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1.
Phytochemistry ; 202: 113290, 2022 Oct.
Article in English | MEDLINE | ID: mdl-35803303

ABSTRACT

Nigella sativa (Black cumin) has many applications in food and pharmaceutical industries. Thymoquinone has been considered as a main effective compound in N. sativa seeds and attracted researchers' attention mainly due to its medicinal potential. In this study, the essential oil components of leaves, flowers and seed developmental stages including half black seeds, soft black seeds and hard black seeds were analyzed in N. sativa. Whereas no terpenes were detected in flowers and leaves, seeds showed an essential oil composition that increased in its thymoquinone content during seed maturation. To study the proposed first step of thymoquinone biosynthesis, the formation of γ-terpinene from geranyl diphosphate (GDP), we identified and functionally characterized a γ-terpinene synthase (NsTPS1) in N. sativa. This monoterpene synthase was identified in RNA sequence data derived from seeds. After heterologous expression in Escherichia coli, partially purified NsTPS1 converted GDP to γ-terpinene. NsTPS1 is the first functionally characterized terpene synthase from N. sativa and displays a higher similarity to other terpene synthases from Ranunculaceae than known γ-terpinene synthases from more distant plant species. Characterization of NsTPS1 elucidates the first dedicated step in the biosynthesis of thymoquinone in N. sativa and paves the way towards metabolic engineering for high-level thymoquinone production.


Subject(s)
Nigella sativa , Oils, Volatile , Cyclohexane Monoterpenes , Seeds
2.
Proc Natl Acad Sci U S A ; 118(52)2021 12 28.
Article in English | MEDLINE | ID: mdl-34930840

ABSTRACT

Thymol and carvacrol are phenolic monoterpenes found in thyme, oregano, and several other species of the Lamiaceae. Long valued for their smell and taste, these substances also have antibacterial and anti-spasmolytic properties. They are also suggested to be precursors of thymohydroquinone and thymoquinone, monoterpenes with anti-inflammatory, antioxidant, and antitumor activities. Thymol and carvacrol biosynthesis has been proposed to proceed by the cyclization of geranyl diphosphate to γ-terpinene, followed by a series of oxidations via p-cymene. Here, we show that γ-terpinene is oxidized by cytochrome P450 monooxygenases (P450s) of the CYP71D subfamily to produce unstable cyclohexadienol intermediates, which are then dehydrogenated by a short-chain dehydrogenase/reductase (SDR) to the corresponding ketones. The subsequent formation of the aromatic compounds occurs via keto-enol tautomerisms. Combining these enzymes with γ-terpinene in in vitro assays or in vivo in Nicotiana benthamiana yielded thymol and carvacrol as products. In the absence of the SDRs, only p-cymene was formed by rearrangement of the cyclohexadienol intermediates. The nature of these unstable intermediates was inferred from reactions with the γ-terpinene isomer limonene and by analogy to reactions catalyzed by related enzymes. We also identified and characterized two P450s of the CYP76S and CYP736A subfamilies that catalyze the hydroxylation of thymol and carvacrol to thymohydroquinone when heterologously expressed in yeast and N. benthamiana Our findings alter previous views of thymol and carvacrol formation, identify the enzymes involved in the biosynthesis of these phenolic monoterpenes and thymohydroquinone in the Lamiaceae, and provide targets for metabolic engineering of high-value terpenes in plants.


Subject(s)
Cymenes/metabolism , Cytochrome P-450 Enzyme System/metabolism , Lamiaceae/metabolism , Short Chain Dehydrogenase-Reductases/metabolism , Thymol/analogs & derivatives , Thymol/metabolism , Cymenes/chemistry , Cytochrome P-450 Enzyme System/genetics , Lamiaceae/enzymology , Lamiaceae/genetics , Metabolic Networks and Pathways/genetics , Plant Proteins/genetics , Plant Proteins/metabolism , Short Chain Dehydrogenase-Reductases/genetics , Thymol/chemistry
3.
Plant Environ Interact ; 2(4): 177-193, 2021 Aug.
Article in English | MEDLINE | ID: mdl-37283700

