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Summary Squalene and botryococcene are linear, hydrocarbon triterpenes that have industrial and medicinal values. While natural sources for these compounds exist, there is a pressing need for robust, renewable production platforms. Oilseeds are an excellent target for heterologous production because of their roles as natural storage repositories and their capacity to produce precursors from photosynthetically‐derived carbon. We generated transgenic Arabidopsis thaliana plants using a variety of engineering strategies (subcellular targeting and gene stacking) to assess the potential for oilseeds to produce these two compounds. Constructs used seed‐specific promoters and evaluated expression of a triterpene synthase alone and in conjunction with a farnesyl diphosphate synthase (FPS) plus 1‐deoxyxylulose 5‐phosphate synthase (DXS). Constructs directing biosynthesis to the cytosol to harness isoprenoid precursors from the mevalonic acid (MVA) pathway were compared to those directing biosynthesis to the plastid compartment diverting precursors from the methylerythritol phosphate (MEP) pathway. On average, the highest accumulation for both compounds was achieved by targeting the triterpene synthase, FPS and DXS to the plastid (526.84 μg/g seed for botryococcene and 227.30 μg/g seed for squalene). Interestingly, a higher level accumulation of botryococcene (a non‐native compound) was observed when the biosynthetic enzymes were targeted to the cytosol (>1000 μg/g seed in one line), but not squalene (natively produced in the cytosol). Not only do these results indicate the potential of engineering triterpene accumulation in oilseeds, but they also uncover some the unique regulatory mechanisms controlling triterpene metabolism in different cellular compartments of seeds.

Summary Squalene is a linear intermediate to nearly all classes of triterpenes and sterols and is itself highly valued for its use in wide range of industrial applications. Another unique linear triterpene is botryococcene and its methylated derivatives generated by the alga Botryococcus braunii race B, which are progenitors to fossil fuel deposits. Production of these linear triterpenes was previously engineered into transgenic tobacco by introducing the key steps of triterpene metabolism into the particular subcellular compartments. In this study, the agronomic characteristics (height, biomass accumulation, leaf area), the photosynthetic capacity (photosynthesis rate, conductance, internal CO 2 levels) and triterpene content of select lines grown under field conditions were evaluated for three consecutive growing seasons. We observed that transgenic lines targeting enzymes to the chloroplasts accumulated 50–150 times more squalene than the lines targeting the enzymes to the cytoplasm, without compromising growth or photosynthesis. We also found that the transgenic lines directing botryococcene metabolism to the chloroplast accumulated 10‐ to 33‐fold greater levels than the lines where the same enzymes were targeted to in the cytoplasm. However, growth of these high botryococcene accumulators was highly compromised, yet their photosynthesis rates remained unaffected. In addition, in the transgenic lines targeting a triterpene methyltransferase (TMT) to the chloroplasts of high squalene accumulators, 55%–65% of total squalene was methylated, whereas in the lines expressing a TMT in the cytoplasm, only 6%–13% of squalene was methylated. The growth of these methylated triterpene‐accumulating lines was more compromised than that of nonmethylated squalene lines.

A screen of activation tagged lines of Arabidopsis thaliana retrieved COLD TEMPERATURE GERMINATING10-D(tag) (CTG10-D(tag)) seeds, capable of radicle protrusion in advance of wild type (WT) at suboptimal- and optimal-temperatures. Genomic walking revealed T-DNA in the intragenic region that upregulates expression of the At4g19330 locus previously predicted to encode an F-BOX protein. A combination of surrogate splicing, primer scanning, RACE, and Illumina PolyAdenylation Tag (PAT) sequencing in petunia (Petunia X hybrida) and Arabidopsis were required to demonstrate that the region around At4g19330 was misannotated and is actually compromised of two separate genes. Even though homologous regions nearby and elsewhere on chromosome 4 are confounding elements in the molecular characterization of the locus, we could determine that the 5′ entity encodes a ribonucleoprotein of unknown function whereas the 3′ gene includes the promoter and full coding region of an F-BOX protein. Although both genes were upregulated, only independently-transformed lines over-expressing the F-Box exhibited enhanced completion of seed germination. We named it CTG10, and a single, poorly penetrant, mutant line of ctg10 manifested the expected reduced completion of seed germination. Whereas CTG10-OE lines are hyposensitive to the gibberellin biosynthetic inhibitor paclobutrazol, the ctg10 mutant line is hypersensitive; a phenotype which could be alleviated when transgenically rescued with CTG10. The F-Box moiety promoted association of CTG10 with ASK proteins in yeast two hybrid assays, indicating that it likely assembles into an SCF-type ubiquitin ligase to promote the ubiquitination of one or more substrates. In this capacity CTG10 might target a protein repressing seed germination for polyubiquitination and subsequent proteasomal degradation.

Cleome arabica is a medicinal plant contains diverse bioactive compounds and terpenoids are the major components. However, the isolation and purification of the active triterpenes from this plant involve long and complicated procedures. The present work investigates the triterpenes profiles of different tissues, besides that, describes the isolation, heterologous expression and functional characterization of C. arabica gene coding for triterpenes synthases. The phytochemical investigation through GC-MS revealed significant accumulation of pentacyclic triterpenes in leaves and siliques at mature stage compared to the stems and roots of C. arabica. Among the pentacyclic triterpenes, the lupeol reached the highest level of 320 μg/g DW in leaves at maturity stage compared to the other tissues. The biosynthesis of a pentacyclic triterpene was investigated through isolation and cloning of a full-length oxidosqualene cyclase cDNA (CaOSC) from mature leaves of C. arabica. The bioinformatic analyses revealed that CaOSC was highly homologous with the characterized lupeol synthases and shared 79.3% identity to camelliol C synthase from A. thaliana. Heterologous expression of CaOSC gene in Saccharomyces cerevisiae synthesized lupeol as a single product. The lupeol biosynthesis was exponentially increased after induction through the fermentation process reaching the maximum of 2.33 μg/ml for 240 h. Furthermore, organ-specific expression of lupeol gene was exactly matched the accumulation pattern in different tissues of C. arabica during phenological cycle. Thus, the identified CaOSC will be useful in enhancing triterpene yield for industrial purposes.

The medicinal properties of Valerian (Valeriana officinalis) root preparations are attributed to the anxiolytic sesquiterpenoid valerenic acid and its biosynthetic precursors valerenal and valerenadiene, as well as the anti-inflammatory sesquiterpenoid β-caryophyllene. In order to study and engineer the biosynthesis of these pharmacologically active metabolites, a binary vector co-transformation system was developed for V. officinalis hairy roots. The relative expression levels and jasmonate-inducibility of a number of genes associated with sesquiterpenoid metabolism were profiled in roots: farnesyl pyrophosphate synthase (VoFPS), valerendiene synthase (VoVDS), germacrene C synthase (VoGCS), and a cytochrome P450 (CYP71D442) putatively associated with terpene metabolism based on sequence homology. Recombinant hairy root lines overexpressing VoFPS or VoVDS were generated and compared to control cultures. Overexpression of the VoFPS cDNA increased levels of the corresponding transcript 4- to 8-fold and sesquiterpene hydrocarbon accumulation by 1.5- to 4-fold. Overexpression of the VoVDS cDNA increased the corresponding transcript levels 5- to 9-fold and markedly increased yields of the oxygenated sesquiterpenoids valerenic acid and valerenal. Our findings suggest that the availability of cytoplasmic farnesyl diphosphate and valerenadiene are potential bottlenecks in Valeriana-specific sesquiterpenoid biosynthesis, which is also subject to regulation by methyl jasmonate elicitation.

