type or mrn1. Alterations in the levels of sterols and triterpenols and defects in membrane integrity and permeability were observed in the mrn1. In addition, GUS expression, under the control of the MRN1 gene promoter, was specifically detected in shoot and root apical meristems, which are responsible for primary growth, and the mRNA expression of Arabidopsis clade II OSCs was preferentially observed in roots and siliques containing developing seeds. The eGFP:MRN1 was localized to the endoplasmic reticulum in tobacco protoplasts. Taken together, this report provides evidence that the unusual triterpenoid pathway via marneral synthase is important for the growth and development of Arabidopsis.

Chemical investigation of the glandular trichome exudate from Ceratotheca triloba (Pedaliaceae) led to the identification of nine 1-O-acetyl-2-O-[(R)-3-acetyloxy-fatty acyl]-3-O-malonylglycerols. Among these, 1-O-acetyl-2-O-[(R)-3-acetyloxyicosanoyl]-3-O-malonylglycerol (7) was the most abundant constituent (41%), followed by 1-O-acetyl-2-O-[(R)-(3-acetyloxyoctadecanoyl)-3-O-malonylglycerol (2; 21%). Compounds having iso- and anteiso-type structures in the 3-acetyloxy-fatty acyl groups in the fatty acyl moiety were also characterized as minor constituents. This is the first report of the isolation of malonylated glycerolipids as natural products.

Glycyrrhizin, a triterpenoid saponin derived from the underground parts of Glycyrrhiza plants (licorice), has several pharmacological activities and is also used worldwide as a natural sweetener. The biosynthesis of glycyrrhizin involves the initial cyclization of 2,3-oxidosqualene to the triterpene skeleton β-amyrin, followed by a series of oxidative reactions at positions C-11 and C-30, and glycosyl transfers to the C-3 hydroxyl group. We previously reported the identification of a cytochrome P450 monooxygenase (P450) gene encoding β-amyrin 11-oxidase (CYP88D6) as the initial P450 gene in glycyrrhizin biosynthesis. In this study, a second relevant P450 (CYP72A154) was identified and shown to be responsible for C-30 oxidation in the glycyrrhizin pathway. CYP72A154 expressed in an engineered yeast strain that endogenously produces 11-oxo-β-amyrin (a possible biosynthetic intermediate between β-amyrin and glycyrrhizin) catalyzed three sequential oxidation steps at C-30 of 11-oxo-β-amyrin supplied in situ to produce glycyrrhetinic acid, a glycyrrhizin aglycone. Furthermore, CYP72A63 of Medicago truncatula, which has high sequence similarity to CYP72A154, was able to catalyze C-30 oxidation of β-amyrin. These results reveal a function of CYP72A subfamily proteins as triterpene-oxidizing enzymes and provide a genetic tool for engineering the production of glycyrrhizin.

Triterpenoids are a diverse group of secondary metabolites that are associated with a variety of biological activities. Oleanolic acid, ursolic acid and betulinic acid are common triterpenoids in plants with diverse biological activities, including antifungal, antibacterial, anti-human immunodeficiency virus (HIV) and/or antitumor activities. In the present study, using the gene co-expression analysis tool of Medicago truncatula, we found a strong correlation between CYP716A12 and β-amyrin synthase (bAS), which encodes the enzyme responsible for the initial cyclization of 2,3-oxidosqualene to β-amyrin (the basic structural backbone of most triterpenoid saponins). Through an in vitro assay, we identified CYP716A12 as a β-amyrin 28-oxidase able to modify β-amyrin to oleanolic acid (through erythrodiol and, possibly, oleanolic aldehyde). We also confirmed its activity in vivo, by expressing CYP716A12 in transgenic yeast that endogenously produce β-amyrin. In addition, CYP716A12 was evaluated for its potential α-amyrin- and lupeol-oxidizing activities. Interestingly, CYP716A12 was able to generate ursolic acid (through uvaol and, possibly, ursolic aldehyde) and betulinic acid (through betulin). Hence, CYP716A12 was characterized as a multifunctional enzyme with β-amyrin 28-oxidase, α-amyrin 28-oxidase and lupeol 28-oxidase activities. We also identified homologs of CYP716A12 in grape (CYP716A15 and CYP716A17) that are involved in triterpenoid biosynthesis, which indicates the highly conserved functionality of the CYP716A subfamily among plants. These findings will be useful in the heterologous production of pharmacologically and industrially important triterpenoids, including oleanolic acid, ursolic acid and betulinic acid.

