Sesquiterpene Accumulation Research Articles

Integrated Analysis of the Transcriptome and Metabolome Reveals Genes Involved in the Synthesis of Terpenoids in Rhododendron fortunei Lindl.

Rhododendron, a globally popular ornamental flower, is nevertheless limited in our understanding of the mechanisms underlying its fragrance formation. Notably, terpenoids are the most prevalent volatile metabolite produced by plants. In this study, gas chromatography–mass spectrometry (GC–MS), liquid chromatography–mass spectrometry (LC–MS) and transcriptomics sequencing were conducted to analyze the synthesis mechanisms of terpenoid fragrance compounds of petals in fragrant R. fortunei Lindl. (YJ) and non-fragrant R. “Nova Zembla” (NW). The results identified that (-)-myrtenol, linalool, pinene, myrtenyl acetate, and terpineol were key floral aroma substances in YJ. Furthermore, an analysis of KEGG enrichment and differentially expressed genes (DEGs) revealed that the bud and decay stages exhibited the highest number of enriched DEGs among different aroma types, indicating these as critical stages for the synthesis of terpenoid floral compounds. In this study, a structural gene, denoted as RfFDPS, was identified as a negative regulatory gene for monoterpene accumulation and a positive regulatory gene for sesquiterpene accumulation in YJ. Utilizing subcellular localization technology, we determined that RfFDPS proteins are located in the cytoplasm. A functional analysis through transient expression and gene silencing of RfFDPS demonstrated its ability to regulate the accumulation of monoterpenes and sesquiterpenes. The overexpression of RfFDPS led to an increase in the expression of structural genes related to terpenoid synthesis, resulting in a decrease in monoterpenes and an increase in sesquiterpenes. Conversely, gene silencing had the opposite effect. In conclusion, RfFDPS plays a pivotal role in the synthesis and release of terpenoid volatile compounds in YJ petals, laying a solid theoretical foundation for the cultivation and enhancement of aromatic R. species.

Functional characterization of sesquiterpene synthase in Mongolian medicine Syringa oblata in heartwood formation

Lilac (Syringa oblata) is a well-known horticultural plant, and its aromatic heartwood is widely utilized in Traditional Mongolian Medicine for treating angina. However, limited research on the dynamic changes and mechanisms of aromatic substance formation during heartwood development hinders the analysis and utilization of its medicinal components. In this study, volatile metabolome analysis revealed that sesquiterpenes are the primary metabolites responsible for the aroma in heartwood, with cadinane and eremophilane types being the most prevalent. Among the identified sesquiterpene synthases, SoSTPS1-5 exhibited significantly increased expression in heartwood formation and was selected for further investigation. Molecular docking simulations predicted multiple amino acid binding sites and confirmed its ability to catalyze the formation of eremophilane, copaene, cadinane, germacrane, and elemane-type sesquiterpenes from FPP (farnesyl pyrophosphate). Co-expression and promoter analysis suggested a transcriptional regulatory network primarily involving WRKY transcription factors. Additionally, aiotic and biotic stress inducers, such as Ag+, Fusarium oxysporum, and especially MeJA, were found to activate the expression of SoSTPS1-5 and promote sesquiterpene accumulation. This study provides insights into the basis of medicinal substance formation and the potential mechanisms of sesquiterpene accumulation in lilac heartwood, laying a foundation for future research on the biosynthesis and utilization of its medicinal components.

