Comparative Metabolomics and Molecular Phylogenetics of Melon (Cucumis melo, Cucurbitaceae) Biodiversity.

The broad variability of Cucumis melo (melon, Cucurbitaceae) presents a challenge to conventional classification and organization within the species. To shed further light on the infraspecific relationships within C. melo, we compared genotypic and metabolomic similarities among 44 accessions representative of most of the cultivar-groups. Genotyping-by-sequencing (GBS) provided over 20,000 single-nucleotide polymorphisms (SNPs). Metabolomics data of the mature fruit flesh and rind provided over 80,000 metabolomic and elemental features via an orchestra of six complementary metabolomic platforms. These technologies probed polar, semi-polar, and non-polar metabolite fractions as well as a set of mineral elements and included both flavor- and taste-relevant volatile and non-volatile metabolites. Together these results enabled an estimate of "metabolomic/elemental distance" and its correlation with the genetic GBS distance of melon accessions. This study indicates that extensive and non-targeted metabolomics/elemental characterization produced classifications that strongly, but not completely, reflect the current and extensive genetic classification. Certain melon Groups, such as Inodorous, clustered in parallel with the genetic classifications while other genome to metabolome/element associations proved less clear. We suggest that the combined genomic, metabolic, and element data reflect the extensive sexual compatibility among melon accessions and the breeding history that has, for example, targeted metabolic quality traits, such as taste and flavor.

Insights into the chemosensory basis of flavor in table grapes.

BACKGROUND: The full flavor of grape berries is determined by the interaction of sugars, acids, volatile compounds, and other berry properties, such as astringency. Sugars and acids are important for berry taste, whereas volatile compounds are important for the unique berry flavors, e.g., monoterpenes for the Muscat varieties. RESULTS: We explored the basis for 'fruity' flavor perception in table grapes. Samples were collected from 134 new table grape lines and commercial varieties and tested chemically for their volatile profiles and organoleptically by tasting panels. At the sensory level, flavor impression was strongly correlated with berry preference, whereas among 'fruity', 'neutral', 'herbaceous,' and 'Muscat', only the 'fruity' flavor was correlated with berry preference. At the chemical level, 114 volatile compounds were detected in the 81 breeding lines and cultivars examined, and grouped into 'core' and 'unique' categories. The typical berry flavor seemed to depend on the major volatile aldehydes - 1-hexanal and (E)-2-hexenal - accounting for up to an average 85% of the berry's core volatile concentration. We found four volatile compounds - α-bergamotene, geranyl formate, aristolene and α-penansinene - previously undetected, to our knowledge, in fresh grape berries. CONCLUSIONS: High 'fruity' flavor scores were related to three independent factors: (i) presence of unique volatile compounds, such as the sesquiterpene α-copaene, (ii) higher total concentration of volatile compounds, (iii) optimal maturity associated to high total soluble solids (TSS) levels, interacting with berry volatile composition. These combined sensory and analytical data on the flavor of table grapes improve our understanding of the complex interface between chemical and sensory perception in fruit. © 2019 Society of Chemical Industry.

Alkaloid chemodiversity in Mandragora spp. is associated with loss-of-functionality of MoH6H, a hyoscyamine 6ß-hydroxylase gene.

Mandrakes (Mandragora spp., Solanaceae) are known to contain tropane alkaloids and have been used since antiquity in traditional medicine. Tropane alkaloids such as scopolamine and hyoscyamine are used in modern medicine to treat pain, motion sickness, as eye pupil dilators and antidotes against organo-phosphate poisoning. Hyoscyamine is converted to 6ß-hydroxyhyoscyamine (anisodamine) and scopolamine by hyoscyamine 6ß-hydroxylase (H6H), a 2-oxoglutarate dependent dioxygenase. We describe here a marked chemo-diversity in the tropane alkaloid content in Mandragora spp. M. officinarum and M. turcomanica lack anisodamine and scopolamine but display up to 10 fold higher hyoscyamine levels as compared with M. autumnalis. Transcriptomic analyses revealed that H6H is highly conserved among scopolamine-producing Solanaceae. MoH6H present in M. officinarum differs in several amino acid residues including a homozygotic mutation in the substrate binding region of the protein and its prevalence among accessions was confirmed by Cleaved-Amplified-Polymorphic-Sequence analyses. Functional expression revealed that MaH6H, a gene isolated from M. autumnalis encodes an active H6H enzyme while the MoH6H sequence isolated from M. officinarum was functionally inactive. A single G to T mutation in nucleotide 663 of MoH6H is associated with the lack of anisodamine and scopolamine in M. officinalis.