Nicotiana benthamiana was used as a model to investigate the spatial and developmental relationship between sterol synthesis rates and sterol content in plants. Stigmasterol levels were approximately twice the level in roots as that found in aerial tissues, while its progenitor sterol sitosterol was the inverse. When incorporation of radiolabeled precursors into sterols was used as measure of in vivo synthesis rates, acetate incorporation was similar across all tissue types, but approximately twofold greater in roots than any other tissue. In contrast, mevalonate incorporation exhibited the greatest differential with the rate of incorporation in roots approximately one-tenth that in apical shoots. Similar to acetate, incorporation of farnesol was higher in roots but remained fairly constant in aerial tissues, suggesting less regulation of the downstream sterol biosynthetic steps. Consistent with the precursor incorporation data, analysis of gene transcript and measurements of putative rate-limiting enzyme activities for 3-hydroxy-3-methylglutaryl-coenzyme A synthase (EC 2.3.3.10) and reductase (EC 1.1.1.34) showed the greatest modulation of levels, while the activity levels for isopentenyl diphosphate isomerase (EC 5.3.3.2) and prenyltransferases (EC 2.5.1.10 and EC 2.5.1.1) also exhibited a strong but moderate correlation with the development age of the aerial tissues of the plants. Overall, the data suggest a multitude of means from transcriptional to posttranslational control affecting sterol biosynthesis and accumulation across an entire plant, and point to some particular control points that might be manipulated using molecular genetic approaches to better probe the role of sterols in plant growth and development.

Mouse FIT2 protein redirects the cytoplasmic terpene biosynthetic machinery to lipid-droplet-forming domains in the ER and this relocalization supports the efficient compartmentalization and accumulation of sesquiterpenes in plant cells. Mouse (Mus musculus) fat storage-inducing transmembrane protein 2 (MmFIT2), an endoplasmic reticulum (ER)-resident protein with an important role in lipid droplet (LD) biogenesis in mammals, can function in plant cells to promote neutral lipid compartmentalization. Surprisingly, in affinity capture experiments, the Nicotiana benthamiana 5-epi-aristolochene synthase (NbEAS), a soluble cytoplasm-localized sesquiterpene synthase, was one of the most abundant proteins that co-precipitated with GFP-tagged MmFIT2 in transient expression assays in N. benthamiana leaves. Consistent with results of pull-down experiments, the subcellular location of mCherry-tagged NbEAS was changed from the cytoplasm to the LD-forming domains in the ER, only when co-expressed with MmFIT2. Ectopic co-expression of NbEAS and MmFIT2 together with mouse diacylglycerol:acyl-CoA acyltransferase 2 (MmDGAT2) in N. benthamiana leaves substantially increased the numbers of cytoplasmic LDs and supported the accumulation of the sesquiterpenes, 5-epi-aristolochene and capsidiol, up to tenfold over levels elicited by Agrobacterium infection alone. Taken together, our results suggest that MmFIT2 recruits sesquiterpene synthetic machinery to ER subdomains involved in LD formation and that this process can enhance the efficiency of sesquiterpene biosynthesis and compartmentalization in plant cells. Further, MmFIT2 and MmDGAT2 represent cross-kingdom lipogenic protein factors that may be used to engineer terpene accumulation more broadly in the cytoplasm of plant vegetative tissues.

Summary Triterpenes are thirty‐carbon compounds derived from the universal five‐carbon prenyl precursors isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP). Normally, triterpenes are synthesized via the mevalonate (MVA) pathway operating in the cytoplasm of eukaryotes where DMAPP is condensed with two IPPs to yield farnesyl diphosphate (FPP), catalyzed by FPP synthase (FPS). Squalene synthase (SQS) condenses two molecules of FPP to generate the symmetrical product squalene, the first committed precursor to sterols and most other triterpenes. In the green algae Botryococcus braunii, two FPP molecules can also be condensed in an asymmetric manner yielding the more highly branched triterpene, botryococcene. Botryococcene is an attractive molecule because of its potential as a biofuel and petrochemical feedstock. Because B. braunii, the only native host for botryococcene biosynthesis, is difficult to grow, there have been efforts to move botryococcene biosynthesis into organisms more amenable to large‐scale production. Here, we report the genetic engineering of the model monocot, Brachypodium distachyon, for botryococcene biosynthesis and accumulation. A subcellular targeting strategy was used, directing the enzymes (botryococcene synthase [BS] and FPS) to either the cytosol or the plastid. High titres of botryococcene (>1 mg/g FW in T0 mature plants) were obtained using the cytosolic‐targeting strategy. Plastid‐targeted BS + FPS lines accumulated botryococcene (albeit in lesser amounts than the cytosolic BS + FPS lines), but they showed a detrimental phenotype dependent on plastid‐targeted FPS, and could not proliferate and survive to set seed under phototrophic conditions. These results highlight intriguing differences in isoprenoid metabolism between dicots and monocots.

Linear, branch-chained triterpenes, including squalene (C30), botryococcene (C30), and their methylated derivatives (C31–C37), generated by the green alga Botryococcus braunii race B have received significant attention because of their utility as chemical and biofuel feedstocks. However, the slow growth habit of B. braunii makes it impractical as a production system. In this study, we evaluated the potential of generating high levels of botryococcene in tobacco (Nicotiana tabacum) plants by diverting carbon flux from the cytosolic mevalonate pathway or the plastidic methylerythritol phosphate pathway by the targeted overexpression of an avian farnesyl diphosphate synthase along with two versions of botryococcene synthases. Up to 544 µg g−1 fresh weight of botryococcene was achieved when this metabolism was directed to the chloroplasts, which is approximately 90 times greater than that accumulating in plants engineered for cytosolic production. To test if methylated triterpenes could be produced in tobacco, we also engineered triterpene methyltransferases (TMTs) from B. braunii into wild-type plants and transgenic lines selected for high-level triterpene accumulation. Up to 91% of the total triterpene contents could be converted to methylated forms (C31 and C32) by cotargeting the TMTs and triterpene biosynthesis to the chloroplasts, whereas only 4% to 14% of total triterpenes were methylated when this metabolism was directed to the cytoplasm. When the TMTs were overexpressed in the cytoplasm of wild-type plants, up to 72% of the total squalene was methylated, and total triterpene (C30+C31+C32) content was elevated 7-fold. Altogether, these results point to innate mechanisms controlling metabolite fluxes, including a homeostatic role for squalene.

Ginkgo biloba is one of the oldest living tree species and has been extensively investigated as a source of bioactive natural compounds, including bioactive flavonoids, diterpene lactones, terpenoids and polysaccharides which accumulate in foliar tissues. Despite this chemical diversity, relatively few enzymes associated with any biosynthetic pathway from ginkgo have been characterized to date. In the present work, predicted transcripts potentially encoding enzymes associated with the biosynthesis of diterpenoid and terpenoid compounds, including putative terpene synthases, were first identified by mining publicly-available G. biloba RNA-seq data sets. Recombinant enzyme studies with two of the TPS-like sequences led to the identification of GbTPS1 and GbTPS2, encoding farnesol and bisabolene synthases, respectively. Additionally, the phylogenetic analysis revealed the two terpene synthase genes as primitive genes that might have evolved from an ancestral diterpene synthase.