ABSTRACT

Plants use a wide array of secondary metabolites including terpenes as defense against herbivore and pathogen attack, which can be constitutively expressed or induced. Here, we investigated aspects of the chemical and molecular basis of resistance against the exotic rust fungus Austropuccinia psidii in Melaleuca quinquenervia, with a focus on terpenes. Foliar terpenes of resistant and susceptible plants were quantified, and we assessed whether chemotypic variation contributed to resistance to infection by A. psidii. We found that chemotypes did not contribute to the resistance and susceptibility of M. quinquenervia. However, in one of the chemotypes (Chemotype 2), susceptible plants showed higher concentrations of several terpenes including α-pinene, limonene, 1,8-cineole, and viridiflorol compared with resistant plants. Transcriptome profiling of these plants showed that several TPS genes were strongly induced in response to infection by A. psidii. Functional characterization of these TPS showed them to be mono- and sesquiterpene synthases producing compounds including 1,8-cineole, ß-caryophyllene, viridiflorol and nerolidol. The expression of these TPS genes correlated with metabolite data in a susceptible plant. These results suggest the complexity of resistance mechanism regulated by M. quinquenervia and that modulation of terpenes may be one of the components that contribute to resistance against A. psidii.

4.
Plants (Basel) ; 9(5)2020 Apr 26.
Article in English | MEDLINE | ID: mdl-32357450

ABSTRACT

Terpene synthases make up a large family of enzymes that convert prenyl diphosphates into an enormous variety of terpene skeletons. Due to their electrophilic reaction mechanism-which involves the formation of carbocations followed by hydride shifts and skeletal rearrangements-terpene synthases often produce complex mixtures of products. In the present study, we investigate amino acids that determine the product specificities of the maize terpene synthases TPS4 and TPS10. The enzymes showed 57% amino acid similarity and produced different mixtures of sesquiterpenes. Sequence comparisons and structure modeling revealed that out of the 43 amino acids forming the active site cavity, 17 differed between TPS4 and TPS10. While combined mutation of these 17 residues in TPS4 resulted in an enzyme with a product specificity similar to TPS10, the additional mutation of two amino acids next to the active site led to a nearly complete conversion of TPS4 into TPS10. These data demonstrate that the different product specificities of TPS4 and TPS10 are determined not only by amino acids forming the active site cavity, but also by neighboring residues that influence the conformation of active site amino acids.

5.
Sci Rep ; 8(1): 14634, 2018 10 02.
Article in English | MEDLINE | ID: mdl-30279427

ABSTRACT

Honey bees are highly prone to infectious diseases, causing colony losses in the worst case. However, they combat diseases through a combination of their innate immune system and social defence behaviours like foraging for health-enhancing plant products (e.g. nectar, pollen and resin). Plant secondary metabolites are not only highly active against bacteria and fungi, they might even enhance selective foraging and feeding decisions in the colony. Here, we tested six major plant terpenes and their corresponding acetates, characterizing six natural Thymus vulgaris chemotypes, for their antimicrobial activity on bacteria associated with European foulbrood. Comparison of the inhibitory activity revealed the highest activity for carvacrol and thymol whereas the acetates mostly did not inhibit bacterial growth. All terpenes and acetates are present in the nectar and pollen of thyme, with pollen containing concentrations higher by several orders of magnitude. The physiological response was tested on forager and freshly emerged bees by means of antennal electroantennography. Both responded much stronger to geraniol and trans-sabinene hydrate compared to carvacrol and thymol. In conclusion, bee-forageable thyme product terpenes (mainly from pollen) yield effective antibiotic activity by reducing the growth of bee disease-associated bacteria and can be detected with different response levels by the honey bees' antennae. This is a further step forward in understanding the complex pathogen-pollinator-plant network.


Subject(s)
Bees/microbiology , Gram-Positive Bacteria/drug effects , Monoterpenes/pharmacology , Thymus Plant/metabolism , Animals , Plant Leaves/chemistry , Plant Nectar/chemistry , Pollen/chemistry
6.
BMC Plant Biol ; 17(1): 160, 2017 Oct 04.
Article in English | MEDLINE | ID: mdl-28978322

ABSTRACT

BACKGROUND: Terpene rich leaves are a characteristic of Myrtaceae. There is significant qualitative variation in the terpene profile of plants within a single species, which is observable as "chemotypes". Understanding the molecular basis of chemotypic variation will help explain how such variation is maintained in natural populations as well as allowing focussed breeding for those terpenes sought by industry. The leaves of the medicinal tea tree, Melaleuca alternifolia, are used to produce terpinen-4-ol rich tea tree oil, but there are six naturally occurring chemotypes; three cardinal chemotypes (dominated by terpinen-4-ol, terpinolene and 1,8-cineole, respectively) and three intermediates. It has been predicted that three distinct terpene synthases could be responsible for the maintenance of chemotypic variation in this species. RESULTS: We isolated and characterised the most abundant terpene synthases (TPSs) from the three cardinal chemotypes of M. alternifolia. Functional characterisation of these enzymes shows that they produce the dominant compounds in the foliar terpene profile of all six chemotypes. Using RNA-Seq, we investigated the expression of these and 24 additional putative terpene synthases in young leaves of all six chemotypes of M. alternifolia. CONCLUSIONS: Despite contributing to the variation patterns observed, variation in gene expression of the three TPS genes is not enough to explain all variation for the maintenance of chemotypes. Other candidate terpene synthases as well as other levels of regulation must also be involved. The results of this study provide novel insights into the complexity of terpene biosynthesis in natural populations of a non-model organism.


