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Terpene synthases are a mechanistically intriguing family of enzymes that catalyze complex, multistep reactions that are capable of generating hundreds of structurally diverse hydrocarbon and oxygenated scaffolds of biological and commercial importance. Interestingly, distantly related terpene synthases from fungi to plants all contain an invariant three-dimensional fold, and molecular comparisons of their active sites indicate that they are enriched with relatively inert amino acid residues that do not react directly with the reaction intermediates. Therefore, catalytic specificity appears to rely on the contour and dynamics of the active site created by the positioning of amino acid backbones and side chains on this catalytic surface and by supporting layers of residues surrounding the synthase active site cavity. Despite the high degree of structural relatedness among terpene synthases, previous studies suggest that no clear relationship between phylogenic organization and catalytic specificities is easily deciphered. We now report on the reciprocal interconversion of catalytic specificities between two distinct yet evolutionarily related terpene synthases based on the systematic identification and mutational replacement of variable residues within and surrounding the active site. Furthermore, we uncover previously undocumented biosynthetic activity during the interconversion, activity that could have been present in a common ancestor of these two highly related synthases. These results provide a simplified means for mapping structural features that are responsible for functional attributes and a strategy for identifying residues that differentiate divergent biosynthetic properties in phylogenetically related terpene synthases.

Terpenes constitute a distinct class of natural products that attract insects, defend against phytopathogenic microbes and combat human diseases. However, like most natural products, they are usually made by plants and microbes in small amounts and as complex mixtures. Chemical synthesis is often costly and inefficient, and may not yield enantiomerically pure terpenes, whereas large-scale microbial production requires expensive feedstocks. We engineered high-level terpene production in tobacco plants by diverting carbon flow from cytosolic or plastidic isopentenyl diphosphate through overexpression in either compartment of an avian farnesyl diphosphate synthase and an appropriate terpene synthase. Isotopic labeling studies suggest little, if any, metabolite exchange between these two subcellular compartments. The strategy increased synthesis of the sesquiterpenes patchoulol and amorpha-4,11-diene more than 1,000-fold, as well as the monoterpene limonene 10-30 fold, and seems equally suited to generating higher levels of other terpenes for research, industrial production or therapeutic applications.

Pogostemon cablin (patchouli), like many plants within the Lamiaceae, accumulates large amounts of essential oil. Patchouli oil is unique because it consists of over 24 different sesquiterpenes, rather than a blend of different mono-, sesqui- and di-terpene compounds. To determine if this complex mixture of sesquiterpenes arises from an equal number of unique sesquiterpene synthases, we developed a RT-PCR strategy to isolate and functionally characterize the respective patchouli oil synthase genes. Unexpectedly, only five terpene synthase cDNA genes were isolated. Four of the cDNAs encode for synthases catalyzing the biosynthesis of one major sesquiterpene, including a gamma-curcumene synthase, two germacrene D synthases, and a germacrene A synthase. The fifth cDNA encodes for a patchoulol synthase, which catalyzes the conversion of FPP to patchoulol plus at least 13 additional sesquiterpene products. Equally intriguing, the yield of the different in vitro reaction products resembles quantitatively and qualitatively the profile of sesquiterpenes found in patchouli oil extracted from plants, suggesting that a single terpene synthase is responsible for the bulk and diversity of terpene products produced in planta.

Nicotiana tabacum (tobacco) 5-epi-aristolochene synthase (TEAS) serves as an useful model for understanding the enzyme mechanisms of sesquiterpene biosynthesis. Despite extensive bio-chemical and structural characterization of TEAS, a more detailed analysis of the reaction product spectrum is lacking. This study reports the discovery and quantification of several alternative sesquiterpene products generated by recombinant TEAS in the single-vial GC–MS assay. The combined use of chiral and non-polar stationary phases for gas chromatography separations proved critical for resolving the numerous sesquiterpene products of TEAS for mass spectral analysis and identification. Co-injection studies with available authentic standards from both synthetic and natural sources further corroborated the assignment of several compounds, resulting in an annotated reaction mechanism accounting for their biosynthesis. Moreover, a previously undocumented farnesyl trans–cis isomerization pathway was observed.