Terpenes comprise a distinct class of natural products that serve a diverse range of physiological functions, provide for interactions between plants and their environment and represent a resource for many kinds of practical applications. To better appreciate the importance of terpenes to overall growth and development, and to create a production capacity for specific terpenes of industrial interest, we have pioneered the development of strategies for diverting carbon flow from the native terpene biosynthetic pathways operating in the cytosol and plastid compartments of tobacco for the generation of specific classes of terpenes. In the current work, we demonstrate how difficult it is to divert the 5-carbon intermediates DMAPP and IPP from the mevalonate pathway operating in the cytoplasm for triterpene biosynthesis, yet diversion of the same intermediates from the methylerythritol phosphate pathway operating in the plastid compartment leads to the accumulation of very high levels of the triterpene squalene. This was assessed by the co-expression of an avian farnesyl diphosphate synthase and yeast squalene synthase genes targeting metabolism in the cytoplasm or chloroplast. We also evaluated the possibility of directing this metabolism to the secretory trichomes of tobacco by comparing the effects of trichome-specific gene promoters to strong, constitutive viral promoters. Surprisingly, when transgene expression was directed to trichomes, high-level squalene accumulation was observed, but overall plant growth and physiology were reduced up to 80 % of the non-transgenic controls. Our results support the notion that the biosynthesis of a desired terpene can be dramatically improved by directing that metabolism to a non-native cellular compartment, thus avoiding regulatory mechanisms that might attenuate carbon flux within an engineered pathway.

We report the genome size and the GC content,and perform a phylogenetic analysis on Botryococcusbraunii Ku¨tz., a green, colony-forming, hydrocarbon-rich alga that is an attractive source for biopetro-leum. While the chemistry of the hydrocarbonsproduced by the B race of B. braunii has beenstudied for many years, there is a deficiency of infor-mation concerning the molecular biology of thisalga. In addition, there has been some discrepancyas to the phylogenetic placement of the Berkeley (orShowa) strain of the B race. To clarify its classifica-tion, we isolated the Berkeley strain nuclear SSU(18S) rRNA gene and b-actin cDNA and used thesesequences for phylogenetic analysis to determinethat the Berkeley strain belongs to the Trebouxio-phyceae class. This finding is in agreement withother B races of B. braunii, indicating the Berkeleystrain is a true B race of B. braunii. To better under-stand molecular aspects of B. braunii, we obtainedthe Berkeley strain genome size as a first step ingenome sequencing. Using flow cytometry, we deter-mined the B. braunii Berkeley genome size to be166.2 ± 2.2 Mb. We also estimated the GC contentof the Berkeley strain as 54.4 ± 1.2% for expressedgene sequences.Key index words:18S rRNA sequences;Botryococcusbraunii;Chlorophyta;GCcontent;genomesize;hydrocarbons; phylogenetic analysis; Trebouxio-phyceaeAbbreviations: 18S rRNA, nuclear SSU(18S)ribosomalRNA; cDNA, complementary DNA; RT–PCR,reverse transcription–PCRB. braunii is a green, colonial microalga withunique liquid-hydrocarbon biosynthetic capabilitiesthat have made this organism a promising source ofrenewable hydrocarbon fuels. The cells of a B. brau-nii colony are held together by an extracellularmatrix composed of a polymer core of aldehydesderived from very long-chain fatty acids (Maxwellet al. 1968, Knights et al. 1970). Although some B.braunii liquid hydrocarbons can be found intracellu-larly, most liquid hydrocarbons are retained withinthe colony extracellular matrix.Three distinct races of B. braunii (A, B, and L)are classified by the type of hydrocarbons occurringin the extracellular matrix. The A race accumulatesthe fatty acid–derived alkadienes and alkatrienes,while the L race accumulates the tetraterpene lyco-padiene. The focus of this study, the B race, accu-mulates the triterpenoid hydrocarbons known asbotryococcenes (Banerjee et al. 2002, Metzger andLargeau 2005).B. braunii possesses many characteristics thatmake it attractive as a biofuel feedstock source.B. braunii has been observed forming massive, densefreshwater blooms, and it is suggested that B. brau-nii oils have contributed to oil deposits throughoutthe world (Traverse 1955, Gelpi et al. 1968, Wake

Plant cytochrome P450 (CYP) enzymes are involved in the biosynthesis of many primary and secondary metabolites including phenylpropanoids, alkaloids, terpenoids, lipids, cyanogenic glycosides, and glucosinolates. However, while hundreds of CYP genes have been identified in plant genomes, relatively few have been functionally characterized. We report here the cloning and characterization of a CYP enzyme from tobacco (Nicotiana tabacum L.) that demethylates nicotine to form nornicotine, a precursor to the nitrosamine N'-nitrosonornicotine (NNN). Microsomal demethylase activity was first shown to be induced in leaves treated with the growth regulator ethylene, required molecular oxygen and reduced form of nicotinamide adenine dinucleotide phosphate (NADPH) for full activity, and exhibited a K m of 3.9 μΜ for nicotine and a maximum turnover rate (V max ) of 8.9 pkat mg protein. Equally important, microsomal activity was reduced greatly by several inhibitors of CYP-mediated reactions. Using a polymerase chain reaction based strategy with degenerate primers designed to conserve P450 motifs and mRNA from ethylene-treated leaves, putative gene fragments representing 32 different P450 gene families were isolated and numerous full-length genes were cloned. Employing Genechip r (Affymetrix Inc., Santa Clara, CA) microarray hybridizations, the steady-state mRNA level of two highly related full-length cDNAs and one cDNA fragment were demonstrated to be highly expressed in ethylene-treated vs. control plant material. Microsomes from yeast over-expressing the two full-length cDNAs along with two other highly homologous P450 genes demonstrated that only one cDNA, D121-AA8 (GenBank accession no. DQ205656) encoded for demethylation activity comparable to that found in planta. Molecular modeling was used to identify putative active site residues and for comparisons to other putative demethylase cDNAs.

A method for the recovery of full-length cDNAs from predicted terpene synthase genes containing introns is described. The approach utilizes Agrobacterium-mediated transient expression coupled with a reverse transcription-polydeoxyribonucleotide chain reaction assay to facilitate expression cloning of processed transcripts. Subsequent expression of intronless cDNAs in a suitable prokaryotic host provides for direct functional testing of the encoded gene product. The method was optimized by examining the expression of an intron-containing β-glucuronidase gene agroinfiltrated into petunia (Petunia hybrida) leaves, and its utility was demonstrated by defining the function of two previously uncharacterized terpene synthases. A tobacco (Nicotiana tabacum) terpene synthase-like gene containing six predicted introns was characterized as having 5-epi-aristolochene synthase activity, while an Arabidopsis (Arabidopsis thaliana) gene previously annotated as a terpene synthase was shown to possess a novel sesquiterpene synthase activity for α-barbatene, thujopsene, and β-chamigrene biosynthesis.