Chemical investigation of the glandular trichome exudate from Geranium carolinianum L. (Geraniaceae) led to the characterization of unique disaccharide derivatives, n-octyl 4-O-isobutyryl-α-L-rhamnopyranosyl-(1→2)-6-O-isobutyryl-β-D-glucopyranoside (1), n-octyl 4-O-isobutyryl-α-L-rhamnopyranosyl-(1→2)-6-O-(2-methylbutyryl)-β-D-glucopyranoside (2) and n-octyl 4-O-(2-methylbutyryl)-α-L-rhamnopyranosyl-(1→2)-6-O-isobutyryl-β-D-glucopyranoside (3), named caroliniasides A-C, respectively. These structures were determined by spectral means. n-Alkyl glycoside derivatives have been isolated from the glandular trichome exudates for the first time. This rare type of secondary metabolites could be applicable to chemotaxonomic perspective because they are found in glandular trichome exudates of plants belonging to the genus Geranium, according to our studies.

The C-26 amino group of tomatine, a representative Solanaceae steroidal alkaloid, is introduced in an early step of its biosynthesis from cholesterol. We recently proposed a transamination mechanism for the C-26 amination as opposed to the previously proposed mechanism involving a nitrogen nucleophilic displacement. In the present study, a deuterium labeled C-26 aldehyde, (24,24,27,27,27- 2 H 5 )-3β-hydroxycholest-5-en-26-al, was synthesized and fed to a tomato ( Solanum lycopersicum ) seedling. LC–MS analysis of the biosynthesized tomatine indicated that the labeled aldehyde was incorporated into tomatine. The finding strongly supports the intermediacy of the aldehyde and the transamination mechanism during C-26 amination.

Dried roots and stolons of licorice plants (Glycyrrhiza uralensis) are among the most important drugs (Glycyrrhizae radix) in traditional oriental medicine; they are also important commercial products used worldwide in sweetening and flavoring. Here, we describe the establishment of an in vitro stolon culture system for G. uralensis. Stolon formation was induced in single node stems (with axillary buds) grown in Murashige and Skoog (MS) liquid culture medium, supplemented with 0.01 μM α-naphthaleneacetic acid (NAA). Stolons were cultured at 26°C in the dark on a rotary shaker, gyrating at 100 rpm. The same NAA concentration produced the highest rates of stolon proliferation (6.58-fold in 4 weeks). A 6% sucrose concentration also enhanced stolon proliferation (6.34-fold in 4 weeks). GC/MS analysis confirmed the accumulation of small amounts of glycyrrhizin (14 μg g−1 dry weight) in cultured stolons. Interestingly, betulinic acid and oleanoic acid production in vitro were higher than in field-grown stolons. Adventitious root and shoot regeneration from cultured stolons were readily achieved under illuminated conditions in MS medium containing 0.01 μM of NAA and 0.2% gerlite. Regenerated plants produced glycyrrhizin (7,600 μg g−1 dry weight) in their roots. Our in vitro stolon culture system is suitable for studying glycyrrhizin biosynthesis and for rapid propagation of elite licorice clones.

Plants have two isoprenoid biosynthetic pathways: the cytosolic mevalonate (MVA) pathway and the plastidic 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway. Since the discovery of the MEP pathway, possible metabolic cross-talk between these pathways has prompted intense research. Although many studies have shown the existence of such cross-talk using feeding experiments, it remains to be determined if native cross-talk, rather than exogenously applied metabolites, can compensate for complete blockage of the MVA pathway. Previously, Arabidopsis mutants for HMG1 and HMG2 encoding HMG-CoA reductase (HMGR) were isolated. Although it was shown that HMGR1 is a functional HMGR, the enzyme activity of HMGR2 has not been confirmed. It is demonstrated here that HMG2 encodes a functional reductase with similar activity to HMGR1, using enzyme assays and complementation experiments. To estimate the contribution of native cross-talk, an attempt was made to block the MVA pathway by making double mutants lacking both HMG1 and HMG2, but no double homozygotes were detected in the progeny of self-pollinated HMG1/hmg1 hmg2/hmg2 plants. hmg1 hmg2 male gametophytes appeared to be lethal based on crossing experiments, and microscopy indicated that approximately 50% of the microspores from the HMG1/hmg1 hmg2/hmg2 plant appeared shrunken and exhibited poorly defined endoplasmic reticulum membranes. In situ hybridization showed that HMG1 transcripts were expressed in both the tapetum and microspores, while HMG2 mRNA appeared only in microspores. It is concluded that native cross-talk from the plastid cannot compensate for complete blockage of the MVA pathway, at least during male gametophyte development, because either HMG1 or HMG2 is required for male gametophyte development.