Subject(s)
Alkyl and Aryl Transferases/metabolism , Melaleuca/enzymology , Alkyl and Aryl Transferases/genetics , Alkyl and Aryl Transferases/isolation & purification , Cyclohexane Monoterpenes , Cyclohexanols/metabolism , DNA, Plant , Eucalyptol , Gene Expression Profiling , Genes, Plant , Melaleuca/chemistry , Melaleuca/genetics , Monoterpenes/metabolism , Sequence Analysis, DNA , Terpenes/metabolism , Trees/chemistry , Trees/enzymology
7.
BMC Genomics ; 18(1): 599, 2017 08 10.
Article in English | MEDLINE | ID: mdl-28797221

ABSTRACT

BACKGROUND: Chamomile (Matricaria recutita L.) has a long history of use in herbal medicine with various applications, and the flower heads contain numerous secondary metabolites which are medicinally active. In the major crop plants, next generation sequencing (NGS) approaches are intensely applied to exploit genetic resources, to develop genomic resources and to enhance breeding. Here, genotyping-by-sequencing (GBS) has been used in the non-model medicinal plant chamomile to evaluate the genetic structure of the cultivated varieties/populations, and to perform genome wide association study (GWAS) focusing on genes with large effect on flowering time and the medicinally important alpha-bisabolol content. RESULTS: GBS analysis allowed the identification of 6495 high-quality SNP-markers in our panel of 91 M. recutita plants from 33 origins (2-4 genotypes each) and 4 M. discoidea plants as outgroup, grown in the greenhouse in Gatersleben, Germany. M. recutita proved to be clearly distinct from the outgroup, as was demonstrated by different cluster and principal coordinate analyses using the SNP-markers. Chamomile genotypes from the same origin were mostly genetically similar. Model-based cluster analysis revealed one large group of tetraploid genotypes with low genetic differentiation including 39 plants from 14 origins. Tetraploids tended to display lower genetic diversity than diploids, probably reflecting their origin by artificial polyploidisation from only a limited set of genetic backgrounds. Analyses of flowering time demonstrated that diploids generally flowered earlier than tetraploids, and the analysis of alpha-bisabolol identified several tetraploid genotypes with a high content. GWAS identified highly significant (P < 0.01) SNPs for flowering time (9) and alpha-bisabolol (71). One sequence harbouring SNPs associated with flowering time was described to play a role in self-pollination in Arabidopsis thaliana, whereas four sequences harbouring SNPs associated with alpha-bisabolol were identified to be involved in plant biotic and abiotic stress response in various plants species. CONCLUSIONS: The first genomic resource for future applications to enhance breeding in chamomile was created, andanalyses of diversity will facilitate the exploitation of these genetic resources. The GWAS data pave the way for future research towards the genetics underlying important traits in chamomile, the identification of marker-trait associations, and development of reliable markers for practical breeding.


Subject(s)
Chamomile/genetics , Flowers/growth & development , Genetic Loci/genetics , Genome-Wide Association Study , Genotyping Techniques , Polymorphism, Single Nucleotide/genetics , Sesquiterpenes/metabolism , Breeding , Chamomile/growth & development , DNA, Plant/genetics , DNA, Plant/isolation & purification , Diploidy , Monocyclic Sesquiterpenes , Sequence Analysis , Tetraploidy
8.
Ecol Evol ; 7(8): 2835-2845, 2017 04.
Article in English | MEDLINE | ID: mdl-28428873

