Analysis of apocarotenoid volatiles from lettuce (Lactuca sativa) induced by insect herbivores and characterization of carotenoid cleavage dioxygenase gene.

Plant apocarotenoids have been shown to have a diverse biological role in herbivore-plant interactions. Despite their importance, little is known about herbivores' effect on apocarotenoid emissions in Lactuca sativa. In this study, we examined changes in apocarotenoid emissions in lettuce leaves after infestation by two insects, viz., Spodoptera littoralis larvae and Myzus persicae aphids. We found that ß-ionone and ß-cyclocitral showed higher concentrations than the other apocarotenoids, with a significant increase as per the intensity of infestation of both herbivore species. Furthermore, we performed functional characterization of Lactuca sativa carotenoid cleavage dioxygenase 1 (LsCCD1) genes. Three LsCCD1 genes were overexpressed in E. coli strains, and recombinant proteins were assayed for cleavage activity on an array of carotenoid substrates. The LsCCD1 protein cleaved ß-carotene at the 9,10 (9',10') positions producing ß-ionone. The transcript analysis of LsCCD1 genes revealed differential expression patterns under varying levels of herbivores' infestation, but the results were inconsistent with the pattern of ß-ionone concentrations. Our results suggest that LsCCD1 is involved in the production of ß-ionone, but other regulatory factors might be involved in its induction in response to herbivory. These results provide new insights into apocarotenoid production in response to insect herbivory in lettuce. Supplementary Information: The online version contains supplementary material available at 10.1007/s13205-023-03511-4.

Identification and characterization of three nearly identical linalool/nerolidol synthase from Acorus calamus.

Acorus calamus is a perennial aromatic medicinal plant from the Acorusaceae family, known for its pharmaceutical and medicinal value. A combined chemical, biochemical, and molecular study was conducted to evaluate the differential accumulation of volatile organic compounds (VOCs) in rhizomes and leaves of A. calamus essential oil. Here, we performed VOC profiling and transcriptome-based identification and functional characterization of terpene synthase (TPS) genes. A total of 110 VOCs were detected from the rhizomes and leaves of A. calamus, and some VOCs showed significant differences between them. The further transcriptome-based analysis led to the identification of six putative TPSs genes. In phylogenetic analysis, three TPSs belonged to the TPS-g clade, one to each of the TPS-a, TPS-c, and TPS-e clades. The heterologous E. coli-based expression of recombinant TPSs identified three genes (AcTPS3, AcTPS4, and AcTPS5) as bifunctional linalool/nerolidol synthase. The correlation of TPS gene expression and VOC metabolite profiles supported the function of these genes in A. calamus. Our findings provide a roadmap for future efforts to enhance the molecular mechanisms of terpene biosynthesis and our understanding of Acorus-insect interactions.

Identification of a wild carrot as carrot psylla (Bactericera trigonica) attractant and host plant chemistry.

Carrot psylla is one of the devastating pests of carrot throughout northern Europe and the Mediterranean basin. Here we characterized the behavioral response of psylla females towards different carrot germplasm and identified the chemical cues involved in the host selection of psylla females by oviposition choice experiments and metabolic profiling of leaf volatiles. In choice assays, carrot psylla displayed differential responses to tested 14 germplasm. Among germplasm, wild accessions 21793 and 20465 were highly preferred by carrot psylla, while wild accessions 20465 and the orange cultivar Nairobi were less. In non-choice experiments conducted only with this four-germplasm revealed that the carrot psylla females gave higher preference to the Nairobi and wild accession 20465, indicating the vicinity to other host plants in the same area might affect female preference. Moreover, the nymph development and survival experiments showed the lowest nymphs survival rate on the wild accessions 21793 and 20497. Furthermore, the volatile emissions among different carrot cultivars infested with psylla showed qualitative and quantitative differences versus intact plants. Among these volatiles, apiol, ß-asarone, myristicin, and sabinene showed a relationship with psyllas growth and survival. We also showed that myristicin and sabinene exogenous applications caused a dramatic reduction in the number of eggs laid by psylla and subsequent nymph survival. This is an initial study of the volatiles that mediate attraction and oviposition preference of carrot psylla in response to its host plant. The results from this study provide baseline information for the development of new control strategies against carrot psylla.