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 β-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.

Dolichols are long-chain unsaturated polyisoprenoids with multiple cellular functions, such as serving as lipid carriers of sugars used for protein glycosylation, which affects protein trafficking in the endoplasmic reticulum. The biological functions of dolichols in plants are largely unknown. We isolated an Arabidopsis thaliana mutant, lew1 (for leaf wilting1), that showed a leaf-wilting phenotype under normal growth conditions. LEW1 encoded a cis-prenyltransferase, which when expressed in Escherichia coli catalyzed the formation of dolichol with a chain length around C80 in an in vitro assay. The lew1 mutation reduced the total plant content of main dolichols by ∼85% and caused protein glycosylation defects. The mutation also impaired plasma membrane integrity, causing electrolyte leakage, lower turgor, reduced stomatal conductance, and increased drought resistance. Interestingly, drought stress in the lew1 mutant induced higher expression of the unfolded protein response pathway genes BINDING PROTEIN and BASIC DOMAIN/LEUCINE ZIPPER60 as well as earlier expression of the stress-responsive genes RD29A and COR47. The lew1 mutant was more sensitive to dark treatment, but this dark sensitivity was suppressed by drought treatment. Our data suggest that LEW1 catalyzes dolichol biosynthesis and that dolichol is important for plant responses to endoplasmic reticulum stress, drought, and dark-induced senescence in Arabidopsis.

The hairy root culture system has several desirable features as an experimental model for functional analyses of the genes involved in root metabolism and/or physiology. We developed a binary vector set for efficient target gene overexpression and RNA interference (RNAi) in transformed (hairy) roots. The vectors, pHR-OX and pHR-RNAi, contain a cluster of rol (rooting locus) genes, together with the single GATEWAYTM conversion cassette (in pHR-OX vectors) or inverted repeats of the GATEWAYTM conversion cassette separated by an intron sequence (in pHR-RNAi vectors), flanked by a dual CaMV 35S promoter and nopaline synthase polyadenylation signal, on the same T-DNA. Transformation experiments with pHR-OX vectors using Arabidopsis, potato, and tobacco as model plants revealed that by inoculating Agrobacterium tumefaciens harboring these vectors, a large number of independently transformed roots could be induced from explants in a short period of time, and subsequent establishment of root culture lines was possible. A model experiment that focused on the sterol biosynthetic pathway in Arabidopsis validated the utility of pHR-RNAi vectors for gene functional analysis in cultured roots. Use of our vector system may facilitate identification of regulatory or biosynthetic genes for the production of valuable secondary metabolites in plant roots.

Isoprene is a volatile compound emitted from leaves of many plant species in large quantities, which has an impact on atmospheric chemistry due to its massive global emission rate (5 × 1014 carbon g year−1) and its high reactivity with the OH radical, resulting in an increase in the half-life of methane. Isoprene emission is strongly induced by the increase in isoprene synthase activity in plastids at high temperature in the day time, which is regulated at its gene expression level in leaves, while the physiological meaning of isoprene emission for plants has not been clearly demonstrated. In this study, we have functionally overexpressed Populus alba isoprene synthase in Arabidopsis to observe isoprene emission from transgenic plants. A striking difference was observed when both transgenic and wild-type plants were treated with heat at 60°C for 2.5 h, i.e. transformants revealed clear heat tolerance compared with the wild type. High isoprene emission and a decrease in the leaf surface temperature were observed in transgenic plants under heat stress treatment. In contrast, neither strong light nor drought treatments showed an apparent difference. These data suggest that isoprene emission plays a crucial role in a heat protection mechanism in plants.