ABSTRACT

Maize (Zea mays) emits volatile terpenes in response to insect feeding and egg deposition to defend itself against harmful pests. However, maize cultivars differ strongly in their ability to produce the defense signal. To further understand the agroecological role and underlying genetic mechanisms for variation in terpene emission among maize cultivars, we studied the production of an important signaling component (E)-caryophyllene in a South American maize landrace Braz1006 possessing stemborer Chilo partellus egg inducible defense trait, in comparison with the European maize line Delprim and North American inbred line B73. The (E)-caryophyllene production level and transcript abundance of TPS23, terpene synthase responsible for (E)-caryophyllene formation, were compared between Braz1006, Delprim, and B73 after mimicked herbivory. Braz1006-TPS23 was heterologously expressed in E. coli, and amino acid sequences were determined. Furthermore, electrophysiological and behavioral responses of a key parasitic wasp Cotesia sesamiae to C. partellus egg-induced Braz1006 volatiles were determined using coupled gas chromatography electroantennography and olfactometer bioassay studies. After elicitor treatment, Braz1006 released eightfold higher (E)-caryophyllene than Delprim, whereas no (E)-caryophyllene was detected in B73. The superior (E)-caryophyllene production by Braz1006 was positively correlated with high transcript levels of TPS23 in the landrace compared to Delprim. TPS23 alleles from Braz1006 showed dissimilarities at different sequence positions with Delprim and B73 and encodes an active enzyme. Cotesia sesamiae was attracted to egg-induced volatiles from Braz1006 and synthetic (E)-caryophyllene. The variation in (E)-caryophyllene emission between Braz1006 and Delprim is positively correlated with induced levels of TPS23 transcripts. The enhanced TPS23 activity and corresponding (E)-caryophyllene production by the maize landrace could be attributed to the differences in amino acid sequence with the other maize lines. This study suggested that the same analogous genes could have contrasting expression patterns in different maize genetic backgrounds. The current findings provide valuable insight not only into genetic mechanisms underlying variation in defense signal production but also the prospect of introgressing the novel defense traits into elite maize varieties for effective and ecologically sound protection of crops against damaging insect pests.

9.
Plant Cell ; 28(10): 2651-2665, 2016 10.
Article in English | MEDLINE | ID: mdl-27662898

ABSTRACT

Plant volatiles not only have multiple defense functions against herbivores, fungi, and bacteria, but also have been implicated in signaling within the plant and toward other organisms. Elucidating the function of individual plant volatiles will require more knowledge of their biosynthesis and regulation in response to external stimuli. By exploiting the variation of herbivore-induced volatiles among 26 maize (Zea mays) inbred lines, we conducted a nested association mapping and genome-wide association study (GWAS) to identify a set of quantitative trait loci (QTLs) for investigating the pathways of volatile terpene production. The most significant identified QTL affects the emission of (E)-nerolidol, linalool, and the two homoterpenes (E)-3,8-dimethyl-1,4,7-nonatriene (DMNT) and (E,E)-4,8,12-trimethyltrideca-1,3,7,11-tetraene (TMTT). GWAS associated a single nucleotide polymorphism in the promoter of the gene encoding the terpene synthase TPS2 with this QTL Biochemical characterization of TPS2 verified that this plastid-localized enzyme forms linalool, (E)-nerolidol, and (E,E)-geranyllinalool. The subsequent conversion of (E)-nerolidol into DMNT maps to a P450 monooxygenase, CYP92C5, which is capable of converting nerolidol into DMNT by oxidative degradation. A QTL influencing TMTT accumulation corresponds to a similar monooxygenase, CYP92C6, which is specific for the conversion of (E,E)-geranyllinalool to TMTT The DMNT biosynthetic pathway and both monooxygenases are distinct from those previously characterized for DMNT and TMTT synthesis in Arabidopsis thaliana, suggesting independent evolution of these enzymatic activities.


Subject(s)
Arabidopsis/metabolism , Acyclic Monoterpenes , Arabidopsis/genetics , Arabidopsis Proteins/metabolism , Genome-Wide Association Study , Monoterpenes/metabolism , Quantitative Trait Loci/genetics , Sesquiterpenes/metabolism
10.
Plant Physiol ; 170(2): 742-51, 2016 Feb.
Article in English | MEDLINE | ID: mdl-26620527

ABSTRACT

While most commonly associated with its role in gibberellin phytohormone biosynthesis, ent-kaurene also serves as an intermediate in more specialized diterpenoid metabolism, as exemplified by the more than 800 known derived natural products. Among these are the maize kauralexins. However, no ent-kaurene synthases (KSs) have been identified from maize. The maize gibberellin-deficient dwarf-5 (d5) mutant has been associated with a loss of KS activity. The relevant genetic lesion has been previously mapped, and was found here to correlate with the location of the KS-like gene ZmKSL3. Intriguingly, this forms part of a tandem array with two other terpene synthases (TPSs). Although one of these, ZmTPS1, has been previously reported to encode a sesquiterpene synthase, and both ZmTPS1 and that encoded by the third gene, ZmKSL5, have lost the N-terminal γ-domain prototypically associated with KS(L)s, all three genes fall within the KS(L) or TPS-e subfamily. Here it is reported that all three genes encode enzymes that are targeted to the plastid in planta, where diterpenoid biosynthesis is initiated, and which all readily catalyze the production of ent-kaurene. Consistent with the closer phylogenetic relationship of ZmKSL3 with previously identified KSs from cereals, only transcription of this gene is affected in d5 plants. On the other hand, the expression of all three of these genes is inducible, suggesting a role in more specialized metabolism, such as that of the kauralexins. Thus, these results clarify not only gibberellin phytohormone, but also diterpenoid phytoalexin biosynthesis in this important cereal crop plant.