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.

Distribution of primary and specialized metabolites in Nigella sativa seeds, a spice with vast traditional and historical uses.

Black cumin (Nigella sativa L., Ranunculaceae) is an annual herb commonly used in the Middle East, India and nowadays gaining worldwide acceptance. Historical and traditional uses are extensively documented in ancient texts and historical documents. Black cumin seeds and oil are commonly used as a traditional tonic and remedy for many ailments as well as in confectionery and bakery. Little is known however about the mechanisms that allow the accumulation and localization of its active components in the seed. Chemical and anatomical evidence indicates the presence of active compounds in seed coats. Seed volatiles consist largely of olefinic and oxygenated monoterpenes, mainly p-cymene, thymohydroquinone, thymoquinone, γ-terpinene and α-thujene, with lower levels of sesquiterpenes, mainly longifolene. Monoterpene composition changes during seed maturation. γ-Terpinene and α-thujene are the major monoterpenes accumulated in immature seeds, and the former is gradually replaced by p-cymene, carvacrol, thymo-hydroquinone and thymoquinone upon seed development. These compounds, as well as the indazole alkaloids nigellidine and nigellicine, are almost exclusively accumulated in the seed coat. In contrast, organic and amino acids are primarily accumulated in the inner seed tissues. Sugars and sugar alcohols, as well as the amino alkaloid dopamine and the saponin α-hederin accumulate both in the seed coats and the inner seed tissues at different ratios. Chemical analyses shed light to the ample traditional and historical uses of this plant.

Benzaldehyde is a precursor of phenylpropylamino alkaloids as revealed by targeted metabolic profiling and comparative biochemical analyses in Ephedra spp.

Ephedrine and pseudoephedrine are phenylpropylamino alkaloids widely used in modern medicine. Some Ephedra species such as E. sinica Stapf (Ephedraceae), a widely used Chinese medicinal plant (Chinese name: Ma Huang), accumulate ephedrine alkaloids as active constituents. Other Ephedra species, such as E. foeminea Forssk. (syn. E. campylopoda C.A. Mey) lack ephedrine alkaloids and their postulated metabolic precursors 1-phenylpropane-1,2-dione and (S)-cathinone. Solid-phase microextraction analysis of freshly picked young E. sinica and E. foeminea stems revealed the presence of increased benzaldehyde levels in E. foeminea, whereas 1-phenylpropane-1,2-dione was detected only in E. sinica. Soluble protein preparations from E. sinica and E. foeminea stems catalyzed the conversion of benzaldehyde and pyruvate to (R)-phenylacetylcarbinol, (S)-phenylacetylcarbinol, (R)-2-hydroxypropiophenone (S)-2-hydroxypropiophenone and 1-phenylpropane-1,2-dione. The activity, termed benzaldehyde carboxyligase (BCL) required the presence of magnesium and thiamine pyrophosphate and was 40 times higher in E. sinica as compared to E. foeminea. The distribution patterns of BCL activity in E. sinica tissues correlates well with the distribution pattern of the ephedrine alkaloids. (S)-Cathinone reductase enzymatic activities generating (1R,2S)-norephedrine and (1S,1R)-norephedrine were significantly higher in E. sinica relative to the levels displayed by E. foeminea. Surprisingly, (1R,2S)-norephedrine N-methyltransferase activity which is a downstream enzyme in ephedrine biosynthesis was significantly higher in E. foeminea than in E. sinica. Our studies further support that benzaldehyde is the metabolic precursor to phenylpropylamino alkaloids in E. sinica.

Naturally occurring norephedrine oxazolidine derivatives in khat (Catha edulis).

Khat (Catha edulis Forsk.) is a perennial shrub whose young leaves are chewed for their psychostimulating and anorectic properties. The main active principles of khat are believed to be the phenylpropylamino alkaloids, primarily (-)-cathinone [(S)-α-aminopropiophenone], (+)-cathine [(1S)(2S)-norpseudoephedrine], and (-)-norephedrine [(1R)(2S)-norephedrine]. GC-MS analyses of young leaf extracts indicated the presence of two oxazolidine derivatives, 2,4-dimethyl-5-phenyloxazolidine and 4-methyl-2-(trans-1-pentenyl)-5-phenyloxazolidine. To ascertain the chemical identity of these compounds, we synthesized the putative compounds by condensation of norephedrine and acetaldehyde or trans-2-hexenal, respectively. Spectroscopic analyses (GC-MS, NMR) of the structures of these synthetic compounds showed them to have identical retention indexes and mass spectra characteristic to 2,4-dimethyl-5-phenyloxazolidine and 4-methyl-2-(trans-1-pentenyl)-5-phenyloxazolidine. Marked differences in the ratios between each of these two norephedrine oxazolidine derivatives and total phenylpropylamino alkaloids were found among thirteen different khat accessions further indicating polymorphism in alkaloid ratios and content in C. edulis.