The final step of capsidiol biosynthesis is catalyzed by 5-epiaristolochene dihydroxylase (EAH), a cytochrome P450 enzyme that catalyzes the regio- and stereospecific insertion of two hydroxyl moieties into the bicyclic sesquiterpene 5-epiaristolochene (EA). Detailed kinetic studies using EA and the two possible monohydroxylated intermediates demonstrated the release of 1beta-hydroxy-EA ((OH)EA) at high EA concentrations and a 10-fold catalytic preference for 1beta(OH)EA versus 3alpha(OH)EA, indicative of a preferred reaction order of hydroxylation at C-1, followed by that at C-3. Sequence alignments and homology modeling identified active-site residues tested for their contribution to substrate specificity and overall enzymatic activity. Mutants EAH-S368C and EAH-S368V exhibited wild-type catalytic efficiencies for 1beta(OH)EA biosynthesis, but were devoid of the successive hydroxylation activity for capsidiol biosynthesis. In contrast to EAH-S368C, EAH-S368V catalyzed the relative equal biosynthesis of 1beta(OH)EA, 2beta(OH)EA, and 3beta(OH)EA from EA with wild-type efficiency. Moreover, EAH-S368V converted approximately 1.5% of these monohydroxylated products to their respective ketone forms. Alanine and threonine mutations at position 368 were significantly compromised in their conversion rates of EA to capsidiol and correlated with 3.6- and 5.7-fold increases in their Km values for the 1beta(OH)EA intermediate, respectively. A role for Ile486 in the successive hydroxylations of EA was also suggested by the EAH-I468A mutant, which produced significant amounts 1beta(OH)EA, but negligible amounts of capsidiol from EA. The altered product profile of the EAH-I486A mutant correlated with a 3.6-fold higher Km for EA and a 4.4-fold slower turnover rate (kcat) for 1beta(OH)EA. These kinetic and mutational studies were correlated with substrate docking predictions to suggest how Ser368 and Ile486 might contribute to active-site topology, substrate binding, and substrate presentation to the oxo-Fe-heme reaction center.

A quantitative assay for the analysis of sesquiterpene synthases, wherein each reaction mixture is formulated in glass gas chromatography vials, overlaid with organic solvent such as ethyl acetate, and subsequently vortexed to extract hydrocarbon reaction products into the organic phase after a suitable incubation period, was developed. The product-enriched organic phase is then sampled in an automated fashion and injected directly into a gas chromatograph-mass spectrometer without further workup for analysis and quantification of hydrocarbon products. Application of the vial assay to the analysis of amorpha-4,11-diene synthase (ADS), a sesquiterpene synthase, demonstrated the sensitivity of the assay for detection of major and minor reaction products and most notably for the identification of several sesquiterpene products that had escaped previous detection. A steady-state kinetic analysis of tobacco 5-epi-aristolochene synthase (TEAS), another sesquiterpene synthase, validated the quantitative nature of the assay, providing an alternative means to the established method of using radiolabeled substrate, extraction, and scintillation counting. This simplified assay provides a standardized method to facilitate analysis of terpene synthases and diverse mutant enzyme libraries by supplanting the common practice of using larger scale reactions, multiple extractions, and evaporative concentration of the organic phase prior to gas chromatography-mass spectrometry (GC-MS) analysis.

