Monoterpene synthases responsible for the terpene profile of anther glands in Eucalyptus polybractea R.T. Baker (Myrtaceae)

2020 ◽  
Author(s):  
Jason Q D Goodger ◽  
Demi Gamble ◽  
John Humphries ◽  
Ian E Woodrow
Keyword(s):  
Recombinant Protein Expression ◽  
Detailed Examination ◽  
Dry Mass ◽  
Site Directed Mutagenesis ◽  
Tissue Type ◽  
Terpene Synthase ◽  
Flower Buds ◽  
Terpene Biosynthesis ◽  
Flower Opening ◽  
Monoterpene Synthases

Abstract Research on terpene biosynthesis in the genus Eucalyptus (Myrtaceae) is poorly developed, but recently large numbers of terpene synthase (TPS) genes have been identified. Few of these have been characterized or their expression localized to specific tissues. A prime candidate for detailed examination of TPS gene expression is the bisexual eucalypt flower—composed of male and female reproductive organs, and vegetative tissues that may express different TPS genes. We aimed to characterize and compare the terpene profile and TPS genes expressed in anthers and gynoecia in the high oil-yielding E. polybractea. We hypothesized that gynoecia will produce greater amounts of defensive terpenes, whereas anthers will have a terpene profile that is biased towards a role in pollination. Microscopy of isolated anthers showed them to possess a single, prominent oil gland. Chemical analysis of whole floral structures at different stages of development showed total oil per unit dry mass increased as flower buds expanded, with highest concentrations in mature flower buds just prior to flower opening. The oil profile of gynoecia was dominated by the monoterpene 1,8-cineole, whereas that of isolated anthers was enriched with the monoterpene α-pinene. Through transcriptomic analysis and recombinant protein expression, we were able to identify monoterpene synthases responsible for the different profiles. Synthases for α-pinene and 1,8-cineole were expressed in each tissue type, but the relative expression of the former was higher in anthers. Sequence comparison and site-directed mutagenesis of the α-pinene synthase allowed us to identify amino acids which influence the α-pinene to β-pinene ratio of the product profile. We suggest the terpene constituents of anthers may have multiple roles including attracting pollinators through emission of volatile α-pinene, deterrence of palynivores through emission of volatile 1,8-cineole and adhesion of pollen to pollinators via the release of sticky α-pinene onto the anther surface.

scholarly journals Biosynthesis of the microtubule-destabilizing diterpene pseudolaric acid B from golden larch involves an unusual diterpene synthase

2017 ◽  
Vol 114 (5) ◽  
pp. 974-979 ◽  
Author(s):  
Sibongile Mafu ◽  
Prema Sambandaswami Karunanithi ◽  
Teresa Ann Palazzo ◽  
Bronwyn Lee Harrod ◽  
Selina Marakana Rodriguez ◽  
...  
Keyword(s):  
Anticancer Agents ◽  
Quantum Chemical ◽  
Site Directed Mutagenesis ◽  
Terpene Synthase ◽  
Proof Of Concept ◽  
Terpene Biosynthesis ◽  
Quantum Chemical Analysis ◽  
Pseudolaric Acid B ◽  
Mechanistic Analysis ◽  
Diterpene Synthase

The diversity of small molecules formed via plant diterpene metabolism offers a rich source of known and potentially new biopharmaceuticals. Among these, the microtubule-destabilizing activity of pseudolaric acid B (PAB) holds promise for new anticancer agents. PAB is found, perhaps uniquely, in the coniferous tree golden larch (Pseudolarix amabilis, Pxa). Here we describe the discovery and mechanistic analysis of golden larch terpene synthase 8 (PxaTPS8), an unusual diterpene synthase (diTPS) that catalyzes the first committed step in PAB biosynthesis. Mining of the golden larch root transcriptome revealed a large TPS family, including the monofunctional class I diTPS PxaTPS8, which converts geranylgeranyl diphosphate into a previously unknown 5,7-fused bicyclic diterpene, coined “pseudolaratriene.” Combined NMR and quantum chemical analysis verified the structure of pseudolaratriene, and co-occurrence with PxaTPS8 and PAB inP.amabilistissues supports the intermediacy of pseudolaratriene in PAB metabolism. Although PxaTPS8 adopts the typical three-domain structure of diTPSs, sequence phylogeny places the enzyme with two-domain TPSs of mono- and sesqui-terpene biosynthesis. Site-directed mutagenesis of PxaTPS8 revealed several catalytic residues that, together with quantum chemical calculations, suggested a substantial divergence of PxaTPS8 from other TPSs leading to a distinct carbocation-driven reaction mechanism en route to the 5,7-trans-fused bicyclic pseudolaratriene scaffold. PxaTPS8 expression in microbial and plant hosts provided proof of concept for metabolic engineering of pseudolaratriene.