20-Hydroxyecdysone (20HE), a molting hormone of insects, is also distributed among a variety of plant families. 20HE is thought to play a role in protecting plants from insect herbivores. In insects, biosynthesis of 20HE from cholesterol proceeds via 7-dehydrocholesterol and 3β,14α-dihydroxy-5β-cholest-7-en-6-one (5β-ketodiol), the latter being converted to 20HE through sequential hydroxylation catalyzed by four P450 enzymes, which have been cloned and identified. In contrast, little is known about plant 20HE biosynthesis, and no biosynthetic 20HE gene has been reported thus far. We recently proposed involvement of 3β-hydroxy-5β-cholestan-6-one (5β-ketone) in 20HE biosynthesis in the hairy roots of Ajuga reptans var. atropurpurea (Lamiaceae). In this study, an Ajuga EST library was generated from the hairy roots and P450 genes were deduced from the library. Five genes with a high expression level (CYP71D443, CYP76AH19, CYP76AH20, CYP76AH21 and CYP716D27) were screened for a possible involvement in 20HE biosynthesis. As a result, CYP71D443 was shown to have C-22 hydroxylation activity for the 5β-ketone substrate using a yeast expression system. The hydroxylated product, 22-hydroxy-5β-ketone, had a 22R configuration in agreement with that of 20HE. Furthermore, labeling experiments indicated that (22R)-22-hydroxy-5β-ketone was converted to 20HE in Ajuga hairy roots. Based on the present results, a possible 20HE biosynthetic pathway in Ajuga plants involved CYP71D443 is proposed.

Sterols are important as structural components of plasma membranes and precursors of steroidal hormones in both animals and plants. Plant sterols show a wide structural variety and significant structural differences from those of animals. To elucidate the origin of structural diversity in plant sterols, their biosynthesis has been extensively studied [Benveniste (2004) Annu. Rev. Plant. Biol. 55: 429, Schaller (2004) Plant Physiol. Biochem. 42: 465]. The differences in the biosynthesis of sterols between plants and animals begin at the step of cyclization of 2,3-oxidosqualene, which is cyclized to lanosterol in animals and to cycloartenol in plants. However, here we show that plants also have the ability to synthesize lanosterol directly from 2,3-oxidosqualene, which may lead to a new pathway to plant sterols. The Arabidopsis gene At3g45130, designated LAS1, encodes a functional lanosterol synthase in plants. A phylogenetic tree showed that LAS1 belongs to the previously uncharacterized branch of oxidosqualene cyclases, which differs from the cycloartenol synthase branch. Panax PNZ on the same branch was also shown to be a lanosterol synthase in a yeast heterologous expression system. The higher diversity of plant sterols may require two biosynthetic routes in steroidal backbone formation.

3-Hydroxy-3-methylglutaryl-CoA reductase (HMGR) catalyzes the first committed step in the cytosolic isoprenoid biosynthesis pathway in higher plants. To understand the contribution of HMGR to plant development, we isolated T-DNA insertion mutants for HMG1 and HMG2. The hmg1 and hmg2 mutants were both more sensitive than the wild type (WT) to lovastatin, an inhibitor of HMGR. The hmg2 mutant showed no visible phenotype under normal growth conditions. In contrast, the hmg1 mutant exhibited dwarfing, early senescence, and sterility. Expression of senescence-associated genes 12 (SAG12), a marker gene for senescence, was induced in the hmg1 mutant at an earlier stage than in the WT. Levels of trans-cytokinins--hormones known to inhibit senescence--were not lower in hmg1. The mutant did not have the typical appearance of brassinosteroid (BR)-deficient mutants, except for a dwarf phenotype, because of the suppression of cell elongation. The expression of several genes involved in cell elongation was suppressed in hmg1. WT plants treated exogenously with inhibitors of sterol biosynthesis had similar gene expression and sterility characteristics as the hmg1 mutants. Pleiotropic phenotypes were rescued by feeding with squalene, the precursor of sterols and triterpenoids. The sterol levels in hmg1 mutants were lower than in the WT. These findings suggest that HMG1 plays a critical role in triterpene biosynthesis, and that sterols and/or triterpenoids contribute to cell elongation, senescence, and fertility.