The extracellular matrix of the alga Botryococcus braunii, Race B, consists mainly of botryococcenes, which have potential as a hydrocarbon fuel. Botryococcenes are structurally similar to squalene raising the possibility of a common enzyme for the biosynthesis of both. While B. braunii squalene synthase (SS) enzyme activity has been documented, botryococcene synthase (BS) enzyme activity has not been. In the current study, an assay for BS activity has been developed and used to show that many of the assay conditions for BS enzyme activity are similar to those of SS. However, SS enzyme activity is stimulated by Tween 80 while BS enzyme activity is inhibited. Moreover, BS enzyme activity was correlated with the accumulation of botryococcenes during a B. braunii culture growth cycle, which was distinctly different from the profile of SS enzyme activity. While the current results indicate a conservation of enzymological features amongst the BS and SS enzymes, raising the possibility of one enzyme capable of catalyzing both activities, they are also consistent with these two activities arising from separate and distinct enzymes.

Publisher Summary This chapter discusses the gene library synthesis by structure-based combinatorial protein engineering (SCOPE). SCOPE is a process for the synthesis of gene libraries that lay the genetic foundation for the exploration of the relationship between structure and function in the encoded proteins. The comparative analysis of both structurally and functionally of protein primary, secondary, and tertiary structure generates numerous hypotheses to probe the relationship between molecular structure and the ensuing functional readout. SCOPE was applied to create a library representing all possible combinations of nine point mutations in the terpene cyclase, tobacco 5-epi-aristolochene synthase (TEAS). The location of mutations in the amino acid and nucleotide sequences of TEAS are presented. Over 600 colonies from discrete mixtures, representing about half of the complexity of the TEAS library, were picked and their sequences determined. It is found that aside from the low-level appearance of wild-type sequence and random mutations likely arising from PCR errors, the actual distribution of mutations in a given mixture was as designed experimentally.

Eyewitnesses are sometimes asked to identify a culprit from a line-up of people associated with a crime scene. An enzyme — iridoid synthase — that catalyses an unusual reaction has been identified by a similar approach. See Letter p.138 This paper reports the identification of iridoid synthase, a key enzyme in the biosynthesis of iridoids, a large family of bicyclic natural products that possess anticancer, anti-inflammatory, antifungal and antibacterial activities. The enzyme produces a bicyclic ring system via a novel NAD(P)H-dependent cascade reaction. Knowledge of its structure and function should contribute towards efforts aimed at modifying and reconstituting iridoid and related pathways in crop plants or microbes for agricultural and pharmaceutical purposes.

Publisher Summary This chapter summarizes the recent advances in the understanding of plant isoprenoid metabolism and molecular biology. Isoprenoids are a family of highly diverse, though structurally related, compounds performing a wide variety of functions in the life processes of plants and nearly every biological entity. Studies designed to elucidate the in vivo biology of isoprenoid metabolism and applied investigations aimed toward enhanced nutritional quality of foods and those exploring the possibilities of new compounds produced through the rational design of isoprenoid synthetic enzymes are discussed in this chapter. These areas include an understanding of the developmental regulation of biosynthetic activities, of metabolic branch points in the control of flux, of possible feedback-regulatory properties of metabolic intermediates, and of mechanisms for the transport of products from the site of biosynthesis.

The field of natural products research has been experiencing a renaissance of interest for quite some time now, led in large part by a combination of practical realities, exciting new discoveries, and the development of some intriguing new concepts. Chemical, agricultural, food, and personal care products industries have all benefited from this arena of research. Nonetheless, the drug development pipeline has probably benefited the most from our fascination with natural products, and largely because of the structural complexity and diversity of compounds biosynthesized by natural sources as compared with those compounds arising from strictly an organic synthesis origin. The notion that natural products provide a greater opportunity for drug discovery and development than pure synthetic approaches has also been argued for some time now and is evident in the results presented by Minami et al. (1) in a recent issue of PNAS. The recent reports by Newman et al. (2, 3) also clearly document how approximately two-thirds of the drugs developed over the last 25 years have been derived from or inspired by particular natural products. Feher and Schmidt (4) provided an additional compelling argument as to why natural products have served so well for drug development. These investigators reported that the structural complexity found in drugs, as measured by the average number of 2.3 chiral centers per drug molecule, more closely resembled that found in natural products (average of 6.2 chiral centers per molecule) than the relative flat molecules (0.4 chiral centers per compound) found in libraries prepared by synthetic, combinatorial means (Fig. 1).