scholarly journals Differential gene expression associated with a floral scent polymorphism in the evening primrose Oenothera harringtonii (Onagraceae)

2021 ◽  
Author(s):  
Lindsey L. Bechen ◽  
Matthew G. Johnson ◽  
Geoffrey T. Broadhead ◽  
Rachel A. Levin ◽  
Rick P. Overson ◽  
...  
Keyword(s):  
Gene Expression ◽  
Differential Gene Expression ◽  
Floral Scent ◽  
Terpene Synthase ◽  
Rna Seq ◽  
Loss Of Function ◽  
Terpene Biosynthesis ◽  
Terpene Synthases ◽  
Evening Primrose ◽  
Differential Gene

AbstractBackgroundPlant volatiles play an important role in both plant-pollinator and plant-herbivore interactions. Intraspecific polymorphisms in volatile production are ubiquitous, but studies that explore underlying differential gene expression are rare. Oenothera harringtonii populations are polymorphic in floral emission of the monoterpene (R)-(-)-linalool; some plants emit (R)-(-)-linalool (linalool+ plants) while others do not (linalool-plants). However, the genes associated with differential production of this floral volatile in Oenothera are unknown. We used RNA-Seq to broadly characterize differential gene expression involved in (R)-(-)-linalool biosynthesis. To identify genes that may be associated with the polymorphism for this trait, we used RNA-Seq to compare gene expression in six different Oenothera harringtonii tissues from each of three linalool+ and linalool-plants.ResultsThree clusters of differentially expressed genes were enriched for terpene synthase activity: two were characterized by tissue-specific upregulation and one by upregulation only in plants with flowers that produce (R)-(-)-linalool. A molecular phylogeny of all terpene synthases identified two putative (R)-(-)-linalool synthase transcripts in Oenothera harringtonii, a single allele of which is found exclusively in linalool+ plants.ConclusionsBy using a naturally occurring polymorphism and comparing different tissues, we were able to identify genes putatively involved in the biosynthesis of (R)-(-)-linalool. Expression of these genes in linalool-plants suggests a regulatory polymorphism, rather than a population-specific loss-of-function allele. Additional terpene biosynthesis-related genes that are up-regulated in plants that emit (R)-(-)-linalool may be associated with herbivore defense, suggesting a potential economy of scale between plant reproduction and defense.

scholarly journals Terpene synthase genes in eukaryotes beyond plants and fungi: Occurrence in social amoebae

2016 ◽  
Vol 113 (43) ◽  
pp. 12132-12137 ◽  
Author(s):  
Xinlu Chen ◽  
Tobias G. Köllner ◽  
Qidong Jia ◽  
Ayla Norris ◽  
Balaji Santhanam ◽  
...  
Keyword(s):  
Early Stage ◽  
Terpene Synthase ◽  
Functional Study ◽  
Terpene Biosynthesis ◽  
Terpene Synthases ◽  
Multicellular Development ◽  
Stage Of Development ◽  
The Social ◽  
Volatile Terpenes ◽  
Social Amoebae