Isolation and identification of endophytic fungi involved in the promotion of sesquiterpene accumulation in Syringa pinnatifolia

Abstract Background The stem of Syringa pinnatifolia is used as traditional Mongolian medicine for the treatment of cardiopulmonary diseases, in which sesquiterpenes are regarded as the primary medicinal compounds. However, only S. pinnatifolia from natural stands is able to produce medicinal-grade stems when compared with that from cultivated stands. Despite the need to develop an effective technique to promote sesquiterpene accumulation in cultivated stands to ensure sustainable resource production, studies on the potential use of fungal endophytes as fungal elicitors were limited. Objective This study explored the potential of selected endophytic fungi isolated from the stems of wild and cultivated S. pinnatifolia and further examined their efficacy in promoting sesquiterpene production in S. pinnatifolia calli. Results Among the 11 fungal groups, Valsa sp, isolated from wild S. pinnatifolia, emerged as a promising fungal elicitor for promoting sesquiterpene accumulation in S. pinnatifolia. This conclusion is drawn from the production of a humulane-type sesquiterpene, known to be one of the most prevalent subtypes of sesquiterpenes in S. pinnatifolia. In addition, it was demonstrated that methyl jasmonate and salicylic acid displayed little effect on sesquiterpene accumulation in S. pinnatifolia. Conclusions The findings of this study lay a foundation for using fungal endophytes to promote sesquiterpene production in S. pinnatifolia. This contribution could subsequently advance the development of the local traditional medicine industry and aid in the protection of wild S. pinnatifolia.

Integrating Multiple Omics Identifies Phaeoacremonium rubrigenum Acting as Aquilaria sinensis Marker Fungus to Promote Agarwood Sesquiterpene Accumulation by Inducing Plant Host Phosphorylation.

ABSTRACTThe present study aimed to explore the factors that promote persistent agarwood accumulation. To this end, we first investigated the morphological changes and volatile compound distribution in five layers of “Guan Xiang” agarwood. The agarwood-normal transition layer (TL), an essential layer of persistent agarwood accumulation, showed clear metabolic differences by microscopy and GC-MS analysis. Microbiome analysis revealed that Phaeocremonium rubrigenum was the predominant biomarker fungus in the TL of “Guan Xiang” agarwood samples. Among the seven isolated fungi, P. rubrigenum exhibited a significantly heightened ability to induce the production in Aquilaria sinensis seedlings, especially for sesquiterpene. Tracing the proteome profile changes in P. rubrigenum-induced A. sinensis calli for 18 ds showed that the fungus-induced sesquiterpene biosynthesis increased mainly through the mevalonate (MVA) pathway. Specifically, the phosphorylation modification level, instead of the protein abundance of transcription factors (TFs), showed corresponding changes during sesquiterpene biosynthesis, thus indicating that induced phosphorylation is the key reason for enhanced sesquiterpene production.IMPORTANCE Agarwood is an expensive resinous portion derived from Aquilaria plants and has been widely used as medicine, incense, and perfume. The factors involved in steady agarwood accumulation remain elusive. Our current study suggests that as a TL marker fungus, P. rubrigenum could persistently promote agarwood sesquiterpene accumulation by inducing phosphorylation of the TFs-MVA network in A. sinensis. Moreover, our work provides strategies to improve agarwood industry management and sheds light on the potential molecular mechanisms of plant adaptation to native microbial conditions.

Volatile organic compound and endogenous phytohormone characteristics during callus browning in Aquilaria sinensis

Callus browning, a common feature in plant cell cultures, has hardly been explored in Aquilaria sinensis. Here, the volatile organic compounds (VOCs) were characterized during the browning of A. sinensis cells using Gas Chromatography-Mass Spectrometry (GC–MS), and total sesquiterpene accumulation was significantly induced and peaked during A. sinensis callus browning, after 60 days of callus growth. Additionally, 14 types of sesquiterpenes were detected in the browning A. sinensis callus, with greater sesquiterpene diversity than in the ones treated with methyl jasmonate and salicylic acid (SA) in a previous study. To further evaluate the mechanism of regulation of agarwood sesquiterpenes during cell browning in A. sinensis, changes on phytohormone concentrations were explored using ultra-performance liquid chromatography-electrospray tandem mass spectrometry system (UPLC-ESI-MS/MS). Both endogenous jasmonic acid and SA, key plant defense hormones, increased during A. sinensis callus browning. 1-aminocyclopropanecarboxylic acid, an ethylene (ET) precursor, decreased significantly after A. sinensis callus browning. Conversely, two primary growth hormones, auxin and cytokinin, exhibited opposite trends, suggesting that they also play roles in agarwood sesquiterpene accumulation. Our results potentially offer novel insights that could facilitate the production valuable phytochemical compounds such as agarwood sesquiterpenes, using plant cell cultures treated with appropriate phytohormone combinations.