Optimization of Culture Conditions for the Efficient Biosynthesis of Trilobatin from Phloretin by Engineered Escherichia coli Harboring the Apple Phloretin-4'-O-glycosyltransferase.

Trilobatin, a dihydrochalcone glucoside and natural sweetener, has diverse biological and therapeutic properties. In the present study, we developed a microbial system to produce trilobatin from phloretin using Escherichia coli (E. coli) overexpressing the phloretin-4'-O-glycosyltransferase from Malus x domestica Borkh. Various optimization strategies were employed for the efficient production of trilobatin using a one-factor-at-a-time method. The effect of UDP-glucose supplementation, substrate, and inducer concentrations, time of substrate feeding as well as protein induction, and different culture media combinations were evaluated and optimized to enhance the production of trilobatin. As a result, the highest trilobatin production, 246.83 µM (107.64 mg L-1), was obtained with an LB-TB medium combination, 22 h of induction with 0.1 mM IPTG followed by 4 h of feeding with 250 µM phloretin and without extracellular UDP-glucose supplementation. These results demonstrate the efficient production of trilobatin and constitute a promising foundation for large-scale production of the dihydrochalcone glycosides in engineered E. coli.

Characterization of terpene synthase genes potentially involved in black fig fly (Silba adipata) interactions with Ficus carica.

The black fig fly (Silba adipata) is one of the major pests of figs worldwide. This study investigated the effect of pollination on black fig fly infestation and volatile emission during fruit development of facultative parthenocarpic Ficus carica. The results from in-field oviposition preference of black fig fly, olfactory analysis, and fruit volatile profiles indicate that the black fig fly gave a strong preference to unpollinated figs that showed higher emissions of volatile organic compounds. Terpenes are known to be important compounds determining many insect-plant interactions, so we report a transcriptome-based identification and functional characterization of a terpene synthase (TPS) gene family in F. carica. The protein expression in Escherichia coli of eight terpene synthases (TPSs) revealed that three were monoterpene synthases belonging to the TPS-b clade, with FcTPS6 catalyzing the formation of 1,8-cineole while the other two converted GPP into linalool. Four sesquiterpene synthases from the TPS-a clade catalyze the formation of germacrene D (FcTPS1), E-ß-caryophyllene (FcTPS2), cadinene (FcTPS3) and δ-elemene (FcTPS5) while one sesquiterpene synthase FcTPS4 from the TPS-b clade showed nerolidol synthase activity. Most of the enzymatic products closely matched the volatile terpenes emitted from fig fruits and all the genes were expressed during fruit development. This study provides new insights into fig-insect interactions and understanding the molecular mechanisms of terpene biosynthesis and could provide the foundations for sustainable pest management strategies.

Broomrape infestation in carrot (Daucus carota): Changes in carotenoid gene expression and carotenoid accumulation in the parasitic weed Phelipanche aegyptiaca and its host.

Carotenogenesis has been intensively studied in carrot roots, and transcriptional regulation is thought to be the major factor in carotenoid accumulation in these organs. However, little is known about the transcriptional regulation of carotenoid biosynthetic genes concerning carotenoid accumulation during infestation by the obligate parasite Phelipanche aegyptiaca. HPLC analysis revealed a decrease in carotenoid levels of the different carrot cultivars when parasitized by P. aegyptiaca. Besides, we isolated and analyzed P. aegyptiaca tubercles parasitizing the various carrot root cultivars and show that they accumulate different carotenoids compared to those in non-infested carrot roots. Expression analysis of PHYTOENE SYNTHASE (PSY1) and CAROTENOID ISOMERASE (CRTISO) as well as the strigolactone apocarotenoid biosynthetic genes DWARF27 (D27), CAROTENOID CLEAVAGE DIOXYGENASE 7 (CCD7) and CCD8 revealed that their transcript levels showed significant variation in P. aegyptiaca infested carrot roots. After parasite infestation, the expression of these genes was strongly reduced, as were the carotenoid levels and this was more pronounced in the uncommon non-orange varieties. We also analyzed the parasite genes encoding D27, CCD7 and CCD8 and show that they are expressed in tubercles. This raises important questions of whether the parasite produces its carotenoids and apocarotenoids including strigolactones and whether the latter might have a role in tubercle development.