Terpenes are structurally diverse natural products involved in many ecological interactions. The pivotal enzymes for terpene biosynthesis, terpene synthases (TPSs), had been described only in plants and fungi in the eukaryotic domain. In this report, we systematically analyzed the genome sequences of a broad range of nonplant/nonfungus eukaryotes and identified putative TPS genes in six species of amoebae, five of which are multicellular social amoebae from the order of Dictyosteliida. A phylogenetic analysis revealed that amoebal TPSs are evolutionarily more closely related to fungal TPSs than to bacterial TPSs. The social amoeba Dictyostelium discoideum was selected for functional study of the identified TPSs. D. discoideum grows as a unicellular organism when food is abundant and switches from vegetative growth to multicellular development upon starvation. We found that expression of most D. discoideum TPS genes was induced during development. Upon heterologous expression, all nine TPSs from D. discoideum showed sesquiterpene synthase activities. Some also exhibited monoterpene and/or diterpene synthase activities. Direct measurement of volatile terpenes in cultures of D. discoideum revealed essentially no emission at an early stage of development. In contrast, a bouquet of terpenes, dominated by sesquiterpenes including β-barbatene and (E,E)-α-farnesene, was detected at the middle and late stages of development, suggesting a development-specific function of volatile terpenes in D. discoideum. The patchy distribution of TPS genes in the eukaryotic domain and the evidence for TPS function in D. discoideum indicate that the TPS genes mediate lineage-specific adaptations.

Foliar Application of Ethephon Decreases Apical Dominance in `Orchid' Petunia (Petunia × hybrida Vilm.-Andr.)

HortScience ◽  
1998 ◽  
Vol 33 (3) ◽  
pp. 491e-491
Author(s):  
Darren L. Haver ◽  
Ursula K. Schuch
Keyword(s):  
Apical Dominance ◽  
Petunia Hybrida ◽  
Foliar Application ◽  
Average Length ◽  
Foliar Spray ◽  
Dry Mass ◽  
Flower Buds ◽  
Flower Bud ◽  
Lateral Shoot ◽  
Application Rates

Ethephon was applied as a foliar spray to 36-day-old petunia seedlings to determine its effectiveness at reducing apical dominance by increasing lateral shoot development. Ethephon application at rates of 125, 250 and 500 mg·L–1 to whole shoots of Petunia × hybrida `Orchid' decreased apical dominance compared to the control. The average length of a lateral shoot increased 56% as ethephon application rates increased from 0 to 500 mg·L–1. In Expt. I, ethephon-treated plants had a greater number of laterals than control plants. The number of nodes produced before the first flower bud was initiated increased from 15 to 21 as rates of ethephon increased from 0 to 500 mg·L–1. Ethephon delayed anthesis up to 10 days when applied at 500 mg·L–1 and up to 8 days when applied at 250 mg·L–1. The number of visible flower buds increased in all ethephon-treated plants compared to the control. Ethephon at 250 mg·L–1, increased shoot and root dry mass 37.9% and 20.4%, respectively, compared to untreated controls. Roots appeared healthy in both experiments, but phytotoxicity (mild chlorosis) occurred in Expt. II on plants treated with 500 mg·L–1. The experiment was repeated twice with similar results.

Mechanistic Investigations on Microbial Type I Terpene Synthases through Site-directed Mutagenesis

Synthesis ◽  
2021 ◽  
Author(s):  
Houchao Xu ◽  
Jeroen Dickschat
Keyword(s):  
Crystal Structures ◽  
Site Directed Mutagenesis ◽  
Terpene Synthase ◽  
Directed Mutagenesis ◽  
Type I ◽  
Terpene Synthases ◽  
Conserved Motifs ◽  
Sequence Identity ◽  
The Past ◽  
Mechanistic Investigations

During the past three decades many terpene synthases have been characterised from all kingdoms of life. The type I of these enzymes from bacteria, fungi and protists commonly exhibit several highly conserved motifs and single residues, and the available crystal structures show a shared -helical fold, while the overall sequence identity is generally low. Several enzymes have been studied by site-directed mutagenesis, giving valuable insights into terpene synthase catalysis and the intriguing mechanisms of terpene synthases. Some mutants are also preparatively useful and give higher yields than the wildtype or a different product that is otherwise difficult to access. The accumulated knowledge obtained from these studies is presented and discussed in this review.