A SlMYB75-centred transcriptional cascade regulates trichome formation and sesquiterpene accumulation in tomato.

Tomato trichomes act as a mechanical and chemical barrier against pests. An R2R3 MYB transcription factor gene, SlMYB75, is highly expressed in type II, V, and VI trichomes. SlMYB75 protein is located in the nucleus and possesses transcriptional activation activity. Down-regulation of SlMYB75 increased the formation of type II, V, and VI trichomes, accumulation of δ-elemene, β-caryophyllene, and α-humulene in glandular trichomes, and tolerance to spider mites in tomato. In contrast, overexpression of SlMYB75 inhibited trichome formation and sesquiterpene accumulation, and increased plant sensitivity to spider mites. RNA-Seq analyses of the SlMYB75 RNAi line indicated massive perturbation of the transcriptome, with a significant impact on several classes of transcription factors. Expression of the MYB genes SlMYB52 and SlTHM1 was strongly reduced in the RNAi line and increased in the SlMYB75-overexpressing line. SlMYB75 protein interacted with SlMYB52 and SlTHM1 and activated their expression. SlMYB75 directly targeted the promoter of the cyclin gene SlCycB2, increasing its activity. The auxin response factor SlARF4 directly targeted the promoter of SlMYB75 and inhibited its expression. SlMYB75 also bound to the promoters of the terpene synthase genes SlTPS12, SlTPS31, and SlTPS35, inhibiting their transcription. Our findings indicate that SlMYB75 perturbation affects several transcriptional circuits, resulting in altered trichome density and metabolic content.

Fungal-Type Terpene Synthases in Marchantia polymorpha Are Involved in Sesquiterpene Biosynthesis in Oil Body Cells.

The liverwort Marchantia polymorpha possesses oil bodies in idioblastic oil body cells scattered in its thallus. Oil bodies are subcellular organelles in which specific sesquiterpenes and bisbibenzyls are accumulated. Therefore, a specialized system for the biosynthesis and accumulation of these defense compounds specifically in oil bodies has been implied. A recent study on M. polymorpha genome sequencing revealed 10 genes that shared high similarities with fungal-type terpene synthases (TPSs). Eight of these fungal-type TPS-like genes in M. polymorpha (MpFTPSL1-6, -9 and -10) are located within a 376-kb stretch on chromosome 6 and share similarities of over 94% at the nucleotide level. Therefore, these genes have likely originated from recent gene duplication events. The expression of a subset of MpFTPSLs was induced under non-axenic growth on vermiculite, which increased the amounts of sesquiterpenes and number of oil bodies. The tdTomato fluorescent protein-based in-fusion reporter assay with MpFTPSL2 promoter revealed fluorescent signals specifically in oil body cells of the thallus, indicating that MpFTPSL2 functions in oil body cells. Recombinant MpFTPSL2 expression in Escherichia coli led to sesquiterpene synthesis from farnesyl pyrophosphate. Moreover, suppression of a subset of MpFTPSLs through RNA interference reduced sesquiterpene accumulation in thalli grown on vermiculite. Taken together, these results suggest that at least a subset of MpFTPSLs is involved in sesquiterpene synthesis in oil body cells.