The enzyme squalene synthase (SS) represents the first commitment of carbon from the general isoprenoid pathway toward sterol biosynthesis and is a potential point for regulation of sterol biosynthesis. The isolation and characterization of tobacco (Nicotiana tabacum) squalene synthase (TSS) cDNA and genomic DNA clones, as well as determination of the steady state level of TSS mRNA in response to elicitor treatment, were investigated. cDNA clones for TSS were isolated from poly (A)+ RNA using a reverse transcription/polymerase chain reaction (RT/PCR) method. A 1233-bp cDNA clone was generated that contained an open reading frame of 411 amino acids giving a predicted molecular mass of 46.9 kDa. Comparison of the TSS deduced amino acid sequence with currently described SS from different species showed the highest identify with Nicotiana benthamiana (97%), followed by Glycyrrhiza glabra (81%), Arabidopsis thaliana (74%), rat (40%), and yeast (37%). Expression of a soluble form of the TSS enzyme with enzymatic activity in Escherichia coli was achieved by truncating 24 hydrophobic amino acids at the carboxy terminus. Characterization of genomic TSS (gTSS) revealed a gene of 7.086 kb with a complex organization of small exons and large introns not typical of plant genes. Southern blot hybridization indicated only two copies of the SS gene in the tobacco genome. Treatment of tobacco cell suspension cultures with a fungal elicitor dramatically reduced TSS enzymatic activity, lowering it to zero within 24 h. Analysis of TSS mRNA levels, by RNA blot hybridization and primer extension assays, in elicitor-treated cells indicated that the transcript level remained largely unchanged over this 24-h period. These results suggest that the suppression of TSS enzyme activity in elicitor-treated cells may result from a posttranscriptional modification of TSS.

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.

An Escherichia coli expression system was used to generate hexahistidyl-tagged plant sesquiterpene cyclases, which were readily purified by a single affinity chromatographic step. Genes for Hyoscyamus muticus vetispiradiene synthase (HVS), a chimeric 5-epi-aristolochene synthase (CH3), and a chimeric sesquiterpene cyclase possessing multifunctional epi-aristolochene and vetispiradiene activity (CH4) were expressed in bacterial cells, which resulted in the sesquiterpene cyclases accumulating to 50% of the total protein and 35% of the soluble protein. From initial velocity experiments, the Michaelis constant for HVS was 3.5 microM, while CH3 and CH4 exhibited smaller values of 0.7 and 0.4 microM, respectively. Steady-state catalytic constants were from 0.02 to 0.04 s-1. A combination of pre-steady-state rapid quench experiments, isotope trapping experiments, and experiments to measure the burst rate constant as a function of substrate concentration revealed that turnover in all three cyclases is limited by a step after the initial chemical step involving rupture of the carbon-oxygen bond in farnesyl diphosphate (FPP). Rate constants for the limiting step were 10-70-fold smaller than for the initial chemical step. Dissociation constants for the enzyme-substrate complex (20-70 microM) were determined from the pre-steady-state experiments and were significantly larger than the observed Michaelis constants. A mechanism that involves an initial, rapid equilibration of enzyme with substrate to form an enzyme-substrate complex, followed by a slower conversion of FPP to an enzyme-bound hydrocarbon and a subsequent rate-limiting step, is proposed for the three enzymes. Interestingly, the multifunctional chimeric enzyme CH4 exhibited both a tighter binding of FPP and a faster conversion of FPP to products than either of its wild-type parents.

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.