Plants have the capacity to produce a wide array of terpene compounds – compounds that provide ecological and physiological benefit to plants. Terpenes also have important practical applications such as flavors, fragrances and medicines. Liat Sharon-Asa and colleagues have recently isolated and characterized a sesquiterpene synthase from Citrus that is providing a new window into the biochemical mechanics of these enzymatic wizards and hints for the future metabolic engineering of terpene biosynthesis.

Squalene synthase (SS) represents a putative branch point in the isoprenoid biosynthetic pathway capable of diverting carbon flow specifically to the biosynthesis of sterols and, hence, is considered a potential regulatory point for sterol metabolism. For example, when plant cells grown in suspension culture are challenged with fungal elicitors, suppression of sterol biosynthesis has been correlated with a reduction in SS enzyme activity. The current study sought to correlate changes in SS enzyme activity with changes in the level of the corresponding protein and mRNA. Using an SS-specific antibody, the initial suppression of SS enzyme activity in elicitor-challenged cells was not reflected by changes in the absolute level of the corresponding polypeptide, implicating a post-translational control mechanism for this enzyme activity. In comparison, the absolute level of the SS mRNA did decrease approximately 5-fold in the elicitor-treated cells, which is suggestive of decreased transcription of the SS gene. Study of SS in intact plants was also initiated by measuring the level of SS enzyme activity, the level of the corresponding protein, and the expression of SS gene promoter-reporter gene constructs in transgenic plants. SS enzyme activity, polypeptide level, and gene expression were all localized predominately to the shoot apical meristem, with much lower levels observed in leaves and roots. These later results suggest that sterol biosynthesis is localized to the apical meristems and that apical meristems may be a source of sterols for other plant tissues.

The tropane alkaloids, hyoscyamine and scopolamine, are medicinal compounds that are the active components of several therapeutics. Hyoscyamine and scopolamine are synthesized in the roots of specific genera of the Solanaceae in a multistep pathway that is only partially elucidated. To facilitate greater understanding of tropane alkaloid biosynthesis, a de novo transcriptome assembly was developed for Deadly Nightshade (Atropa belladonna). Littorine is a key intermediate in hyoscyamine and scopolamine biosynthesis that is produced by the condensation of tropine and phenyllactic acid. Phenyllactic acid is derived from phenylalanine via its transamination to phenylpyruvate, and mining of the transcriptome identified a phylogenetically distinct aromatic amino acid aminotransferase (ArAT), designated Ab-ArAT4, that is coexpressed with known tropane alkaloid biosynthesis genes in the roots of A. belladonna. Silencing of Ab-ArAT4 disrupted synthesis of hyoscyamine and scopolamine through reduction of phenyllactic acid levels. Recombinant Ab-ArAT4 preferentially catalyzes the first step in phenyllactic acid synthesis, the transamination of phenylalanine to phenylpyruvate. However, rather than utilizing the typical keto-acid cosubstrates, 2-oxoglutarate, pyruvate, and oxaloacetate, Ab-ArAT4 possesses strong substrate preference and highest activity with the aromatic keto-acid, 4-hydroxyphenylpyruvate. Thus, Ab-ArAT4 operates at the interface between primary and specialized metabolism, contributing to both tropane alkaloid biosynthesis and the direct conversion of phenylalanine to tyrosine.

Phytoalexins play an important role in the inducible defense responses of plants against diseases caused by fungi. Some enzymes, involved in the respective biosynthetic pathways, catalyze key steps. Farnesyl pyrophosphate (FPP) is a key intermediate in the biosynthesis of terpenes. It may be converted into several cyclic and some acyclic sesquiterpenic derivatives, sterol precursors, or to geranylgeranyl pyrophosphate according to the requirements of the plant cells. Specific cyclization reactions of FPP are catalyzed by particular sesquiterpene cyclases. The 5-epi-aristolochene synthase (EAS) enzyme of tobacco (Nicotiana tabacum) and pepper (Capsicum annuum) produce the 5-epi-aristolochene, which is the immediate precursor of the bicyclic phytoalexin capsidiol. Oligonucleotides homologous to 3′-end specific regions of tobacco and Hyoscyamus muticus inducible sesquiterpene cyclase genes were used with the system for rapid amplification of cDNA ends (3′-RACE) technique to prepare pepper EAS-cDNA fragments (PEAS-cDNA). Three specific PEAS-cDNAs were isolated (PEAS1, PEAS18, and PEAS55). Northern blots of total RNA samples from pepper plant tissues challenged with cellulase or Phytophthora capsici showed different expression levels of the respective transcripts. PEAS1 was used to identify the corresponding elicitor-stimulated gene (gPEAS1). The nucleotide sequence of the proposed gPEAS1 promoter showed putative stimuli- and tissue-specific responsive elements. A pepper 5-epi-aristolochene synthase gene family of 5–8 members was demonstrated by Southern blot.

Summary The first unique step in the synthesis of the tobacco phytoalexin capsidiol is cyclization of farnesyl pyrophosphate catalyzed by 5-epi-aristolochene synthase (EAS), a sesquiterpene cyclase. Earlier work demonstrated that the elicitor-inducibility of this enzyme activity corresponded to the transcriptional activation of at least one gene, EAS4, of a rather complex gene family consisting of > 10 members. To investigate the mechanism(s) controlling expression of this gene, fragments of the EAS4 promoter were examined for binding by proteins in nuclear and whole-cell extracts. A strong protein binding site (TAC box; ACTCTACAGTACTC) was identified between ‐245 and ‐232 by the electrophoretic mobility shift assay, and DNAase I and methylation interference footprinting. Several distinctly migrating bands representing protein‐ TAC box complexes were also observed in the mobility shift assays, and the relative abundance of these bands varied in extracts from cells at different stages of EAS induction. The TAC box binding factor (TacBBF) was purified > 450-fold from crude whole-cell extracts by a combination of DNA affinity and cation exchange chromatography. The purified fractions were enriched for polypeptides of 17 and 19 kDa and the DNA binding properties of these preparations were characterized. Mutation of 2 bp in the TAC box prevented protein binding in vitro and

International audience; The induction of sesquiterpene cyclase, a key phytoalexin biosynthetic enzyme, and the accumulation of phytoalexins in relation to the induction of a hypersensitive response (HR) and cell necrosis in tobac- co (Nicotiana tabacum L.) were investigated. When tobacco leaves were inoculated with virulent or avirulent isolates of Ralstonia solanacearum, steady-state levels of mRNA complementary to cDNA of the sensitivity- related (sts) gene str319 were dramatically induced. This cDNA clone is greater than 90% homologous with a gene coding for 5-epi-aristolochene synthase (EAS), previously described as a branch-point enzyme regulat- ing the synthesis of capsidiol, the major sesquiterpenoid phytoalexin found in tobacco. Accumulation of EAS transcripts in leaves after inoculation with virulent and avirulent strains of R. solanacearum, or after treatment with necrotizing or non-necrotizing elicitins was rapid but transient, and restricted to the site of infiltration. Two highly similar sesquiterpene cyclase activities, 5-epi- aristolochene synthase and a vetispiradiene synthase-like activity, were found in extracts of elicitin-challenged and R. solanacearum-inoculated tobacco. Under all condi- tions tested, the induction of cyclase activity was closely correlated with induction of the cyclase mRNA level. In contrast, high levels of capsidiol were found only after treatment with the necrosis-inducing elicitin cryptogein, or after infiltration with HR-inducing bacterial strains. Low levels of capsidiol did accumulate after application of capsicein, an elicitin that induces little or no necrosis on tobacco, or after infection with a virulent bacterium. Hence, capsidiol accumulation, not 5-epi-aristolochene synthase gene expression or total sesquiterpene cyclase enzyme activity, appears to be a good marker for the HR of tobacco.