Cloning and functional characterization of farnesyl diphosphate synthase in Senecio scandens

As a secondary metabolite, sesquiterpenes are not only have important functions in plant defense and signaling, but also play potential roles in basic materials for pharmaceuticals, cosmetic and flavor. As a traditional Chinese herbal medicine, Senecio scandens exhibits effects of anti-inflammatory and immunosuppressive, as well as invigorating the blood and removing extravasated blood. Over 600 sesquiterpenes with diverse structures were isolated from S. scandens and related species in the same genus. To characterize sesquiterpenes synthesis, two FPS genes(SsFPS1 and SsFPS2) were identified in S. scandens through transcriptomic analysis. Bioinformatic analysis showed that both SsFPSs have conserved motifs for FPS function. Both SsFPSs exhibited constitutive gene expression in S. scandens tissues and SsFPS2 accumulated higher transcript in leaves and roots than SsFPS1. Meanwhile consistent with constitutive sesquiterpene accumulation in S.scandens tissues, most of these sesquiterpenes were detected in leaves and roots more than stems and flowers. Recombinant expression through Escherichia coli metabolic engineering, SsFPS1 or SsFPS2 was co-transformed with ZmTPS11(maize β-macrocarpene synthase) into BL21 competent cells. The results showed that the content of β-macrocarpene was increased by co-transformation with SsFPSs. It is demonstrated that SsFPS1 and SsFPS2 catalyzed E,E-FPP formation and provided FPP precursor for downstream sesquiterpene synthases. Characterization of SsFPSs provided the foundation for the exploration of biosynthesis of sesquiterpenoid with diverse structures and potential pharmaceutical values in S.scandens, and provide an important theoretical basis for the development of S. scandens abundant resources.

Transcriptomic analysis of postharvest toon buds and key enzymes involved in terpenoid biosynthesis during cold storage

Toon buds have been used in China as an important woody vegetable for its unique flavor. However, the changes of volatile terpenoids related to flavor and their biosynthesis in postharvest toon buds after cold storage remain largely unknown. Therefore, a transcriptomic database must be constructed to analyze the molecular mechanism of terpenoid biosynthesis in toon buds during cold storage. The chemical profiles of volatile terpenoids over postharvest storage periods were comparatively analyzed by gas chromatography–mass spectrometry. Results showed that the total content of volatile terpenoids and their oxidates, except for monoterpenes and a small portion of sesquiterpenes, increased significantly after cold storage. The transcriptome database derived from different cold storage times was established using BGIseq500 technology. Approximately 6.5 g of clean nucleotides were obtained and de novo assembled into 152 127 non-redundant unigenes, approximately 72.36% of which could be aligned to public databases. Many candidate genes involved in terpenoid biosynthesis were identified. Furthermore, 29 513 unigenes were demonstrated to be differentially expressed under different cold storage times. These differentially expressed genes were functionally annotated by gene ontology enrichment and Kyoto Encyclopedia of Genes and Genomes enrichment analyses. The expression of 20 putative unigenes involved in terpenoid pathways was confirmed by qRT-PCR, the results of which were consistent with the RNA-seq data. These genes with different expression patterns correlated well with the changes in the volatile terpenoids in the toon buds during low-temperature storage. In summary, our results showed that the high expression levels of terpenoid backbone biosynthesis pathways contributed to sesquiterpene biosynthesis after cold storage, which led to high sesquiterpene accumulation in toon buds during low-temperature storage.

Expression of the Fusarium graminearum terpenome and involvement of the endoplasmic reticulum-derived toxisome