Subject(s)
Alkyl and Aryl Transferases/genetics , Gibberellins/metabolism , Plant Growth Regulators/metabolism , Plant Proteins/genetics , Zea mays/enzymology , Alkyl and Aryl Transferases/metabolism , Base Sequence , Diterpenes, Kaurane/chemistry , Diterpenes, Kaurane/metabolism , Phylogeny , Plant Proteins/metabolism , Sequence Alignment , Sesquiterpenes/metabolism , Zea mays/genetics , Phytoalexins
11.
BMC Genomics ; 16: 450, 2015 Jun 11.
Article in English | MEDLINE | ID: mdl-26062733

ABSTRACT

BACKGROUND: Terpenoids are abundant in the foliage of Eucalyptus, providing the characteristic smell as well as being valuable economically and influencing ecological interactions. Quantitative and qualitative inter- and intra- specific variation of terpenes is common in eucalypts. RESULTS: The genome sequences of Eucalyptus grandis and E. globulus were mined for terpene synthase genes (TPS) and compared to other plant species. We investigated the relative expression of TPS in seven plant tissues and functionally characterized five TPS genes from E. grandis. Compared to other sequenced plant genomes, Eucalyptus grandis has the largest number of putative functional TPS genes of any sequenced plant. We discovered 113 and 106 putative functional TPS genes in E. grandis and E. globulus, respectively. All but one TPS from E. grandis were expressed in at least one of seven plant tissues examined. Genomic clusters of up to 20 genes were identified. Many TPS are expressed in tissues other than leaves which invites a re-evaluation of the function of terpenes in Eucalyptus. CONCLUSIONS: Our data indicate that terpenes in Eucalyptus may play a wider role in biotic and abiotic interactions than previously thought. Tissue specific expression is common and the possibility of stress induction needs further investigation. Phylogenetic comparison of the two investigated Eucalyptus species gives insight about recent evolution of different clades within the TPS gene family. While the majority of TPS genes occur in orthologous pairs some clades show evidence of recent gene duplication, as well as loss of function.


Subject(s)
Alkyl and Aryl Transferases/genetics , Eucalyptus/enzymology , Plant Proteins/genetics , Alkyl and Aryl Transferases/metabolism , Eucalyptus/classification , Eucalyptus/genetics , Evolution, Molecular , Genome, Plant , Multigene Family , Phylogeny , Plant Proteins/metabolism
12.
Org Biomol Chem ; 13(21): 6021-30, 2015 Jun 07.
Article in English | MEDLINE | ID: mdl-25940560

ABSTRACT

Multiproduct terpene synthases TPS4-B73 and TPS5-Delprim from maize (Zea mays) catalyze the conversion of farnesyl diphosphate (FDP) and geranyl diphosphate (GDP) into a complex mixture of sesquiterpenes and monoterpenes, respectively. Various isotopic and geometric isomers of natural substrates like (2Z)-[2-(2)H]- and [2,4,4,9,9,9-(2)H6]-(GDP) and (2Z,6E)-[2-(2)H]- and [2,4,4,13,13,13-(2)H6]-(FDP) were synthesized analogous to presumptive reaction intermediates. On incubation with labeled (2Z) substrates, TPS4 and TPS5 showed much lower kinetic isotope effects than the labeled (2E) substrates. Interestingly, the products arising from the deuterated (2Z)-precursors revealed a distinct preference for cyclic products and exhibited an enhanced turnover on comparison with natural (2E)-substrates. This increase in the efficiency due to (2Z) configuration emphasizes the rate limiting effect of the initial (2E) → (2Z) isomerization step in the reaction cascade of the multiproduct terpene synthases. Apart from turnover advantages, these results suggest that substrate geometry can be used as a tool to optimize the biosynthetic reaction cascade towards valuable cyclic terpenoids.