Analysis of apocarotenoid volatiles during the development of Ficus carica fruits and characterization of carotenoid cleavage dioxygenase genes.

In plants the oxidative cleavage of carotenoid substrates produces volatile apocarotenoids, including ß-ionone, 6-methyl-5-hepten-2-ol, and α-ionone; these compounds are important in herbivore-plant communication. Combined chemical, biochemical, and molecular studies were conducted to evaluate the differential accumulation of carotenoids and volatile apocarotenoids during the development of pollinated and parthenocarpic fig fruits. Pollinated fig fruits showed less emission of apocarotenoid volatiles than the parthenocarpic figs, while in the case of carotenoid pigments, pollinated figs manifested higher accumulation. The apocarotenoids, 6-methyl-5-hepten-2-ol and ß-cyclogeraniol, showed a marked increase after the two weeks of hand-pollination in pollinated and parthenocarpic figs; but afterwards these volatile levels decreased during further fruit development. In addition, we report a transcriptome-based identification and functional characterization of the carotenoid cleavage dioxygenase (FcCCD) genes. These genes were overexpressed in Escherichia coli strains previously engineered to produce different carotenoids. The recombinant FcCCD1A enzyme showed specificity for the 9,10 (9',10') double bond position of cyclic carotenoids to generate α-ionone and ß-ionone, while FcCCD1B cleaved lycopene and an acyclic moiety of δ-carotene, producing 6-methyl-5-hepten-2-one. The qRT-PCR analysis of FcCCD genes revealed differential gene expression during fig fruit development. Our results suggest a role for the FcCCD1genes in apocarotenoid biosynthesis in fig fruits.

Profiling of volatile terpenes from almond (Prunus dulcis) young fruits and characterization of seven terpene synthase genes.

Almond (Prunus dulcis) is an agricultural and economically important fruit tree from the Rosaceae family used in the food industry. The monoterpenes and sesquiterpenes perform important ecological functions such as insecticidal and antifeedant activities against various insects. The young fruits of the different almond varieties were found to produce considerable amounts of terpene volatiles, including linalool and geraniol. To identify terpene synthases (TPSs) involved in the production of these volatile terpenes, existing genome databases of the Rosaceae were screened for almond genes with significant sequence similarity to other plants TPSs. Bioinformatics analysis led to the identification of seven putative TPSs genes with complete open reading frames. We characterized the enzymes encoded by these seven complementary DNAs: the monoterpene synthases PdTPS1, PdTPS3, PdTPS5, and PdTPS6 belong to the TPS-b clade, which catalyzes the formation of ß-phellandrene, geraniol, linalool, and farnesene, respectively. The sesquiterpene synthases PdTPS2 and PdTPS4, which belong to the TPS-a clade mainly catalyze the formation of bergamotene, while another sesquiterpene synthase, PdTPS7, from the TPS-g clade showed nerolidol synthase activity. The qRT-PCR analysis revealed that the various tissues of almond varieties showed differential transcription for all these PdTPSs genes.

A Pyrus communis gene for p-hydroxystyrene biosynthesis, has a role in defense against the pear psylla Cacopsylla biden.