The sesquiterpenoid deoxynivalenol (DON) is an important trichothecene mycotoxin produced by the cereal pathogen Fusarium graminearum. DON is synthesized in specialized subcellular structures called toxisomes. The first step in DON synthesis is catalyzed by the sesquiterpene synthase (STS), Tri5 (trichodiene synthase), resulting in the cyclization of farnesyl diphosphate (FPP) to produce the sesquiterpene trichodiene. Tri5 is one of eight putative STSs in the F. graminearum genome. To better understand the F. graminearum terpenome, the volatile and soluble fractions of fungal cultures were sampled. Stringent regulation of sesquiterpene accumulation was observed. When grown in trichothecene induction medium, the fungus produces trichothecenes as well as several volatile non-trichothecene related sesquiterpenes, whereas no volatile terpenes were detected when grown in non-inducing medium. Surprisingly, a Δtri5 deletion strain grown in inducing conditions not only ceased accumulation of trichothecenes, but also failed to produce the non-trichothecene related sesquiterpenes. To test whether Tri5 from F. graminearum may be a promiscuous STS directly producing all observed sesquiterpenes, Tri5 was cloned and expressed in E. coli and shown to produce primarily trichodiene in addition to minor, related cyclization products. Therefore, while Tri5 expression in F. graminearum is necessary for non-trichothecene sesquiterpene biosynthesis, direct catalysis by Tri5 does not explain the sesquiterpene deficient phenotype observed in the Δtri5 strain. To test whether Tri5 protein, separate from its enzymatic activity, may be required for non-trichothecene synthesis, the Tri5 locus was replaced with an enzymatically inactive, but structurally unaffected tri5N225D S229T allele. This allele restores non-trichothecene synthesis but not trichothecene synthesis. The tri5N225D S229T allele also restores toxisome structure which is lacking in the Δtri5 deletion strain. Our results indicate that the Tri5 protein, but not its enzymatic activity, is also required for the synthesis of non-trichothecene related sesquiterpenes and the formation of toxisomes. Toxisomes thus not only may be important for DON synthesis, but also for the synthesis of other sesquiterpene mycotoxins such as culmorin by F. graminearum.

Seasonal and Differential Sesquiterpene Accumulation in Artemisia annua Suggest Selection Based on Both Artemisinin and Dihydroartemisinic Acid may Increase Artemisinin in planta.

Commercial Artemisia annua crops are the sole source of artemisinin (ART) worldwide. Data on seasonal accumulation and peak of sesquiterpenes, especially ART in commercial A. annua, is lacking while current breeding programs focus only on ART and plant biomass, but ignores dihydroartemisinic acid (DHAA) and artemisinic acid (AA). Despite past breeding successes, plants richer in ART are needed to decrease prices of artemisinin-combination therapy (ACT). Our results show that sesquiterpene concentrations vary greatly along the growing season and that sesquiterpene profiles differ widely among chemotypes. Field studies with elite Brazilian, Chinese, and Swiss germplasms established that ART peaked in vegetative plants from late August to early September, suggesting that ART is related to the photoperiod, not flowering. DHAA peaks with ART in Chinese and Swiss plants, but decreases, as ART increases, in Brazilian plants, while AA remained stable through the season in these genotypes. Chinese plants peaked at 0.9% ART, 1.6% DHAA; Brazilian plants at 0.9% ART, with less than 0.4% DHAA; Swiss plants at 0.8% ART and 1% DHAA. At single-date harvests, seeded Swiss plants produced 0.55–1.2% ART, with plants being higher in DHAA than ART; Brazilian plants produced 0.33–1.5% ART, with most having higher ART than DHAA. Elite germplasms produced from 0.02–0.43% AA, except Sandeman-UK (0.4–1.1% AA). Our data suggest that different chemotypes, high in ART and DHAA, have complementary pathways, while competing with AA. Crossing plants high in ART and DHAA may generate hybrids with higher ART than currently available in commercial germplasms. Selecting for high ART and DHAA (and low AA) can be a valuable approach for future selection and breeding to produce plants more efficient in transforming DHAA into ART in planta and during post-harvest. This novel approach could change the breeding focus of A. annua and other pharmaceutical species that produce more than one desired metabolite in the same pathway. Obtaining natural variants with high ART content will empower countries and farmers who select, improve, and cultivate A. annua as a commercial pharmaceutical crop. This selection approach could enable ART to be produced locally where it is most needed to fight malaria and other parasitic neglected diseases.