Subject(s)
Alkyl and Aryl Transferases/metabolism , Monoterpenes/metabolism , Sesquiterpenes/metabolism , Zea mays/enzymology , Cyclization , Diphosphates/chemistry , Diphosphates/metabolism , Diterpenes/chemistry , Diterpenes/metabolism , Monoterpenes/chemistry , Polyisoprenyl Phosphates/chemistry , Polyisoprenyl Phosphates/metabolism , Sesquiterpenes/chemistry , Stereoisomerism , Substrate Specificity , Zea mays/chemistry , Zea mays/metabolism
13.
PLoS One ; 10(5): e0123226, 2015.
Article in English | MEDLINE | ID: mdl-25978451

ABSTRACT

Phenotypic mosaic trees offer an ideal system for studying differential gene expression. We have investigated two mosaic eucalypt trees from two closely related species (Eucalyptus melliodora and E. sideroxylon), which each support two types of leaves: one part of the canopy is resistant to insect herbivory and the remaining leaves are susceptible. Driving this ecological distinction are differences in plant secondary metabolites. We used these phenotypic mosaics to investigate genome wide patterns of foliar gene expression with the aim of identifying patterns of differential gene expression and the somatic mutation(s) that lead to this phenotypic mosaicism. We sequenced the mRNA pool from leaves of the resistant and susceptible ecotypes from both mosaic eucalypts using the Illumina HiSeq 2000 platform. We found large differences in pathway regulation and gene expression between the ecotypes of each mosaic. The expression of the genes in the MVA and MEP pathways is reflected by variation in leaf chemistry, however this is not the case for the terpene synthases. Apart from the terpene biosynthetic pathway, there are several other metabolic pathways that are differentially regulated between the two ecotypes, suggesting there is much more phenotypic diversity than has been described. Despite the close relationship between the two species, they show large differences in the global patterns of gene and pathway regulation.


Subject(s)
Eucalyptus/genetics , Transcriptome/genetics , Gene Expression Regulation, Plant/genetics , Models, Theoretical , Plant Leaves/genetics
14.
J Chem Ecol ; 41(3): 213-23, 2015 Mar.
Article in English | MEDLINE | ID: mdl-25893788

ABSTRACT

The sesquiterpene (E)-ß-caryophyllene is emitted from maize (Zea mays) leaves and roots in response to herbivore attack. This compound serves as a signal for the attraction of herbivore enemies and is present in most European maize varieties. However, most North American maize lines have lost the ability to produce (E)-ß-caryophyllene. Previously, we showed that restoring the ability to synthesize (E)-ß-caryophyllene in a non-producing maize line improved its resistance against the root herbivore Diabrotica virgifera virgifera. However, it is largely unknown whether this modification affects the resistance to other pests. In this study, we investigated the response of constitutively (E)-ß-caryophyllene-producing transgenic lines to infection by a hemibiotrophic fungus Colletotrichum graminicola. Our results showed that restoring (E)-ß-caryophyllene synthesis in a Hi-II genetic background enhanced the susceptibility of the plant to C. graminicola infection rather than increasing its resistance. This modification did not alter the baseline levels of plant defense hormones and metabolites. Nor did (E)-ß-caryophyllene production modify the expression of anti-fungal defense genes. Instead, the addition of (E)-ß-caryophyllene seemed to directly stimulate fungal growth. In an in vitro antifungal assay, we found that (E)-ß-caryophyllene stimulated hyphal growth of C. graminicola and Fusarium graminearum. Thus, although restoring (E)-ß-caryophyllene production in a non-producing maize line may improve the resistance of the plant against herbivores, it may compromise its resistance to major fungal pathogens. This might explain the loss of (E)-ß-caryophyllene during maize breeding in environments where C. graminicola and Fusarium graminearum are prevalent.


Subject(s)
Antifungal Agents/metabolism , Colletotrichum/physiology , Sesquiterpenes/metabolism , Zea mays/metabolism , Zea mays/microbiology , Antifungal Agents/pharmacology , Benzoxazines/chemistry , Benzoxazines/metabolism , Colletotrichum/drug effects , Colletotrichum/growth & development , Fusarium/drug effects , Fusarium/growth & development , Glucosides/chemistry , Inbreeding , Plant Diseases/microbiology , Polycyclic Sesquiterpenes , Sesquiterpenes/pharmacology , Zea mays/physiology
15.
Chem Commun (Camb) ; 51(18): 3797-800, 2015 Mar 04.
Article in English | MEDLINE | ID: mdl-25658388

ABSTRACT

Multiproduct terpene synthases TPS4-B73 and TPS5-Delprim from Zea mays exhibit isotopically sensitive branching in the formation of mono- and sesquiterpene volatiles. The impact of the kinetic isotope effects and the stabilization of the reactive intermediates by hyperconjugation along with the shift of products from alkenes to alcohols are discussed.