Summary With the aim of studying the production of chloroplast isoprenoid derivatives, chimeric gene constructs comprising partial and full-length forms of the Arabidopsis thaliana hmg1 gene, encoding 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR), in sense and antisense forms were fused to a chloroplast transit peptide (CTP) sequence from a pea ribulose bisphosphate carboxylase/oxygenase (Rubisco) small subunit gene. These plasmids were placed downstream from the constitutive CaMV 35S promoter and introduced into tobacco ( Nicotiana tabacum cv. Xanthi) by Agrobacterium -mediated transformation. Southern and northern blot analysis confirmed the transformation and transcription of the respective constructs within transgenic tobacco plants. In addition total and chloroplast specific HMGR enzyme activities, and the levels of chlorophyll, carotenoids and total sterols were analyzed. Results of these analyses suggest that neither partial nor full-length sense constructs had any effect on chloroplast isoprenoid production. However, the corresponding antisense constructs decreased general isoprenoid levels. The significance of these results is discussed.

Treatment of tobacco (Nicotiana tabacum) cell-suspension cultures with cryptogein, an elicitin protein from Phytophthora cryptogea, resulted in the release of a factor(s) that diffused through a 1000-D cutoff dialysis membrane and was capable of inducing sesquiterpene cyclase enzyme activity (a key phytoalexin biosynthetic enzyme in solanaceous plants) when added to fresh cell-suspension cultures. The diffusible factor(s) was released from cells over a 20-h period and induced a more rapid induction of cyclase enzyme activity than did direct treatment of the cultures with pure elicitin protein. The diffusible factor also induced a more rapid accumulation of transcripts encoding for sesquiterpene cyclase, acidic and basic chitinase, and hsr203 (a putative hypersensitive response gene) than did elicitin treatment. The diffusible factor(s) was resistant to protease, pectinase, Dnase, and RNase treatments, was not extractable into organic solvents, and was not immunoprecipitable when challenged with polyclonal antibodies prepared against elicitin protein. The diffusible factor(s) could not induce the release of more factor, suggesting that it was a terminal signal. These results are consistent with the notion that cells directly challenged or stimulated by pathogen-derived elicitors release diffusible secondary signal molecules that orchestrate the induction of complementary defense responses in neighboring cells.

3-Hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) catalyzes the irreversible conversion of 3-hydroxy-3-methylglutaryl coenzyme A to mevalonate and is considered a key regulatory step controlling isoprenoid metabolism in mammals and fungi. The rate-limiting nature of this enzyme for isoprenoid biosynthesis in plants remains controversial. To investigate whether HMGR activity could be limiting in plants, we introduced a constitutively expressing hamster HMGR gene into tabacco (Nicotiana tabaccum L.) plants to obtain unregulated HMGR activity. The impact of the resulting enzyme activity on the biosynthesis and accumulation of particular isoprenoids was evaluated. Expression of the hamster HMGR gene led to a 3- to 6-fold increase in the total HMGR enzyme activity. Total sterol accumulation was consequently increased 3- to 10-fold, whereas end-product sterols such as sitosterol, campesterol, and stigmasterol were increased only 2-fold. The level of cycloartenol, a sterol biosynthetic intermediate, was increased more than 100-fold. Although the synthesis of total sterols appears to be limited normally by HMGR activity, these results indicate that the activity of one or more later enzyme(s) in the pathway must also be involved in determining the relative accumulation of end-product sterols. The levels of other isoprenoids such as carotenoids, phytol chain of chlorophyll, and sesquiterpene phytoalexins were relatively unaltered in the transgenic plants. It appears from these results that compartmentation, channeling, or other rate-determining enzymes operate to control the accumulation of these other isoprenoid end products.

Marchantia polymorpha is a basal terrestrial land plant, which like most liverworts accumulates structurally diverse terpenes believed to serve in deterring disease and herbivory. Previous studies have suggested that the mevalonate and methylerythritol phosphate pathways, present in evolutionarily diverged plants, are also operative in liverworts. However, the genes and enzymes responsible for the chemical diversity of terpenes have yet to be described. In this study, we resorted to a HMMER search tool to identify 17 putative terpene synthase genes from M. polymorpha transcriptomes. Functional characterization identified four diterpene synthase genes phylogenetically related to those found in diverged plants and nine rather unusual monoterpene and sesquiterpene synthase-like genes. The presence of separate monofunctional diterpene synthases for ent-copalyl diphosphate and ent-kaurene biosynthesis is similar to orthologs found in vascular plants, pushing the date of the underlying gene duplication and neofunctionalization of the ancestral diterpene synthase gene family to >400 million years ago. By contrast, the mono- and sesquiterpene synthases represent a distinct class of enzymes, not related to previously described plant terpene synthases and only distantly so to microbial-type terpene synthases. The absence of a Mg2+ binding, aspartate-rich, DDXXD motif places these enzymes in a noncanonical family of terpene synthases.

A convenient in vitro transcription system using monocot and dicot whole cell extracts and circular DNA templates has been developed. The system consists of incubating template and whole cell extract to generate initiation complexes, followed by addition of nucleotide triphosphates to support elongation, and primer extension assay to detect authentic transcripts. This in vitro transcription system required circularized templates and was essentially inactive with linearized templates. Accurate in vitro transcription of a rice phenylalanine ammonia-lyase (PAL) promoter-beta-glucuronidase (GUS) gene fusion and a tobacco sesquiterpene cyclase promoter-GUS gene fusion was examined in their homologous whole cell extracts, and the optimal concentrations for several reaction components, including DNA template, whole cell extract, monovalent and divalent cations, were determined for specific initiation from the in vivo start site. Transcription was inhibited by low concentrations of alpha-amanitin, demonstrating that the reaction was mediated by RNA polymerase II. Accurate transcription initiation was dependent on the TATA-box motif within the respective promoters. Based on the effect of delayed addition of sarkosyl at a concentration sufficient to inhibit transcription initiation but not elongation, three to four rounds of transcription were initiated in standard assays.