Styrene analogs are known to be naturally synthesized in the leaves of pears and in other plant species, including several trees in the Styracaceae family. Styrene analogs are potential contributors to the aroma of wine, perfumes, pharmaceuticals, and other fermented foods and beverages. In addition, styrene analogs perform important ecological functions such as insecticidal and antifeedant activities against insects. We showed here that exogenous applications of styrene and p-hydroxystyrene caused a dramatic reduction the number of eggs laid by psylla and of subsequent nymph survival. Despite their importance specific reactions that lead to the biosynthesis of the styrene analogs in pear are unknown. To identify genes involved in the synthesis of these metabolites, existing genome databases of the Rosaceae were screened for pear genes with significant sequence similarity to bacterial phenolic acid decarboxylase. Herein described are the isolation and characterization of a pear phenolic acid decarboxylase, designated PyPAD1, which catalyzed the decarboxylation of p-coumaric acid and ferulic acid to p-hydroxystyrene and 3-methoxy-4-hydroxystyrene respectively. Its apparent Km values for p-coumaric acid and ferulic acid were 34.42 and 84.64 µM, respectively. The PyPAD1 preferred p-coumaric acid to ferulic acid as a substrate by a factor of 2.4 when comparing catalytic efficiencies in vitro. Expression analysis of PyPAD1 showed that the gene was transcribed in all five pear genotypes examined. However, transcript abundance was increased in correlation with the presence of p-hydroxystyrene in resistant cultivars Py-701 and Py-760 and in the sensitive cultivar Spadona when grafted on these resistant cultivars. Thus, PyPAD1 appears to be responsible for the decarboxylation of the p-coumaric acid, and for the production of metabolites that are active against pear psylla.

Biosynthesis of methyleugenol and methylisoeugenol in Daucus carota leaves: Characterization of eugenol/isoeugenol synthase and O-Methyltransferase.

Carrot (Daucus carota subsp. sativus) is a widely cultivated root vegetable of high economic importance. The aroma of carrot roots and aboveground organs is mainly defined by terpenes. We found that leaves of orange carrot cultivar also produce considerable amounts of the phenylpropenes methyleugenol and methylisoeugenol. Notably, methyleugenol is most abundant in young leaves, while methylisoeugenol is the dominant phenylpropene in mature leaf tissue. The goal of the present study was to shed light on the biochemistry and molecular biology of these compounds' biosynthesis and accumulation. Using the available genomic and transcriptomic data, we isolated a cDNA encoding eugenol/isoeugenol synthase (DcE(I)GS1), an NADPH-dependent enzyme that converts coniferyl acetate to eugenol. This enzyme exhibits dual product specificity and yields propenylphenol isoeugenol alongside allylphenol eugenol. Furthermore, we identified a cDNA encoding S-adenosyl-L-methionine:eugenol/isoeugenol O-methyltransferase 1 (DcE(I)OMT1) that produces methyleugenol and methylisoeugenol via methylation of the para-OH-group of their respective precursors. Both DcE(I)GS1 and DcE(I)OMT1 were expressed in seeds, roots, young and mature leaves, and the DcE(I)OMT1 transcript levels were the highest in leaves. The DcE(I)GS1 protein is 67% identical to anise t-anol/isoeugenol synthase and displays an apparent Km of 247 µM for coniferyl acetate. The catalytic efficiency of DcEOMT1 with eugenol is more than five-fold higher than that with isoeugenol, with Km values of 40 µM for eugenol, and of 115 µM for isoeugenol. This work expands the current knowledge of the enzymes involved in phenylpropene biosynthesis and would enable studies into structural elements defining the regioselectivity of phenylpropene synthases.

Profiling of the Terpene Metabolome in Carrot Fruits of Wild ( Daucus carota L. ssp. carota) Accessions and Characterization of a Geraniol Synthase.

Fruits from wild carrot ( Daucus carota L. ssp. carota) have been used for medicinal purposes since ancient times. The oil of its seeds, with their abundant monoterpenes and sesquiterpenes, has drawn attention in recent years because of its potential pharmaceutical application. A combined chemical, biochemical, and molecular study was conducted to evaluate the differential accumulation of terpene volatiles in carrot fruits of wild accessions. This work reports a similarity-based cloning strategy identification and functional characterization of one carrot monoterpene terpene synthase, WtDcTPS1. Recombinant WtDcTPS1 protein produces mainly geraniol, the predominant monoterpene in carrot seeds of wild accession 23727. The results suggest a role for the WtDcTPS1 gene in the biosynthesis of carrot fruit aroma and flavor compounds.

Characterization of three chalcone synthase-like genes from apple (Malus x domestica Borkh.).