Potatoes (Solanum tuberosum) contain α-solanine and α-chaconine, two well-known toxic steroidal glycoalkaloids (SGAs). Sprouts and green tubers accumulate especially high levels of SGAs. Although SGAs were proposed to be biosynthesized from cholesterol, the biosynthetic pathway for plant cholesterol is poorly understood. Here, we identify sterol side chain reductase 2 (SSR2) from potato as a key enzyme in the biosynthesis of cholesterol and related SGAs. Using in vitro enzyme activity assays, we determined that potato SSR2 (St SSR2) reduces desmosterol and cycloartenol to cholesterol and cycloartanol, respectively. These reduction steps are branch points in the biosynthetic pathways between C-24 alkylsterols and cholesterol in potato. Similar enzymatic results were also obtained from tomato SSR2. St SSR2-silenced potatoes or St SSR2-disrupted potato generated by targeted genome editing had significantly lower levels of cholesterol and SGAs without affecting plant growth. Our results suggest that St SSR2 is a promising target gene for breeding potatoes with low SGA levels.

3β-Hydroxy-5β-cholestan-6-one was identified in the EtOAc extract of Ajuga hairy roots by micro-analysis using LC-MS/MS in the multiple reaction mode (MRM). Furthermore, administration of (2,2,4,4,7,7-(2)H6)- and (2,2,4,4,6,7,7-(2)H7)-cholesterols to the hairy roots followed by LC-MS/MS analysis of the EtOAc extract of the hairy roots indicated that cholesterol was converted to the 5β-ketone with hydrogen migration from the C-6 to the C-5 position. These findings, in conjunction with the previous observation that the ketone was efficiently converted to 20-hydroxyecdysone, strongly suggest that the 5β-ketone is an intermediate immediately formed after cholesterol during 20-hydroxyecdysone biosynthesis in Ajuga sp. In addition, the mechanism of the 5β-ketone formation from cholesterol is discussed.

Hairy roots of Ajuga reptans var. atropurpurea produce clerosterol, 22-dehydroclerosterol, and cholesterol as sterol constituents, and 20-hydroxyecdysone, cyasterone, isocyasterone, and 29-norcyasterone as ecdysteroid constituents. To better understand the biosynthesis of these steroidal compounds, we carried out feeding studies of variously 2H- and 13C-labeled sterol substrates with Ajuga hairy roots. In this article, we review our studies in this field. Feeding of labeled desmosterols, 24-methylenecholesterol, and 13C2-acetate established the mechanism of the biosynthesis of the two C29-sterols and a newly accumulated codisterol, including the metabolic correlation of C-26 and C-27 methyl groups. In Ajuga hairy roots, 3alpha-, 4alpha-, and 4beta-hydrogens of cholesterol were all retained at their original positions after conversion into 20-hydroxyecdysone, in contrast to the observations in a fern and an insect. Furthermore, the origin of 5beta-H of 20-hydroxyecdysone was found to be C-6 hydrogen of cholesterol exclusively, which is inconsistent with the results in the fern and the insect. These data strongly support the intermediacy of 7-dehydrocholesterol 5alpha,6alpha-epoxide. Moreover, 7-dehydrocholesterol, 3beta-hydroxy-5beta-cholest-7-en-6-one (5beta-ketol), and 3beta,14alpha-dihydroxy-5beta-cholest-7-en-6-one (5beta-ketodiol) were converted into 20-hydroxyecdysone. Thus, the pathway cholesterol--7-dehydrocholesterol--7-dehydrocholesterol 5alpha,6alpha-epoxide--5beta-ketol--5beta-k etodiol is proposed for the early stages of 20-hydroxyecdysone biosynthesis. 3beta-Hydroxy-5beta-cholestan-6-one was also incorporated into 20-hydroxyecdysone, suggesting that the introduction of a 7-ene function is not necessarily next to cholesterol. C-25 Hydroxylation during 20-hydroxyecdysone biosynthesis was found to proceed with ca. 70% retention and 30% inversion. Finally, clerosterol was shown to be a precursor of cyasterone and isocyasterone.

The fate of 6alpha- and 6beta-hydrogens of lathosterol during the transformation into 20-hydroxyecdysone was chased by feeding [3alpha,6beta-2H2]- and [3alpha,6alpha-2H2]-lathosterols to hairy roots of Ajuga reptans var. atropurpurea. The behavior of 6beta-hydrogen, which mostly migrated to the C-5 position of 20-hydroxyecdysone, was in agreement with that of C-6 hydrogen of cholesterol. The results strongly supported the view that cholesterol and lathosterol are first metabolized into 7-dehydrocholesterol, which is then converted into 20-hydroxyecdysone via 7-dehydrocholesterol 5alpha,6alpha-epoxide in the hairy roots.