Laboratory assays were conducted to determine the sensitivity of tomato and tobacco cell suspension cultures and tomato and pepper cotyledons to clomazone. A comparison of fresh weight and carotenoid content indicated up to a three-fold difference between the clomazone-tolerant to- bacco and clomazone-susceptible tomato cell suspension cul- tures. In contrast, an approximate 60-fold difference between the tolerant pepper and susceptible tomato cotyledons was observed when total chlorophyll and carotenoid contents were measured. The effect of clomazone and its possible metabolites on in vivo and in vitro extractable IPPisomerase (EC 53.3.2) and prenyl transferase (EC 2.5.1.29) activity was investigated. There was no clear inhibitory effect of cloma- zone or possible clomazone metabolites upon enzyme activity in tomato or tobacco cell suspension cultures or on light or dark grown tomato or pepper cotyledons. No specific enzy- matic target site of clomazone was identified in correlation with the reduction in total chlorophyll or carotenoid content. Nomenclature: Clomazone, 2-((2-chlorophenyl)methyl)-4,4- dimethyl-3-isoxazolidinone; pepper, Capsicum annum L. cv Yolo Wonder L; tobacco, Nicotiana tabacum L. 'KY-14'; tomato, Lycopersicon esculentum Mill. 'Niagara.'

A role for calcium/calcium-binding proteins in a mechanism of signaling elicitor-inducible phytoalexin biosynthesis was investigated. Two classes of calcium/calmodulin antagonists, phenothiazines and naphthalenesulfonamides, inhibited sesquiterpene phytoalexin accumulation in tobacco (Nicotiana tabacum) cell-suspension cultures when added 1 h before elicitor. The antagonists also inhibited the induction of sesquiterpene cyclase enzyme activity, a key regulatory enzyme for sesquiterpene biosynthesis. The antagonists suppressed the induction of sesquiterpene cyclase only if added before or simultaneously with elicitor. Additionally, the antagonists inhibited (a) accumulation of the cyclase protein as measured in immunoblots; (b) the in vivo synthesis rate of the cyclase protein, measured as the incorporation of [(35)S]methionine into immunoprecipitable cyclase protein; and (c) the cyclase mRNA translational activity, measured as the incorporation of [(35)S]methionine into immunoprecipitable cyclase protein synthesized by in vitro translation of RNA isolated from antagonist-treated, elicitor-induced cells. In contrast, elicitor-inducible phenylalanine ammonia lyase enzyme activity, the level of the enzyme protein, the in vivo synthesis rate, and the mRNA translational activity were not affected by any of the antagonist treatments. Uptake and incorporation of [(35)S]methionine into total cellular proteins and total in vitro translation products were also not indiscriminately altered by the antagonist treatments. The current results suggest that calcium and/or calmodulin-like proteins may be elements of a signal transduction pathway mediating elicitor-induced accumulation of phytoalexins in tobacco.

The olefin reaction products of the potato and tobacco sesquiterpene cyclases were partially separated from each other by argenation chromatography. Arachidonic acid-elicited potato tuber discs or cellulase-elicited tobacco cell suspension cultures metabolized the olefin reaction products of potato and tobacco sesquiterpene cyclases into different sets of compounds. These results provide evidence that the two olefin cyclase products are chemically different. The Km of the potato sesquiterpene cyclase for farnesyl diphosphate was 7μM and 1.0 mM for magnesium chloride. Inorganic diphosphate inhibited enzyme activity with an ID50 of approximately 4 mM.

Squalene synthetase, an integral membrane protein and the first committed enzyme for sterol biosynthesis, was solubilized and partially purified from tobacco (Nicotiana tabacum) cell suspension cultures. Tobacco microsomes were prepared and the enzyme was solubilized from the lipid bilayer using a two-step procedure. Microsomes were initially treated with concentrations of octyl-β-d-thioglucopyranoside and glycodeoxycholate below their critical micelle concentration, 4.5 and 1.1 millimolar, respectively, to remove loosely associated proteins. Complete solubilization of the squalene synthetase enzyme activity was achieved after a second treatment at detergent concentrations above or at their critical micelle concentration, 18 and 2.2 millimolar, respectively. The detergent-solubilized enzyme was further purified by a combination of ultrafiltration, gel permeation, and Fast Protein Liquid Chromatography anion exchange. A 60-fold purification and 20% recovery of the enzyme activity was achieved. The partially purified squalene synthetase protein was used to generate polyclonal antibodies from mice that efficiently inhibited synthetase activity in an in vitro assay. The apparent molecular mass of the squalene synthetase protein as determined by immunoblot analysis of the partially purified squalene synthetase protein separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis was 47 kilodaltons. The partially purified squalene synthetase activity was optimal at pH 6.0, exhibited a Km for farnesyl diphosphate of 9.5 micromolar, and preferred NADPH as a reductant rather than NADH.

Magnolia grandiflora (Southern Magnolia) is a primitive evergreen tree that has attracted attention because of its horticultural distinctiveness, the wealth of natural products associated with it, and its evolutionary position as a basal angiosperm. Three cDNAs corresponding to terpene synthase (TPS) genes expressed in young leaves were isolated, and the corresponding enzymes were functionally characterized in vitro. Recombinant Mg25 converted farnesyl diphosphate (C15) predominantly to β-cubebene, while Mg17 converted geranyl diphosphate (C5) to α-terpineol. Efforts to functionally characterize Mg11 were unsuccessful. Transcript levels for all three genes were prominent in young leaf tissue and significantly elevated for Mg25 and Mg11 messenger RNAs in stamens. A putative amino-terminal signal peptide of Mg17 targeted the reporter green fluorescent protein to both chloroplasts and mitochondria when transiently expressed in epidermal cells of Nicotiana tabacum leaves. Phylogenetic analyses indicated that Mg25 and Mg11 belonged to the angiosperm sesquiterpene synthase subclass TPS-a, while Mg17 aligned more closely to the angiosperm monoterpene synthase subclass TPS-b. Unexpectedly, the intron-exon organizations for the three Magnolia TPS genes were different from one another and from other well-characterized TPS gene sets. The Mg17 gene consists of six introns arranged in a manner similar to many other angiosperm sesquiterpene synthases, but Mg11 contains only four introns, and Mg25 has only a single intron located near the 5′ terminus of the gene. Our results suggest that the structural diversity observed in the Magnolia TPS genes could have occurred either by a rapid loss of introns from a common ancestor TPS gene or by a gain of introns into an intron-deficient progenote TPS gene.

An elicitor-inducible sesquiterpene cyclase, which catalyzes the conversion of farnesyl diphosphate to 5-epi-aristolochene (IM Whitehead, DR Threlfall, DF Ewing [1989] Phytochemistry 28:775-779) and representing a committed step in the phytoalexin biosynthetic pathway in tobacco, was purified by a combination of hydrophobic interaction, anion exchange, hydroxylapatite, and chromatofocusing chromatography. From 2 kilograms of elicited tobacco (Nicotiana tabacum) cells, approximately 500 micrograms of cyclase protein was purified, representing greater than a 130-fold increase in the specific activity of the enzyme and a 4% recovery of the starting activity. The purified enzyme resolved as two major polypeptides of 60 and 62 kilodaltons by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Biochemical characterization of the enzyme activity included an absolute requirement for magnesium, an isoelectric point of 4.5 to 4.9, and a Km for farnesyl diphosphate of 2 to 5 micromolar. The purified cyclase protein was used to generate mouse polyclonal antibodies which efficiently inhibited cyclase activity in an in vitro assay. Electrophoresis of extracts from elicitor-treated cells or purified cyclase enzyme on native polyacrylamide gels separated the cyclase enzyme into four polypeptides as shown by immunoblot analysis using poly- and monoclonal antibodies. Proportionate cyclase enzyme activity comigrated with those polypeptides. No cyclase polypeptides were detectable in extracts of control cells by immunoblot analysis. However, immunoblot analysis of proteins from elicitor-treated cells using four independent monoclonal antibody lines and the polyclonal antibodies detected the same polypeptides, regardless of whether the proteins were separated by native or SDS-PAGE. The results suggest an induction of multiple cyclase polypeptides in elicitor-treated cells resulting from either the expression of multiple genes or multiple post-translational processing events.