Genomic and cDNA clones for vetispiradiene synthase, a sesquiterpene cyclase found in Hyoscyamus muticus, were isolated using a combination of reverse transcription-polymerase chain reactions and conventional cloning procedures. RNA blot hybridization demonstrated an induction of mRNA consistent with the induction of cyclase enzyme activity in elicitor-treated cells, DNA blot hybridization indicated a gene family of 6 to 8 members, and bacterial expression of 3 cDNA clones indicated that each coded for a vetispiradiene synthase enzyme activity catalyzing the synthesis of a single reaction product. Intron-exon organization of the vetispiradiene synthase gene was identical with that previously described for 5-epi-aristolochene synthase (tobacco sesquiterpene cyclase) and casbene synthase (castor bean diterpene cyclase), and the vetispiradiene synthase amino acid sequence was 77% identical with and 81% similar to the tobacco sesquiterpene cyclase. Regions of the vetispiradiene synthase sequence centered around amino acids 60, 100, and 370 were conspicuously different relative to the tobacco sesquiterpene cyclase. The sequence similarity between the tobacco and H. muticus enzymes is suggested to be reflective of the conservation of several partial reactions common to both enzymes, and the differences may be reflective of a partial reaction unique to each enzyme.

A diverse array of compounds are synthesized from the intermediates of the isoprenoid pathway in plants. Many of these compounds are essential to the growth and development of higher plants including growth regulators, carotenoid pigments, sterols, and polyprenols. Additionally, biologically important secondary metabolites involved in the plant defense response are also synthesized from the isoprenoid pathway (18). For example, when tobacco cell suspensions cultures are challenged with a fungal elicitor (8), they respond by suppressing the synthesis of sterols and inducing the synthesis of sesquiterpenes (Fig. 1). The predominant species of sesquiterpene phytoalexins being produced is capsidiol (6). Capsidiol is a bicyclic, di-alcohol sesquiterpenoid that accumulates extracellularly (Fig. 2). In studies to further characterize the response of tobacco cells to elicitor treatment, putative key regulatory enzymes were also measured. When tobacco cell suspension cultures are challenged with a fungal elicitor, the biosynthesis of capsidiol is preceded by a sharp, transient induction of 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase enzyme activity (6,20). HMG CoA reductase catalyses the conversion of HMG CoA to mevalonate (MVA), and is the rate-limiting step for cholesterol biosynthesis in mammalian systems (10). Sesquiterpene cyclase, the enzyme catalyzing the cyclization of farnesyl diphosphate (FPP) to 5-epi-aristolochene, a precursor to capsidiol, is also induced, but with a more protracted time course (21). Sesquiterpene cyclase has been purified from tobacco cells and used to generate both poly-and monoclonal antibodies (22). As illustrated in figure 3, the induction of sesquiterpene cyclase activity is regulated by the synthesis rate of the enzyme protein (as measured by the incorporation of 35S-methionine into the cyclase protein). Furthermore, the induction of enzyme activity is also correlated with an induction in the translational activity of the cyclase mRNA and the transcription rate of the cyclase gene (23).

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 mRNAs encoding two enzymes of phenylpropanoid metabolism, phenylalanine ammonia-lyase (PAL; EC 4.3.1.5) and 4-coumarate:CoA ligase (4CL; EC 6.2.1.12), were induced in cultured parsley cells ( Petroselinum hortense ) either by irradiation with UV light or by treatment with elicitor, a cell-wall fraction of the fungus Phytophthora megasperma f. sp. glycinea . Two-dimensional gel electrophoresis of the encoded PAL and 4CL proteins revealed that the mRNAs induced by either treatment were very similar if not identical. RNA blot hybridization with cDNAs complementary to these mRNAs was used to measure changes in the mRNA amounts at various times after either treatment. Total cellular PAL and 4CL mRNA amounts increased coordinately after UV irradiation to a maximum at 7 hr and then decreased to uninduced levels by 30 hr with the same kinetics as observed previously for the changes in the translational activities. Treatment with the fungal elicitor also caused coordinated, but more rapid, changes in PAL and 4CL mRNA translational activities, with a sharp peak occurring 3 hr after the addition of elicitor. Corresponding changes in mRNA amounts were observed only for 4CL, whereas the amount of PAL mRNA continued to increase at least up to 20 hr after elicitor addition. Our results suggest that parsley cells respond to UV irradiation or addition of fungal elicitor by increased rates of transcription of genes involved in the synthesis of compounds related to UV or disease resistance, respectively.