Leaf Essential Oil from Eugenia luschnathiana and Myrciaria tenella (Myrtaceae) from Two Different Accesses in Southeastern Brazil

The leaf essential oils from Eugenia luschnathiana Klotzsch ex B.D. Jacks. and Myrciaria tenella (DC.) O. Berg growing in two different locations in southeastern Brazil were obtained by hydrodistillation and analyzed by GC and GC-MS. The oil profiles of individual accesses of E. luschnathiana from each location were characterized by the virtual absence of monoterpenes. The sesquiterpenes were of the caryophyllane (32%), guaiane (27%) and cadinane (19%) types in the São Paulo State access and caryophyllane (43%) and cadinane (28%) types in the Rio de Janeiro access. This is the first report on the composition of E. luschnathiana oil. Monoterpenes were predominant in both accesses of M. tenella (57-65%), and sesquiterpenes were mostly represented by the guaiane-eudesmane (16%) or caryophyllene-guaiane types (17%), respectively, in the São Paulo and Rio de Janeiro accesses. In monoterpene composition, the M. tenella oils qualitatively and quantitatively resembled that from a previous access from southern Brazil, whereas the sesquiterpene accumulation showed no similarity to any previous collection in Brazil regarding any favored skeleton type.

Metabolic engineering of sesquiterpene metabolism in yeast.

Terpenes are structurally diverse compounds that are of interest because of their biological activities and industrial value. These compounds consist of chirally rich hydrocarbon backbones derived from terpene synthases, which are subsequently decorated with hydroxyl substituents catalyzed by terpene hydroxylases. Availability of these compounds is, however, limited by intractable synthetic means and because they are produced in low amounts and as complex mixtures by natural sources. We engineered yeast for sesquiterpene accumulation by introducing genetic modifications that enable the yeast to accumulate high levels of the key intermediate farnesyl diphosphate (FPP). Co-expression of terpene synthase genes diverted the enlarged FPP pool to greater than 80 mg/L of sesquiterpene. Efficient coupling of terpene production with hydroxylation was also demonstrated by coordinate expression of terpene hydroxylase activity, yielding 50 mg/L each of hydrocarbon and hydroxylated products. These yeast now provide a convenient format for investigating catalytic coupling between terpene synthases and hydroxylases, as well as a platform for the industrial production of high value, single-entity and stereochemically unique terpenes.

Induction of volatile terpene biosynthesis and diurnal emission by methyl jasmonate in foliage of Norway spruce.

Terpenoids are characteristic constitutive and inducible defense chemicals of conifers. The biochemical regulation of terpene formation, accumulation, and release from conifer needles was studied in Norway spruce [Picea abies L. (Karst)] saplings using methyl jasmonate (MeJA) to induce defensive responses without inflicting physical damage to terpene storage structures. MeJA treatment caused a 2-fold increase in monoterpene and sesquiterpene accumulation in needles without changes in terpene composition, much less than the 10- and 40-fold increases in monoterpenes and diterpenes, respectively, observed in wood tissue after MeJA treatment (D. Martin, D. Tholl, J. Gershenzon, J. Bohlmann [2002] Plant Physiol 129: 1003-1018). At the same time, MeJA triggered a 5-fold increase in total terpene emission from foliage, with a shift in composition to a blend dominated by oxygenated monoterpenes (e.g. linalool) and sesquiterpenes [e.g. (E)-beta-farnesene] that also included methyl salicylate. The rate of linalool emission increased more than 100-fold and that of sesquiterpenes increased more than 30-fold. Emission of these compounds followed a pronounced diurnal rhythm with the maximum amount released during the light period. The major MeJA-induced volatile terpenes appear to be synthesized de novo after treatment, rather than being released from stored terpene pools, because they are almost completely absent from needle oleoresin and are the major products of terpene synthase activity measured after MeJA treatment. Based on precedents in other species, the induced emission of terpenes from Norway spruce foliage may have ecological and physiological significance.