Leaf absorption, withdrawal and remobilization of autumn-applied urea-N in apple

2004 ◽  
Vol 84 (1) ◽  
pp. 259-264 ◽  
Author(s):  
S. Guak ◽  
D. Neilsen ◽  
P. Millard ◽  
N. E. Looney
Keyword(s):  
Malus Domestica ◽  
Low Temperatures ◽  
Fruit Set ◽  
Urea Solution ◽  
Flower Buds ◽  
Flower Bud ◽  
Flower Opening ◽  
Total N ◽  
Bud Growth ◽  
Flower Quality

Six-year-old well-nourished Jonagold/M9 apple (Malus domestica) trees were sprayed 7 d after harvest with a 2% urea solution enriched with 9.9% atom 15N. Through 3 d of the absorption period, leaves absorbed 19.2% of the intercepted urea 15N. This low absorption could be in part due to unfavourable conditions, i.e., low temperatures (daily mean ≈5°C) and windy conditions following treatment. During leaf senescence, 48% of the urea 15N absorbed was withdrawn from leaves and most of that (95%) remained in the treated branch section. Of this portion, 65% of the urea 15N was found in dormant bark, 29% in wood, and 6% in flower buds. In the following spring, 46% of the stored urea 15N was remobilized for growth of the flower buds when sampled at the “pink” stage of bud development. This accounted for 3.8% of total N in these tissues. This contribution did not influence flower quality, estimated by the length of the period between flower opening and petal fall and the level of fruit set. Key words: Malus × domestica, urea-15N, flower bud growth, fruit set

scholarly journals Enhanced limonene production in cyanobacteria reveals photosynthesis limitations

2016 ◽  
Vol 113 (50) ◽  
pp. 14225-14230 ◽  
Author(s):  
Xin Wang ◽  
Wei Liu ◽  
Changpeng Xin ◽  
Yi Zheng ◽  
Yanbing Cheng ◽  
...  
Keyword(s):  
Metabolic Flux ◽  
Carbon Fixation ◽  
Carbon Sink ◽  
Electron Flow ◽  
Fold Increase ◽  
Industrial Applications ◽  
Fine Tuning ◽  
Terpene Synthase ◽  
Terpene Biosynthesis ◽  
Cell Factories

Terpenes are the major secondary metabolites produced by plants, and have diverse industrial applications as pharmaceuticals, fragrance, solvents, and biofuels. Cyanobacteria are equipped with efficient carbon fixation mechanism, and are ideal cell factories to produce various fuel and chemical products. Past efforts to produce terpenes in photosynthetic organisms have gained only limited success. Here we engineered the cyanobacterium Synechococcus elongatus PCC 7942 to efficiently produce limonene through modeling guided study. Computational modeling of limonene flux in response to photosynthetic output has revealed the downstream terpene synthase as a key metabolic flux-controlling node in the MEP (2-C-methyl-d-erythritol 4-phosphate) pathway-derived terpene biosynthesis. By enhancing the downstream limonene carbon sink, we achieved over 100-fold increase in limonene productivity, in contrast to the marginal increase achieved through stepwise metabolic engineering. The establishment of a strong limonene flux revealed potential synergy between photosynthate output and terpene biosynthesis, leading to enhanced carbon flux into the MEP pathway. Moreover, we show that enhanced limonene flux would lead to NADPH accumulation, and slow down photosynthesis electron flow. Fine-tuning ATP/NADPH toward terpene biosynthesis could be a key parameter to adapt photosynthesis to support biofuel/bioproduct production in cyanobacteria.

The potential of jojoba (Simmondsia chinensis) in New South Wales. 2. Some factors affecting yield

1989 ◽  
Vol 29 (3) ◽  
pp. 389 ◽  
Author(s):  
PL Milthorpe ◽  
RL Dunstone
Keyword(s):  
New South ◽  
New South Wales ◽  
Frost Damage ◽  
High Rate ◽  
High Yield ◽  
Simmondsia Chinensis ◽  
Chilling Requirement ◽  
Flower Buds ◽  
Flower Opening ◽  
South Wales