Subject(s)
Alkyl and Aryl Transferases/chemistry , Deuterium/chemistry , Diphosphates/chemistry , Diterpenes/chemistry , Plant Proteins/chemistry , Polyisoprenyl Phosphates/chemistry , Sesquiterpenes/chemistry , Zea mays/enzymology , Kinetics
16.
Planta ; 241(6): 1351-61, 2015 Jun.
Article in English | MEDLINE | ID: mdl-25680349

ABSTRACT

MAIN CONCLUSION: Of the three functional FPPS identified in maize, fpps3 is induced by herbivory to produce FDP important for the formation of the volatile sesquiterpenes of plant defense. Sesquiterpenes are not only crucial for the growth and development of a plant but also for its interaction with the environment. The biosynthesis of sesquiterpenes proceeds over farnesyl diphosphate (FDP), which is either used as a substrate for protein prenylation, converted to squalene, or to volatile sesquiterpenes. To elucidate the regulation of sesquiterpene biosynthesis in maize, we identified and characterized the farnesyl diphosphate synthase (FPPS) gene family which consists of three genes. Synteny analysis indicates that fpps2 and fpps3 originate from a genome duplication in an ancient tetraploid ancestor. The three FPPSs encode active enzymes that produce predominantly FDP from the isopentenyl diphosphate and dimethylallyl diphosphate substrates. Only fpps1 and fpps3 are induced by elicitor treatment, but induced fpps1 levels are much lower and only increased to the amounts of fpps3 levels in intact leaves. Elicitor-induced fpps3 levels in leaves increase to more than 15-fold of background levels. In undamaged roots, transcript levels of fpps1 are higher than those of fpps3, but only fpps3 transcripts are induced in response to herbivory by Diabrotica virgifera virgifera. A kinetic of transcript abundance in response to herbivory in leaves provided further evidence that the regulation of fpps3 corresponds to that of tps23, a terpene synthase, that converts FDP to the volatile (E)-ß-caryophyllene. Our study indicates that the differential expression of fpps1 and fpps3 provides maize with FDP for both primary metabolism and terpene-based defenses. The expression of fpps3 seems to coincide with the herbivore-induced emission of volatile sesquiterpenes that were demonstrated to be important defense signals.


Subject(s)
Biosynthetic Pathways , Geranyltranstransferase/metabolism , Herbivory/physiology , Multigene Family , Polyisoprenyl Phosphates/metabolism , Sesquiterpenes/metabolism , Zea mays/enzymology , Alkyl and Aryl Transferases/genetics , Alkyl and Aryl Transferases/metabolism , Amino Acid Sequence , Biosynthetic Pathways/genetics , Gene Expression Regulation, Enzymologic , Gene Expression Regulation, Plant , Geranyltranstransferase/genetics , Kinetics , Molecular Sequence Data , Phylogeny , Plant Leaves/enzymology , Plant Proteins/chemistry , Plant Proteins/metabolism , Plant Roots/enzymology , RNA, Messenger/genetics , RNA, Messenger/metabolism , Sequence Alignment , Zea mays/genetics
18.
Planta ; 238(1): 191-204, 2013 Jul.
Article in English | MEDLINE | ID: mdl-23624978

ABSTRACT

The identification, isolation and functional characterization of two genes encoding two monoterpene synthases-γ-terpinene synthase (Tctps2) and α-terpineol synthase (Tctps5)-from three chemically distinct Thymus caespititius (Lamiaceae) genotypes were performed. Genomic exon-intron structure was also determined for both terpene synthase genes, revealing an organization with seven exons and six introns. The cDNA of Tctps2 was 2,308 bp long and had an open reading frame of 1,794 bp encoding for a protein with 598 amino acids. Tctps5 was longer, mainly due to intron sequences, and presented high intraspecific variability on the plants analyzed. It encoded for a protein of 602 amino acids from an open reading frame of 1,806 bp comprising a total of 2,507 bp genomic sequence. The amino acid sequence of these two active Tctps genes shared 74 % pairwise identity, ranging between 42 and 94 % similarity with about 50 known terpene synthases of other Lamiaceae species. Gene expression revealed a multi-product Tctps2 and Tctps5 enzymes, producing γ-terpinene and α-terpineol as major components, respectively. These enzymatic results were consistent with the monoterpene profile present in T. caespititius field plants, suggesting a transcriptional regulation in leaves. Herewith reported for the first time for this species, these two newly characterized Tctps genes improve the understanding of the molecular mechanisms of reaction responsible for terpene biosynthesis and chemical diversity found in T. caespititius.