Effects of potential signal transduction antagonists on phytoalexin accumulation in tobacco

Tobacco cell suspensions produce sesquiterpene phytoalexins when treated with cellulase. This response is enhanced by CaCl 2, MgCl 2 or CdCl 2. For CaCl 2 and MgCl 2, enhancement extends for 8 hr after elicitor treatment. Several classes of signal transduction antagonists were tested on this plant system. EGTA inhibits sesquiterpene accumulation with an effective concentration range suggesting that Ca 2 + is the divalent cation being affected. Mg 2 + is as effective as Ca 2 + in relieving the inhibition by EGTA. This type of shared role by Ca 2 + and Mg 2 + has not been described previously and is yet to be characterized. The timing of the EGTA effect suggests a role of Ca 2 + in signal transduction. Calcium ion influx across the plasma membrane is one possibility since inhibitors of Ca 2 + influx inhibit sesquiterpene accumulation induced by cellulase. Verapamil is an inhibitor and its action is relieved by the Ca 2 + ionophore A23187. Among the cations which inhibit Ca 2 + influx, La 3 + is the most effective inhibitor of sesquiterpene accumulation. Calmodulin, or a Ca 2 + -dependent protein kinase, is also implicated in the signal transduction sequence, based on the results with W7, W5 and staurosporine. Neomycin and LiCl, inhibitors of phosphoinositide cycle steps, enhance cellulase-elicited sesquiterpene accumulation, suggesting a complex interaction of this cycle in the phytoalexin response. The results indicate that Ca 2 + and Mg 2 + play several roles in sesquiterpene biosynthesis. The involvement of a multicomponent elicitor signal transduction pathway, with calcium ion functioning at more than one step, is suggested.

Elicitor-Inducible 3-Hydroxy-3-Methylglutaryl Coenzyme A Reductase Activity Is Required for Sesquiterpene Accumulation in Tobacco Cell Suspension Cultures

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.

Inhibition by Salicylhydroxamic Acid, BW755C, Eicosatetraynoic Acid, and Disulfiram of Hypersensitive Resistance Elicited by Arachidonic Acid or Poly-l-Lysine in Potato Tuber

The hypothesis that arachidonic acid (AA) induction of sesquiterpene accumulation and browning in potato (Solanum tuberosum) is mediated by a lipoxygenase metabolite of AA was tested using lipoxygenase inhibitors. Salicylhydroxamic acid (SHAM) and 3-amino-1-(3-trifluoromethylphenyl)-2-pyrazoline hydrochloride (BW755C) delayed the response to AA. Inhibition by eicosatetraynoic acid (ETYA) was more persistent. These results are consistent with previous reports that SHAM and BW755C are reversible inhibitors of lipoxygenase and easily oxidized by potato while ETYA acts as an irreversible inhibitor. Disulfiram (tetraethylthiuram disulfide) also inhibited AA elicitor activity. SHAM was most effective if applied at the time of AA treatment, having no effect if applied 6 hours afterward. SHAM was effective in the presence of MES or MOPS buffers but not in acetate-buffered or unbuffered solutions; neither BW755C nor ETYA exhibited this restriction. However, SHAM, BW755C, and ETYA also were inhibitors of browning and sesquiterpene accumulation elicited in potato by poly-l-lysine, which, unlike AA, is not a lipoxygenase substrate. SHAM effectiveness also was restricted to 6 hours after treatment with poly-l-lysine. While the results with AA support a role for lipoxygenase, those with poly-l-lysine may be evidence that these compounds are having other effects in potato tissue.