A jojoba (Simmondsia chinensis [Link] Schneider) stand at Condobolin. N.S.W.. established from a range of plant material, exhibited great variability in a number of plant characteristics including seed yield. Observations over 4 years indicated that a high bud to node ratio is necessary for high yield. Different lines varied from 44 to 74% in this ratio in the fourth year of study. The survival of buds to form open flowers varied greatly between lines and from year to year. Death of flower buds before opening was attributable to frost damage. Buds swelled as early as June in some lines while others showed no sign of swelling until September. In those lines with early swelling or flower opening a high proportion of the buds were frost damaged, whereas late flowering lines had a high rate of survival. Terminal flower buds formed just prior to winter dormancy survived and flowered in the next spring, even in otherwise early flowering lines. Earlier work has shown that jojoba flower buds remain dormant until a chilling requirement has been met. Jojoba lines should have a long chilling requirement to maintain dormancy in the buds until the danger of frosts is past. Almost all of the flowers that opened set fruit, indicating that pollination is not a problem in the New South Wales environment.

1, 10/1, 11 Cyclization catalyzed by diverged plant sesquiterpene synthases is dependent on a single residue

2021 ◽  
Author(s):  
Xin Fang ◽  
Jin-Quan Huang ◽  
Dong-Mei Li ◽  
Jian-Xu Li ◽  
Jia-Ling Lin ◽  
...  
Keyword(s):  
Phylogenetic Analyses ◽  
Site Directed Mutagenesis ◽  
Terpene Synthase ◽  
Directed Mutagenesis

The exquisite chemodiversity of terpenoids are the product of the large diverse terpene synthase (TPS) superfamily. Here, by using structural and phylogenetic analyses and site-directed mutagenesis, we identified a residue...

Integrated transcriptomic and metabolomic analyses reveal regulation of terpene biosynthesis in the stems of Sindora glabra

2020 ◽  
Author(s):  
Niu Yu ◽  
Zhaoli Chen ◽  
Jinchang Yang ◽  
Rongsheng Li ◽  
Wentao Zou
Keyword(s):  
Signaling Pathway ◽  
Molecular Mechanisms ◽  
Defense Responses ◽  
Terpene Synthase ◽  
Transcriptional Expression ◽  
Plant Defense Responses ◽  
Terpene Biosynthesis ◽  
Jasmonic Acid Signaling ◽  
Salicylic Acid Signaling ◽  
Meja Treatment

Abstract Sesquiterpenes are important defensive secondary metabolites that are synthesized in various plant organs. Methyl jasmonate (MeJA) plays a key role in plant defense responses and secondary metabolism. Sindora glabra produces abundant sesquiterpenes in their trunks and was subjected to investigation after MeJA treatment in order to characterize the molecular mechanisms underlying the regulation of sesquiterpene biosynthesis in plant stems and further our understanding of oleoresin production in trees. A total of 14 types of sesquiterpenes in the stems of mature S. glabra trees were identified. The levels of two sesquiterpenes, α-copaene and β-caryophyllene, significantly increased after MeJA treatment. Differentially expressed genes involved in terpenoid backbone biosynthesis were significantly enriched over time, while the expression of JAZ genes involved in the jasmonic acid signaling pathway and TGA genes involved in the salicylic acid signaling pathway was significantly enriched at later time points after treatment. Two new terpene synthase genes, SgSTPS4 and SgSTPS5, were also identified. Following MeJA treatment, the expression levels of SgSTPS1, SgSTPS2, and SgSTPS4 decreased, while SgSTPS5 expression increased. The major enzymatic products of SgSTPS4 were identified as β-elemene and cyperene, while SgSTPS5 was identified as a bifunctional mono/sesquiterpene synthase that could catalyze FPP to produce nine types of sesquiterpenes, including α-copaene and β-caryophyllene, while SgSTPS5 could also use GPP to produce geraniol. Dramatic changes in the amounts of α-copaene and β-caryophyllene in response to MeJA were correlated with transcriptional expression changes of SgSTPS5 in the wood tissues. In addition, the transcription factors MYB, NAC, ARF, WRKY, MYC, ERF, and GRAS were co-expressed with terpene biosynthesis genes and might potentially regulate terpene biosynthesis. Metabolite changes were further investigated with UPLC-TOF/MS following MeJA treatment. These results contribute to the elucidation of the molecular mechanisms of terpene biosynthesis and regulation as well as to the identification of candidate genes involved in these processes.

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