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.

Germination and seedling growth of mung bean are accompanied by a 7- to 10-fold increase in the ribonuclease content of the cotyledons. The increase occurs during the first 4 days of seedling growth and precedes the senescence of the cotyledons. Separation of the RNases in the cotyledons by polyacrylamide gel electrophoresis indicates the presence of several minor bands in seeds imbibed for 24 hr. On the second day of seedling growth a new major band with an Rf of 0.76 is present. In 4- to 5-day old seedlings this major band accounts for nearly all the RNase activity in the tissue. The characteristics of this RNase show that it is a plant ribonuclease I (pH optimum of 5.0; MW 16,000; activity preferentially inhibited by purine nucleotides; no activity toward DNA; no phosphodiesterase activity). When the seedlings are grown in 66% D2O the RNase activity undergoes a density shift of 0.61% indicating that the increase in enzyme activity is due to the de novo synthesis of the enzyme molecules. A method is described for the isolation of protein bodies from protoplasts of storage parenchyma cells. Fractionation of protoplast lysates on Ficoll gradients results in the recovery of a high proportion (75%) of intact protein bodies. On these gradients RNase activity comigrates with α-mannosidase, a protein body marker enzyme indicating that the newly synthesized RNase accumulates in the protein bodies. We suggest that the synthesis of RNase in the cotyledons and its accumulation in the protein bodies indicates that protein bodies may function in the degradation of cellular macromolecules other than the reserves stored within them.

The synthesis of secondary metabolites in response to stress conditions has been implicated as a major defence response of higher plants1. Cell suspension cultures of parsley (Petroselinum hortense) have been studied extensively as a model system and shown to respond to UV irradiation and treatment with fungal elicitor by the synthesis of flavonoids and furanocoumarins, respectively2,3. The induced synthesis of these compounds might afford the plant protection against the respective environmental hazards, because flavonoids strongly absorb UV light and furanocoumarins possess fungitoxic properties4. The production of each class of compound is preceded by the induction of groups of coordinately regulated enzymes2,5–8, based on rapid changes in amounts and activities of the corresponding messenger RNAs9,10. We now report that these changes are due to transient increases in the transcription rates of the respective genes, which indicates that gene activation has an important role in UV and disease resistance in higher plants.

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.

In the present study, parsley suspension culture cells are treated with a cell wall preparation of Phytophthora megasperma var. glycinia and this interaction is considered as a model system for plant-fungal pathogen interactions with respect to phytoalexin biosynthesis. The relevance of this system for these studies can best be understood by a general discussion of phytoalexins and their biosynthesis in more intensively researched plant families, such as the Leguminosae.

YisP is involved in biofilm formation in Bacillus subtilis and has been predicted to produce C30 isoprenoids. We determined the structure of YisP and observe that it adopts the same fold as squalene and dehydrosqualene synthases. However, the first aspartate-rich motif found in essentially all isoprenoid synthases is aspartate-poor in YisP, and cannot catalyze head-to-head condensation reactions. We find that YisP acts as a phosphatase, catalyzing formation of farnesol from farnesyl diphosphate, and is the first phosphatase to adopt the fold seen in the head-to-head prenyl synthases. Farnesol restores biofilm formation in a Δyisp mutant, and modifies lipid membrane structure similarly to the virulence factor, staphyloxanthin. The work clarifies the role of YisP in biofilm formation and suggests an intriguing possibility that many of the YisP-like homologs found in other bacteria may also have interesting products and functions.