Arachidonic acid-related elicitors of the hypersensitive response in potato and enhancement of their activities by glucans from Phytophthora infestans (Mont.) deBary

The dose response for elicitation of the hypersensitive reaction in potato tuber discs by arachidonic acid (AA) suggested saturation at higher concentrations. Glucans from Phytophthora infestans, inactive themselves as elicitors of the hypersensitive reaction, enhanced sesquiterpene accumulation and hypersensitive browning elicited by AA. Significant activity (seven times control values) was observed with 33 pmol AA/3.0cm potato disc in the presence of glucans. Glucans did not affect accumulation of steroid glycoalkaloids, influence the timing or relative amounts of sesquiterpenes which accumulate, or affect recovery of AA added to potato discs. Glucans enhanced activity whether added to potato discs 18 h prior to AA, at the same time as AA, or 18 h after AA. Elicitor activity in the presence of glucans was evident with 20-carbon unsaturated fatty acids that had little or no elicitor activity in the absence of glucans. The position of double bonds had considerable influence on the specific activity of unsaturated fatty acids. The most active had a minimum of three double bonds in a methylene-interrupted series beginning with Δ5, e.g., Δ5,8,11. A Δ5 double bond conferred significant activity even if it was not part of a methylene-interrupted series. The 20-carbon chain length appeared optimal for elicitor activity. The 22-carbon chain acids had low activity, and 16- and 18-carbon acids were inactive. A free carboxyl group or easily transesterified group appeared necessary for activity. Arachidonyl alcohol had very low activity and arachidonyl cyanide was inactive. AA-containing phosphatidylcholine, lysophosphatidylcholine and monoacylglycerol were at least as active as free AA, AA-containing diacylglycerols were slightly less active than free AA, and triarachidonyl glycerol was inactive.

Phenylalanine ammonia lyase activity and fungitoxic metabolites produced by potato cultivars in response to Fusarium tuber rot

The specific activity of phenylalanine ammonia lyase (PAL), glycoalkaloid and sesquiterpene accumulation were measured in potato tubers ( Solanum tuberosum L.) at 48, 96, and 168 h following wounding or inoculation with a spore suspension of the dry rot pathogen Fusarium roseum var. sambucinum. Activity of PAL was higher in inoculated than in wounded tissues and remained higher up to 168 h after inoculation in both resistant and susceptible cultivars. PAL activities in inoculated tissues of four clones of divergent genotype varied threefold but very high resistance shown by the breeding clone BR6316-7 was not related to its PAL activity. Challenge with the nonpathogen Fusarium reseum resulted in less stimulation of PAL activity than did inoculation with the virulent Fusarium roseum var. sambucinum or with the relatively weak pathogen Alternaria solani. Steroid glycoalkaloid accumulation was repressed in inoculated tissues of both resistant and susceptible clones compared to wounded controls. This repression was least, however, in the highly resistant clone. The terpenoids, rishitin, phytuberin, and a compound tentatively identified as lubimin, were detected in inoculated tissues of both resistant and susceptible cultivars 48 h after inoculation. Total accumulation after 168 h was least in the highly resistant clone and greatest in the most susceptible clone. The three factors measured appear to be involved in a generalized response to the dry rot pathogen for both resistant and susceptible cultivars. Moderate resistance as shown by the cultivar Pioneer may be related to an intensification of this generalized response following inoculation, but very high resistance involves other mechanisms.

Chemical and morphological studies on sites of sesquiterpene accumulation in Pogostemon cablin (patchouli)

Techniques are described for the micro-analysis of the free volatile sesquiterpenes in plant tissue by direct-loading gas liquid chromatography. Evidence is presented for the accumulation of sesquiterpenes, not only in the external glandular trichomes, but also in specialized internal accumulatory cells. The morphology and development of these external and internal structures are described as seen under light and electron microscopy. Also, while the apical dome of the stem neither shows any specialized accumulatory structures nor contains any detectable sesquiterpenes, the second pair of primordial leaves has glandular trichomes and a sesquiterpene concentration approximately twelve times higher than that in any other part of the plant.