The promoter for a tobacco (Nicotiana tabacum) sesquiterpene cyclase gene, a key regulatory step in sesquiterpene phytoalexin biosynthesis, has been analyzed. The EAS4 promoter was fused to the β-glucuronidase (GUS) reporter gene, and the temporal and spatial expression patterns of GUS activity were examined in stably transformed plants and in transient expression assays using electroporated protoplasts of tobacco. No GUS activity was observed in any tissues under normal growth conditions. A low level of GUS activity was detected in wounded leaf, root, and stem tissues, whereas a much higher level was observed when these tissues were challenged with elicitors or microbial pathogens. The GUS expression pattern directed by the EAS4 promoter was identical to the induction patterns observed for the endogenous sesquiterpene cyclase genes. Neither exogenous salicylic acid nor methyl jasmonate induced GUS expression; and H2O2 induced GUS expression to only a limited extent. Although the EAS4 promoter contains cis-sequences resembling previously identified transcriptional control motifs, other cis-sequences important for quantitative and qualitative gene expression were identified by deletion and gain-of-function analyses. The EAS4 promoter differs from previously described pathogen-/elicitor-inducible promoters because it only supports inducible gene expression and directs unique spatial expression patterns.

Just fifteen years ago the community of plant biology was all abuzz with the discoveries of Agrobacterium tumefaciens-mediated transformation and cloning of the first plant genes. Expectations were high as biotechnology industries sprang up everywhere, and the idea of engineering enhanced nutritional value into foodstuffs, altering disease resistance traits and even the capacity for nitrogen fixation into cereal crops were discussed. Some of those early expectations were too grand, and others not grand enough. Nonetheless, plant biotechnology has had a tremendous impact on industries worldwide and scientists continue to develop extremely imaginative and innovative applications. As part of an effort to recognize the commercial emergence of plant biotechnology as its first products are making their way into the marketplace, the Tobacco and Health Research Institute and the University of Kentucky hosted an international symposium entitled ‘Engineering Plants for Commercial Products and Applications’, October 1-4, 1995, in Lexington, Kentucky. The goal of the meeting was to assess the status of the commercialization and development of plant-based products and to look into the crystal ball for future applications. Glenn B. Collins and Robert Shepherd, the co-organizers for the meeting, coordinated an international organizational team which reflected industrial and academic interests in five topic areas: Jean Kridl (Calgene, Davis, CA), Food Applications; Roger Beachy (The Scripps Research Institute, La Jolla, CA), Human and Animal Health; Lothar Willmitzer (Max Planck Institute, Berlin, Germany), Industrial Uses; Simon Bright (Zeneca, Jeallot’s Hill, England), Ecological and Environmental Applications; Robert Fraley (Ceregen, a unit of Monsanto Corp., St. Louis, MO), Commercialization of the Technology; plus Chris Somerville (Carnegie Institute of Washington, Stanford, CA) and Charles Arntzen (Boyce Thompson Institute, Ithaca, NY) who helped establish the tone for the meeting by making keynote addresses. Sessions were arranged such that the organizer of each session provided an overview of the topic area and addressed how their own work had contributed to the area, followed by four or five additional presentations from other scientific experts in the field. A meeting review such as this can never hope to address all the issues and topics raised, and this review is only intended to capture the spirit of this timely and exciting symposium.

Addition of cell wall fragments from Phytophthora species or cellulase from Trichoderma viride, but not pectolyase from Aspergillus japonicus, to tobacco (Nicotiana tabacum) cell suspension cultures induced the accumulation of the extracellular sesquiterpenoid capsidiol. Pulse-labeling experiments with [(14)C]acetate and [(3)H]mevalonate suggested that enzymatic steps preceding mevalonate were limiting capsidiol biosynthesis in the pectolyase-treated cell cultures. Treatment of the cell cultures with either Phytophthora cell wall fragments or cellulase induced 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) and sesquiterpene cyclase activities, enzymes of the sesquiterpene biosynthetic pathway, and phenylalanine ammonia lyase activity, an enzyme of the general phenylpropanoid pathway. Pectolyase treatment induced sesquiterpene cyclase and phenylalanine ammonia lyase activities, but not HMGR activity. These results corroborate the importance of inducible HMGR enzyme activity for sesquiterpene accumulation.

The regulation of an elicitor-inducible sesquiterpene cyclase in tobacco (Nicotiana tabacum) cell suspension cultures was investigated. Sesquiterpene cyclase activity was absent from control cell cultures but induced to a maximum within 15 hours of cellulase addition to the cell cultures. The induction of the cyclase activity was correlated with an absolute amount of the cyclase protein as measured in immunoblots. Both the in vivo synthesis rate, measured as the incorporation of [(35)S]methionine by cell cultures into immunoprecipitable cyclase protein, and the cyclase mRNA translational activity, measured as the incorporation of [(35)S]methionine into immunoprecipitable cyclase protein synthesized by in vitro translation of isolated RNA, were maximal at that time when the increase in cyclase enzyme activity was maximal. Using thiouridine to selectively label and isolate de novo synthesized mRNA, the in vitro translation products encoded by the newly synthesized RNA from elicitor-treated, but not control, cell cultures contained immunoprecipitable cyclase protein. These results suggest that the induction of the sesquiterpene cyclase in elicitor-treated cell cultures is primarily regulated by transcriptional control of the cyclase gene.

The accumulation and biosynthesis of sterols and fungal elicitor-inducible sesquiterpenoids by tobacco (Nicotiana tabacum) cell suspension cultures were examined as a function of a 10 day culture cycle. Sterols accumulated concomitantly with fresh weight gain. The rate of sterol biosynthesis, measured as the incorporation rate of [(14)C]acetate and [(3)H]mevalonate, was maximal when the cultures entered into their rapid phase of growth. Changes in squalene synthetase enzyme activity correlated more closely with thein vivo synthesis rate and accumulation of sterols than 3-hydroxy-3-methylglutaryl CoA reductase (HMGR) enzyme activity. Cell cultures entering into the rapid phase of growth also responded maximally to fungal elicitor as measured by the production of capsidiol, an extracellular sesquiterpenoid. However, the rate of sesquiterpenoid biosynthesis, measured as the incorporation rate of [(14)C]acetate and [(3)H]mevalonate, could not be correlated with elicitor-inducible HMGR or sesquiterpene cyclase enzyme activities, nor elicitor-suppressible squalene synthetase enzyme activity.