Triterpenoid saponins are a diverse group of specialized (secondary) metabolites with many biological properties. The model legume Medicago truncatula has an interesting profile of triterpenoid saponins from which sapogenins are differentiated into hemolytic and non-hemolytic types according to the position of their functional groups and hemolytic properties. Gene co-expression analysis confirmed the presence of candidate P450s whose gene expression correlated highly with that of b-amyrin synthase (bAS). Among these, we identified CYP716A12 and CYP93E2 as key enzymes in hemolytic and non-hemolytic sapogenin biosynthetic pathways. The other candidate P450s showed no b-amyrin oxidation activity. However, among the remaining candidate P450s, CYP72A61v2 expression highly correlated with that of CYP93E2, and CYP72A68v2 expression highly correlated with that of CYP716A12. These correlation values were higher than occurred with bAS expression. We generated yeast strains expressing bAS, CPR, CYP93E2 and CYP72A61v2, and bAS, CPR, CYP716A12 and CYP72A68v2. These transgenic yeast strains produced soyasapogenol B and gypsogenic acid, respectively. We were therefore able to identify two CYP72A subfamily enzymes: CYP72A61v2, which modifies 24-OH-b-amyrin, and CYP72A68v2, which modifies oleanolic acid. Additionally, P450s that seemed not to work together in planta were combinatorially expressed in transgenic yeast. The yeast strains (expressing bAS, CPR, CYP72A63 and CYP93E2 or CYP716A12) produced rare triterpenoids that do not occur in M. truncatula. These results show the potential for combinatorial synthesis of diverse triterpenoid structures and enable identification of the enzymes involved in their biosynthesis.

In the past, we have questioned the accuracy of the stereochemistry of elisabethatriene, a putative biosynthetic intermediate of pseudopterosins, in light of the configuration of elisabethatrienol isolated from Pseudopterogorgia elisabethae, which was represented as 1S,4R,9S,11S. We have reinvestigated the stereochemistry of elisabethatriene. Elisabethatriene with the reported 1S,4R,9R,11S configuration was synthesized starting from (-)-isopulegol in its enantiomeric form. The (1)H- and (13)C-NMR data of the synthesized compound differed from those reported for elisabethatriene. In addition to the fact that elisabethatriene is converted into pseudopterosins, this finding has allowed us to propose that elisabethatriene should have the 1S,4R,9S,11S stereochemistry, which is identical to that of elisabethatrienol.

In plants, more than 200 triterpenes with various carbon skeletons have been found, of which some triterpenes are biologically active. Here we show recent detailed analyses on the biosynthetic genes and the mechanism of triterpene biosynthesis characteristic of higher plants. The studies presented here employed molecular genetic tools and organic chemistry methods to focus on the function of HMG-CoA reductase, the triterpene biosynthesis rate-limiting enzyme, and oxidosqualene cyclase, the enzyme catalyzing the formation of the cyclic triterpene skeletons.

The absorption, distribution and excretion of a benzoylphenylurea derivative, HO-221 were studied after repeated administration of 14C(Pc)-HO-221 once a day for a maximum of 28 days to male rats at a dose of 25 mg/kg. The radioactivity levels in the blood at 24 hr after daily dosing reached a steady state after the 20th dosing, being 2 times higher than after the 7th dosing. The radioactivity levels in all the tissues after the 28th dosing were less than 2 times of those after single dosing. The radioactivity was still detected at 240 hr after administration but only in the kidney and liver. The excretion of radioactivity in the urine and feces 24 hr after daily dosing did not change after the 4th dosing, and after the final repeated dosing was similar to that after single dosing. The metabolism was investigated after administration of 14C(Pc)-HO-221 or 14C(Pn)-HO-221 to male rats. The parent compound accounted for about 70% of the radioactivity in the plasma at 6 hr after administration. M5, M6, M7 and ο-nitrobenzamide were also detected in the plasma. The parent compound and M5 were principally observed in the liver, kid ney and fat. The conjugate of M6 was major metabolite in the urine, accounting for about 46% of the radioactivity in the urine. Unknown metabolites were major components in the bile. The parent compound and M5 were major in the feces. No difference was noted in urinary and fecal metabolites between the single and repeated dosing groups. UDPG transferase activity increased significantly by repeated dosing of HO-221.