Apple (Malus x domestica Brokh.) is a widely cultivated deciduous tree species of significant economic importance. Apple leaves accumulate high levels of flavonoids and dihydrochalcones, and their formation is dependent on enzymes of the chalcone synthase family. Three CHS genes were cloned from apple leaves and expressed in Escherichia coli. The encoded recombinant enzymes were purified and functionally characterized. In-vitro activity assays indicated that MdCHS1, MdCHS2 and MdCHS3 code for proteins exhibiting polyketide synthase activity that accepted either p-dihydrocoumaroyl-CoA, p-coumaroyl-CoA, or cinnamoyl-CoA as starter CoA substrates in the presence of malonyl-CoA, leading to production of phloretin, naringenin chalcone, and pinocembrin chalcone. MdCHS3 coded a chalcone-dihydrochalcone synthase enzyme with narrower substrate specificity than the previous ones. The apparent Km values of MdCHS3 for p-dihydrocoumaryl-CoA and p-coumaryl-CoA were both 5.0 µM. Expression analyses of MdCHS genes varied according to tissue type. MdCHS1, MdCHS2 and MdCHS3 expression levels were associated with the levels of phloretin accumulate in the respective tissues.

Identification and characterization of UDP-glucose:Phloretin 4'-O-glycosyltransferase from Malus x domestica Borkh.

Apples (Malus x domestica Brokh.) are among the world's most important food crops with nutritive and medicinal importance. Many of the health beneficial properties of apple fruit are suggested to be due to (poly)phenolic metabolites, including various dihydrochalcones. Although many of the genes and enzymes involved in polyphenol biosynthesis are known in many plant species, the specific reactions that lead to the biosynthesis of the sweet tasting dihydrochalcones, such as trilobatin, are unknown. To identify candidate genes for involvement in the glycosylation of dihydrochalcones, existing genome databases of the Rosaceae were screened for apple genes with significant sequence similarity to Bacillus subtilis phloretin glycosyltransferase. Herein reported is the identification and functional characterization of a Malus x domestica gene encoding phloretin-4'-O-glycosyltransferase designated MdPh-4'-OGT. Recombinant MdPh-4'-OGT protein glycosylates phloretin in the presence of UDP-glucose into trilobatin in vitro. Its apparent Km values for phloretin and UDP-glucose were 26.1 µM and 1.2 mM, respectively. Expression analysis of the MdPh-4'-OGT gene indicated that its transcript levels showed significant variation in apple tissues of different developmental stages.

Isolation and Functional Characterization of Carotenoid Cleavage Dioxygenase-1 from Laurus nobilis L. (Bay Laurel) Fruits.

Bay laurel (Laurus nobilis L.) is an agriculturally important tree used in food, drugs, and the cosmetics industry. Many of the health beneficial properties of bay laurel are due to volatile terpene metabolites that they contain, including various norisoprenoids. Despite their importance, little is known about the norisoprenoid biosynthesis in Laurus nobilis fruits. We found that the volatile norisoprenoids 6-methyl-5-hepten-2-one, pseudoionone, and ß-ionone accumulated in Laurus nobilis fruits in a pattern reflecting their carotenoid content. A full-length cDNA encoding a potential carotenoid cleavage dioxygenase (LnCCD1) was isolated. The LnCCD1 gene was overexpressed in Escherichia coli, and recombinant protein was assayed for its cleavage activity with an array of carotenoid substrates. The LnCCD1 protein was able to cleave a variety of carotenoids at the 9,10 (9',10') and 5,6 (5',6') positions to produce 6-methyl-5-hepten-2-one, pseudoionone, ß-ionone, and α-ionone. Our results suggest a role for LnCCD1 in Laurus nobilis fruit flavor biosynthesis.

Identification, Functional Characterization, and Evolution of Terpene Synthases from a Basal Dicot.