Addition of elicitor, cell wall fragments of the fungus Phytophthora parasitica, to tobacco cell suspension cultures (Nicotiana tabacum) resulted in the rapid synthesis and secretion of large amounts of antibiotic sesquiterpenoids. Pulse-labeling experiments with [(14)C]acetate and [(3)H] mevalonate demonstrated that the induction of sesquiterpenoid biosynthesis, maximal by 6 to 9 hours after elicitor addition to the cell cultures, was paralleled by a rapid and large decline in the incorporation rate of radioactivity into sterols. Consequently, sterol accumulation was also inhibited upon addition of elicitor to the cell cultures. Sesquiterpene cyclase activity was absent from control cell cultures but induced to a maximum within 10 hours of elicitor addition to the cell cultures. The cyclase activity remained elevated for an additional 30 hours before declining. In contrast, squalene synthetase activity was suppressed to less than 15% of that found in control cells within 7 hours of elicitor addition. Our results suggest that the channeling of isoprenoid intermediates, and especially farnesyl diphosphate, into sesquiterpenoids occurred by a coordinated increase in the sesquiterpene cyclase and a decrease in the squalene synthetase enzyme activities. A reexamination of the data pertaining to the transient induction of 3-hydroxy-3-methylglutaryl coenzyme A reductase activity (EC 1.1.1.34) in elicitor-treated cells suggested that, while the reductase activity was necessary for sesquiterpenoid biosynthesis, it functioned more to maintain a sufficient level of intermediates between mevalonate and farnesyl diphosphate rather than as a rate limiting step controlling the synthesis rate of any one class of isoprenoids.

The biosynthesis of ethylene was examined in suspension-cultured cells of parsley (Petroselinum hortense) treated with an elicitor from cell walls of Phytophthora megasperma. Untreated cells contained 50 nmol g(-1) of the ethylene precursor, 1-aminocyclopropane-1-carboxylic acid (ACC), and produced ethylene at a rate of about 0.5 nmol g(-1) h(-1). Within 2 h after addition of elicitor to the culture medium, the cells started to produce more ethylene and accumulated more ACC. Exogenously added ACC did not increase the rate of ethylene production in control or elicitor-treated cells, indicating that the enzyme converting ACC to ethylene was limiting in both cases. The first enzyme in ethylene biosynthesis, ACC synthase, was very rapidly and transiently induced by the elicitor treatment. Its activity increased more than tenfold within 60 min. Density labelling with (2)H2O showed that this increase was caused by the denovo synthesis of the enzyme protein. Cordycepin and actinomycin D did not affect the induction of ACC synthase, indicating that the synthesis of new mRNA was not required. The peak of ACC-synthase activity preceded the maximal phenylalanine ammonia-lyase (PAL) activity by several hours. Exogenously supplied ethylene or ACC did not induce PAL. However, aminoethoxyvinylglycine, an inhibitor of ACC synthase, suppressed the rise in ethylene production in elicitor-treated cells and partially inhibited the induction of PAL. Exogenously supplied ACC reversed this inhibition. It is concluded that induction of the ethylene biosynthetic pathway is a very early symptom of elicitor action. Although ethylene alone is not a sufficient signal for PAL induction, the enhanced activity of ACC synthase and the ethylene biosynthetic pathway may be important for the subsequent induction of PAL.

Mitochondria from castor bean (Ricinus communis cv Hale) endosperm, purified on sucrose gradients, were used to investigate transport of dicarboxylic acids. The isolated mitochondria oxidized malate and succinate with respiratory control ratios greater than 2 and ADP/O ratios of 2.6 and 1.7, respectively. Net accumulation of (14)C from [(14)C]malate or [(14)C]succinate into the mitochondrial matrix during substrate oxidation was examined by the silicone oil centrifugation technique. In the presence of ATP, there was an appreciable increase in the accumulation of (14)C from [(14)C]malate or [(14)C]succinate accompanied by an increased oxidation rate of the respective dicarboxylate. The net accumulation of dicarboxylate in the presence of ATP was saturable with apparent K(m) values of 2 to 2.5 millimolar. The ATP-stimulated accumulation of dicarboxylate was unaffected by oligomycin but inhibited by uncouplers (2,4-dinitrophenol and carbonyl cyanide m-chlorophenylhydrazone) and inhibitors of the electron transport chain (antimycin A, KCN). Dicarboxylate accumulation was also inhibited by butylmalonate, benzylmalonate, phenylsuccinate, mersalyl and N-ethylmaleimide. The optimal ATP concentration for stimulation of dicarboxylate accumulation was 1 millimolar. CTP was as effective as ATP in stimulating dicarboxylate accumulation, and other nucleotide triphosphates showed intermediate or no effect on dicarboxylate accumulation. Dicarboxylate accumulation was phosphate dependent but, inasmuch as ATP did not increase phosphate uptake, the ATP stimulation of dicarboxylate accumulation was apparently not due to increased availability of exchangeable phosphate.The maximum rate of succinate accumulation (14.5 nanomoles per minute per milligram protein) was only a fraction of the measured rate of oxidation (100-200 nanomoles per minute per milligram protein). Efflux of malate from the mitochondria was shown to occur at high rates (150 nanomoles per minute per milligram protein) when succinate was provided, suggesting dicarboxylate exchange. The uptake of [(14)C]succinate into malate or malonate preloaded mitochondria was therefore determined. In the absence of phosphate, uptake of [(14)C]succinate into mitochondria preloaded with malate was rapid (27 nanomoles per 15 seconds per milligram protein at 4 degrees C) and inhibited by butylmalonate, benzylmalonate, and phenylsuccinate. Uptake of [(14)C]succinate into mitochondria preloaded with malonate showed saturation kinetics with an apparent K(m) of 2.5 millimolar and V(max) of 250 nanomoles per minute per milligram protein at 4 degrees C. The measured rates of dicarboxylate-dicarboxylate exchange in castor bean mitochondria are sufficient to account for the observed rates of substrate oxidation.

Nuclei from cell suspension cultures of parsley (Petroselinum crispum L.) were isolated and the effects of salt concentrations on the in vitro transcriptional activity determined. Increasing concentrations of monovalent and divalent cations stimulated α-amanitin sensitive transcriptional activity, whereas α-amanitin insensitive transcriptional activity was not affected. The relative rates of in vitro synthesis of rRNA and four different mRNAs at selected salt concentrations were greatest when the transcription assays were carried out at the lowest salt concentrations tested. Hence, optimal conditions for total RNA synthesis (high salt concentrations) were not necessarily optimal for the run-off transcription of particular genes (low salt concentrations) in isolated nuclei.

The expression of phaseolin and phytohemagglutinin (PHA) in the developing cotyledons of a normal (Greensleeves) and a PHA-deficient (Pinto 111) cultivar of Phaseolus vulgaris was investigated. Phaseolin mRNA translational activity and abundance were present at similar levels in both cultivars. In contrast, PHA mRNA translational activity and abundance in Pinto 111 were less than 1% of the levels measured in Greensleeves. Using nuclear runoff assays, the transcription rate of phaseolin gene sequences was similar in both cultivars. The transcription rate of PHA gene sequences in Pinto 111 was only 20% of that measured in Greensleeves. Comparison of the transcription rates with the relative mRNA amounts measured in RNA blot hybridizations indicated that the normally expressed storage protein gene mRNAs were very stable with half-lives greater than several days. Because a low level of PHA gene transcription in Pinto 111 was measurable but no PHA mRNA accumulated, these results suggest that the PHA deficiency in Pinto 111 is due to a reduced transcription rate and possibly an instability of the mRNA.