The absorption, distribution and excretion of a benzoylphenylurea derivative, HO-221 were studied in rats after single oral administration of 14C(Pc)-HO-221 or 14C(Pn)-HO-221. 1. The radioactivity level in the blood reached a maximum at 12 hr after administration of 14C(Pc)-HO-221 to male and female rats at a dose of 25 mg/kg. The absorption ratio was about 9 % as caluculated from the mass balance study. The Cm and AUC were not dose-related at the dose range of 8.3 to 25 mg/kg. The absorption from the digestive tracts tended to decrease by fasting. The Cmax and AUC derived from 14C(Pn)-HO-221 were both 2 times higher than those derived from 14C(Pc)-HO-221. 2. The radioactivity levels in the fat, liver, brown fat, adrenal gland and Harderian gland were high after administration of 14C(Pc)-HO-221 to male and female rats. The radioactivity in the tissues was eliminated rapidly, except the kidney and liver. 3. The excretion of radioactivity in the urine and feces was about 2 and 95% of the dose, respectively, within 120 hr after administration of 14C (Pc)-HO-221 to male and female rats. The excretion of radioactivity in the bile was about 5% of the dose. About 30% of the biliary excreta (1% of the dose) was re-absorbed. The excretion of radioactivity in the urine was 3 % of the dose within 120 hr after administration of 14C(Pn)-HO 221. 4. The protein binding ratios in vitro and vivo were more than 99% but the binding was reversible.

The absorption, metabolism and excretion of benzoylphenylurea derivative, HO-221 in male dogs were studied after oral administration of 14C(Pc)-HO-221 at a dose of 25 mg/kg. 1. The radioactivity levels in the plasma reached a maximum at 24 hr after administration and declined with a half-life of 220 hr up to 120 hr. 2. The excretion of radioactivity in the urine and feces was 0.2 and 98.2% of the dose, respectively, within 120 hr after administration. The excretion of radioactivity in the bile was 0.5% of the dose within 48 hr after administration. 3. The protein binding ratios in vitro and vivo were more than 99% both in the case of dog or human plasma, but binding was reversible. 4. The parent compound was a major component in the plasma. M5 and M6 were also observed. Polar metabolites in origin accounted for about 80% of the radioactivity in the urine. The parent compound was a major component in the feces and M5 was detected as a minor metabolite. Polar metabolites in origin were major in the bile, part of which were conjugates.

Biosynthesis of clerosterol, (24 S )-ethylcholesta-5,25-dien-3β-ol ( 1 ), involves transfer of the methyl group from S -adenosylmethionine to the C-28 position of a 24-methylene-sterol precursor. The resulting C-24 cationic species undergoes migration of hydrogen from C-25 to C-24, followed by deprotonation from C-26 to form 1 . We have now investigated the steric course of the methylation in hairy roots of Ajuga reptans var. atropurpurea . Feeding of [28 E - 2 H]- and [28 Z - 2 H]-24-methylenecholesterols and 2 H NMR analysis of clionasterol obtained by partial hydrogenation of the biosynthesized clerosterol have revealed that the methylation takes place from the 28- si face.

Barley (Hordeum vulgare) has a much higher content of bioactive substances than wheat (Triticum aestivum). In order to investigate additive and/or synergistic effect(s) on the phytosterol content of barley chromosomes, we used a series of barley chromosome addition lines of common wheat that were produced by normal crossing. In determining the plant sterol levels in 2-week-old seedlings and dry seeds, we found that the level of stigmasterol in the barley chromosome 3 addition (3H) line in the seedlings was 1.5-fold higher than that in the original wheat line and in the other barley chromosome addition lines, but not in the seeds. Simultaneously, we determined the overall expression pattern of genes related to plant sterol biosynthesis in the seedlings of wheat and each addition line to assess the relative expression of each gene in the sterol pathway. Since we elucidated the CYP710A8 (cytochrome P450 subfamily)-encoding sterol C-22 desaturase as a key characteristic for the higher level of stigmasterol, full-length cDNAs of wheat and barley CYP710A8 genes were isolated. These CYP710A8 genes were mapped on chromosome 3 in barley (3H) and wheat (3A, 3B, and 3D), and the expression of CYP710A8 genes increased in the 3H addition line, indicating that it is responsible for stigmasterol accumulation. Overexpression of the CYP710A8 genes in Arabidopsis increased the stigmasterol content but did not alter the total sterol level. Our results provide new insight into the accumulation of bioactive compounds in common wheat and a new approach for assessing plant metabolism profiles.