Bay laurel (Laurus nobilis) is an agriculturally and economically important dioecious tree in the basal dicot family Lauraceae used in food and drugs and in the cosmetics industry. Bay leaves, with their abundant monoterpenes and sesquiterpenes, are used to impart flavor and aroma to food, and have also drawn attention in recent years because of their potential pharmaceutical applications. To identify terpene synthases (TPSs) involved in the production of these volatile terpenes, we performed RNA sequencing to profile the transcriptome of L. nobilis leaves. Bioinformatic analysis led to the identification of eight TPS complementary DNAs. We characterized the enzymes encoded by three of these complementary DNAs: a monoterpene synthase that belongs to the TPS-b clade catalyzes the formation of mostly 1,8-cineole; a sesquiterpene synthase belonging to the TPS-a clade catalyzes the formation of mainly cadinenes; and a diterpene synthase of the TPS-e/f clade catalyzes the formation of geranyllinalool. Comparison of the sequences of these three TPSs indicated that the TPS-a and TPS-b clades of the TPS gene family evolved early in the evolution of the angiosperm lineage, and that geranyllinalool synthase activity is the likely ancestral function in angiosperms of genes belonging to an ancient TPS-e/f subclade that diverged from the kaurene synthase gene lineages before the split of angiosperms and gymnosperms.

Identification and Characterization of Terpene Synthases Potentially Involved in the Formation of Volatile Terpenes in Carrot (Daucus carota L.) Roots.

Plants produce an excess of volatile organic compounds, which are important in determining the quality and nutraceutical properties of fruit and root crops, including the taste and aroma of carrots (Daucus carota L.). A combined chemical, biochemical, and molecular study was conducted to evaluate the differential accumulation of volatile terpenes in a diverse collection of fresh carrots (D. carota L.). Here, we report on a transcriptome-based identification and functional characterization of two carrot terpene synthases, the sesquiterpene synthase, DcTPS1, and the monoterpene synthase, DcTPS2. Recombinant DcTPS1 protein produces mainly (E)-ß-caryophyllene, the predominant sesquiterpene in carrot roots, and α-humulene, while recombinant DcTPS2 functions as a monoterpene synthase with geraniol as the main product. Both genes are differentially transcribed in different cultivars and during carrot root development. Our results suggest a role for DcTPS genes in carrot aroma biosynthesis.

Gene silencing of CCD7 and CCD8 in Phelipanche aegyptiaca by tobacco rattle virus system retarded the parasite development on the host.

Strigolactones are phytohormones that stimulate seed germination of parasitic plants including Phelipanche aegyptiaca. Strigolactones are derived from carotenoids via a pathway involving the carotenoid cleavage dioxygenases CCD7 and CCD8. We report here identification of PaCCD7 and PaCCD8 orthologous genes from P. aegyptiaca. Expression analysis of PaCCD7 and PaCCD8 genes showed significant variation in their transcript levels in seeds and tubercles of P. aegyptiaca at different developmental stages. These two parasitic PaCCD7 and PaCCD8 genes were silenced in P. aegyptiaca using a trans-silencing approach in Nicotiana benthamiana. The transient knock-down of PaCCD7 and PaCCD8 inhibited tubercle development and the infestation process in host plants. Our results suggest an important role of the strigolactone associated genes (PaCCD7 and PaCCD8) in the parasite life cycle.

Formation of norisoprenoid flavor compounds in carrot (Daucus carota L.) roots: characterization of a cyclic-specific carotenoid cleavage dioxygenase 1 gene.

Carotenoids are isoprenoid pigments that upon oxidative cleavage lead to the production of norisoprenoids that have profound effect on flavor and aromas of agricultural products. The biosynthetic pathway to norisoprenoids in carrots (Daucus carota L.) is still largely unknown. We found the volatile norisoprenoids farnesylacetone, α-ionone, and ß-ionone accumulated in Nairobi, Rothild, and Purple Haze cultivars but not in Yellowstone and Creme de Lite in a pattern reflecting their carotenoid content. A cDNA encoding a protein with carotenoid cleavage dioxygenase activity, DcCCD1, was identified in carrot and was overexpressed in Escherichia coli strains previously engineered to produce different carotenoids. The recombinant DcCCD1 enzyme cleaves cyclic carotenes to generate α- and ß-ionone. No cleavage products were found when DcCCD1 was co-expressed in E. coli strains accumulating non-cyclic carotenoids, such as phytoene or lycopene. Our results suggest a role for DcCCD1 in carrot flavor biosynthesis.