Subject(s)
Alkyl and Aryl Transferases/genetics , Lamiaceae/enzymology , Lamiaceae/genetics , Alkyl and Aryl Transferases/metabolism , Amino Acid Sequence , Base Sequence , Cloning, Molecular , Gene Expression Regulation, Plant , Introns , Lamiaceae/metabolism , Molecular Sequence Data , Phylogeny , Plant Proteins/genetics , Plant Proteins/metabolism , Sequence Homology, Amino Acid
19.
BMC Plant Biol ; 13: 15, 2013 Jan 30.
Article in English | MEDLINE | ID: mdl-23363415

ABSTRACT

BACKGROUND: Maize (Zea mays L.) leaves damaged by lepidopteran herbivores emit a complex volatile blend that can attract natural enemies of the herbivores and may also have roles in direct defense and inter- or intra-plant signaling. The volatile blend is dominated by sesquiterpenes of which the majority is produced by two herbivore-induced terpene synthases, TPS10 and TPS23. However, little is known about the pattern of volatile emission within maize leaves. RESULTS: In this study, we restricted herbivore feeding to small sections of the maize leaf with the aim of determining the patterns of volatile sesquiterpene emission throughout the damaged leaf and in neighboring leaves. Sesquiterpene volatiles were released at high rates from damaged leaves, but at much lower rates from neighboring leaves. Release was restricted to the site of damage or to leaf sections located apical to the damage, but was not seen in sections basal to the damage or on the other side of the midrib. The emission pattern correlated well with the transcript pattern of the respective sesquiterpene synthase genes, tps10 and tps23, implying that biosynthesis likely occurs at the site of emission. The concentrations of jasmonic acid and its leucine derivative were also elevated in terpene-emitting tissues suggesting a role for jasmonates in propagating the damage signal. CONCLUSIONS: In contrast to other defense reactions which often occur systemically throughout the whole plant, herbivore-induced sesquiterpene production in maize is restricted to the wounding site and distal leaf parts. Since the signal mediating this reaction is directed to the leaf tip and cannot propagate parallel to the leaf axis, it is likely connected to the xylem. The increasing gradient of volatiles from the tip of the leaf towards the damage site might aid herbivore enemies in host or prey finding.


Subject(s)
Herbivory , Plant Leaves/metabolism , Plant Leaves/parasitology , Sesquiterpenes/metabolism , Zea mays/metabolism , Zea mays/parasitology , Alkyl and Aryl Transferases/metabolism , Animals , Cyclopentanes/metabolism , Oxylipins/metabolism , Plant Leaves/genetics , Poaceae/metabolism , Poaceae/parasitology , Zea mays/genetics
20.
Plant Biotechnol J ; 11(5): 628-39, 2013 Jun.
Article in English | MEDLINE | ID: mdl-23425633

ABSTRACT

Genetic manipulation of plant volatile emissions is a promising tool to enhance plant defences against herbivores. However, the potential costs associated with the manipulation of specific volatile synthase genes are unknown. Therefore, we investigated the physiological and ecological effects of transforming a maize line with a terpene synthase gene in field and laboratory assays, both above- and below ground. The transformation, which resulted in the constitutive emission of (E)-ß-caryophyllene and α-humulene, was found to compromise seed germination, plant growth and yield. These physiological costs provide a possible explanation for the inducibility of an (E)-ß-caryophyllene-synthase gene in wild and cultivated maize. The overexpression of the terpene synthase gene did not impair plant resistance nor volatile emission. However, constitutive terpenoid emission increased plant apparency to herbivores, including adults and larvae of the above ground pest Spodoptera frugiperda, resulting in an increase in leaf damage. Although terpenoid overproducing lines were also attractive to the specialist root herbivore Diabrotica virgifera virgifera below ground, they did not suffer more root damage in the field, possibly because of the enhanced attraction of entomopathogenic nematodes. Furthermore, fewer adults of the root herbivore Diabrotica undecimpunctata howardii were found to emerge near plants that emitted (E)-ß-caryophyllene and α-humulene. Yet, overall, under the given field conditions, the costs of constitutive volatile production overshadowed its benefits. This study highlights the need for a thorough assessment of the physiological and ecological consequences of genetically engineering plant signals in the field to determine the potential of this approach for sustainable pest management strategies.


Subject(s)
Genetic Engineering , Volatile Organic Compounds/metabolism , Zea mays/genetics , Zea mays/metabolism , Alkyl and Aryl Transferases/metabolism , Animals , Herbivory , Insecta/physiology , Monocyclic Sesquiterpenes , Nematoda/physiology , Plant Development , Plant Roots/physiology , Plants, Genetically Modified , Polycyclic Sesquiterpenes , Risk Assessment , Sesquiterpenes/metabolism , Terpenes/metabolism , Zea mays/enzymology
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