Fourteen cyclic glycolipids, named glomerasides A-N, have been isolated from the glandular trichome exudate of Cerastium glomeratum (Caryophyllaceae). Their structures were determined by spectroscopic analysis of the glycolipids, as well as by application of the Ohrui-Akasaka method to the fatty acid methyl esters derived from the glycolipids and GCMS studies of trimethylsilyl ether derivatives of the methyl esters. The various glomerasides have a glycosidic linkage between the anomeric hydroxy group of the glucose and the C-11, C-10 or C-9 positions of the docosanoyl moiety. They also contained an ester linkage between the C-6 hydroxy group of the glucose ring and the carboxyl group of the oxygenated fatty acid to form their macrocyclic structures. The glucose moiety was optionally acetylated and/or malonylated at the C-2 or C-3 hydroxy groups. Among these compounds, the 1,6'-cyclic ester of 11(R)-(2-O-acetyl-β-d-glucopyranosyloxy)docosanoic acid (glomeraside D) was the most abundant (25%).

Chemical shifts of the diastereotopic methyl groups (C-26 and C-27) of 24-methylenecholesterol have been unambiguously assigned by synthesizing stereochemically defined 13 C-labeled compounds. This allowed us to establish the steric course of hydrogen migration from C-24 to C-25 during the formation of 24-methylenecholesterol from a Δ 24 -precursor in a tissue culture of Catharanthus roseus.

A novel chlorophenol, 4-chloro-2-(hepta-1,3,5-triyn-1-yl)-phenol (1), was isolated as the major phenolic compound from the cells of Helichrysum aureonitens suspension cultures. Compound 1 has been proposed to be an intermediate in the acetylene biosynthetic pathway of other acetylenic compounds in Helichrysum spp. The ethanol extract of cell suspension cultures and compound 1 were evaluated for their cytotoxicity against monkey kidney Vero (Vero cells) and human prostate epithelial carcinoma (DU145) cell lines, also, the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) against Mycobacterium tuberculosis H37Rv were determined as well.

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.

A novel chlorophenol, 4-chloro-2-(hepta-1,3,5-triyn-1-yl)-phenol (1), was isolated as the major phenolic compound from the cells of Helichrysum aureonitens suspension cultures. Compound 1 has been proposed to be an intermediate in the acetylene biosynthetic pathway of other acetylenic compounds in Helichrysum spp. The ethanol extract of cell suspension cultures and compound 1 were evaluated for their cytotoxicity against monkey kidney Vero (Vero cells) and human prostate epithelial carcinoma (DU145) cell lines, also, the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) against Mycobacterium tuberculosis H37Rv were determined as well.

Euphorbia tirucalli L., which is also known as a petroleum plant, produces a large amount of phytosterols and triterpenes. During their biosynthesis, squalene synthase converts two molecules of the hydrophilic substrate farnesyl diphosphate into a hydrophobic product, squalene. An E. tirucalli cDNA clone of a putative squalene synthase gene (EtSS) was isolated by RT-PCR followed by 5′- and 3′-RACE. The restriction fragment polymorphisms revealed by Southern blot analysis suggest that EtSS is a single copy gene. The glycine at the 287th residue from the N-terminal end of domain C has replaced alanine, which is conserved among all the other SS sequences deposited in the Genbank database. The N-terminal 380 residues of the hydrophilic sequence was expressed as a peptide-tagged protein in E. coli, and the resultant bacterial crude extract was incubated with farnesyl diphosphate and NADPH. GC-MS analysis showed that squalene was detected in the in vitro reaction mixture. E. tirucalli transgenic callus lines, in which EtSS was overexpressed, accumulated increased amounts of phytosterols as compared with that of wild type callus. RT-PCR analysis of wild type E. tirucalli plants revealed that the EtSS transcript accumulated in almost equal amounts in the stems and the leaves with a stalk, while a lower amount was detected in the roots. In situ hybridization analysis revealed that prominent antisense-probe signal was detected in the cambia within bundle sheathes. These results indicate that EtSS functions prominently in cambia, which are located adjacent to conductive tubes, and that this gene plays important roles in phytosterol accumulation in petroleum plants.