Utilisation of the MVA pathway to produce elevated levels of the sesquiterpene α-copaene in potato tubers.

Potato flavour is a complex trait resulting from the presence of a combination of volatile and non-volatile compounds. The aim of this work was to investigate the effect of specifically altering the volatile content of tubers and assess its impact on flavour. Tuber-specific over-expression of a potato α-copaene synthase gene resulted in enhanced levels (up to 15-fold higher than controls) of the sesquiterpene α-copaene. A positive correlation (R(2)=0.8) between transgene expression level and α-copaene abundance was observed. No significant changes in the levels of volatiles other than α-copaene were detected. Non-volatile flavour compounds (sugars, glycoalkaloids, major umami amino acids and 5'-ribonucleotides) were also determined. Relationships between flavour compounds and sensory evaluation data were investigated. Evaluators could not detect any aroma differences in the transgenic samples compared with controls and no significant differences in taste attributes were found. Thus although successful engineering of potato tubers to accumulate high levels of the flavour volatile α-copaene was achieved, sensory analysis suggests that α-copaene is not a major component of potato flavour.

Expressed sequence tag analysis of khat (Catha edulis) provides a putative molecular biochemical basis for the biosynthesis of phenylpropylamino alkaloids

Khat (Catha edulis Forsk.) is a flowering perennial shrub cultivated for its neurostimulant properties resulting mainly from the occurrence of (S)-cathinone in young leaves. The biosynthesis of (S)-cathinone and the related phenylpropylamino alkaloids (1S,2S)-cathine and (1R,2S)-norephedrine is not well characterized in plants. We prepared a cDNA library from young khat leaves and sequenced 4,896 random clones, generating an expressed sequence tag (EST) library of 3,293 unigenes. Putative functions were assigned to > 98 percent of the ESTs, providing a key resource for gene discovery. Candidates potentially involved at various stages of phenylpropylamino alkaloid biosynthesis from L-phenylalanine to (1S,2S)-cathine were identified.

Composition and stereochemistry of ephedrine alkaloids accumulation in Ephedra sinica Stapf.

Ephedra sinica Stapf (Ephedraceae) is a widely used Chinese medicinal plant (Chinese name: Ma Huang). The main active constituents of E. sinica are the unique and taxonomically restricted adrenergic agonists phenylpropylamino alkaloids, also known as ephedrine alkaloids: (1R,2S)-norephedrine (1S,2S)-norpseudoephedrine, (1R,2S)-ephedrine, (1S,2S)-pseudoephedrine, (1R,2S)-N-methylephedrine and (1S,2S)-N-methylpseudoephedrine. GC-MS analysis of freshly picked young E. sinica stems enabled the detection of 1-phenylpropane-1,2-dione and (S)-cathinone, the first two putative committed biosynthetic precursors to the ephedrine alkaloids. These metabolites are only present in young E. sinica stems and not in mature stems or roots. The related Ephedra foemina and Ephedra foliata also lack ephedrine alkaloids and their metabolic precursors in their aerial parts. A marked diversity in the ephedrine alkaloids content and stereochemical composition in 16 different E. sinica accessions growing under the same environmental conditions was revealed, indicating genetic control of these traits. The accessions can be classified into two groups according to the stereochemistry of the products accumulated: a group that displayed only 1R stereoisomers, and a group that displayed both 1S and 1R stereoisomers. (S)-cathinone reductase activities were detected in E. sinica stems capable of reducing (S)-cathinone to (1R,2S)-norephedrine and (1S,2S)-norpseudoephedrine in the presence of NADH. The proportion of the diastereoisomers formed varied according to the accession tested. A (1R,2S)-norephedrine N-methyltransferase capable of converting (1R,2S)-norephedrine to (1R,2S)-ephedrine in the presence of S-adenosylmethionine (SAM) was also detected in E. sinica stems. Our studies further support the notion that 1-phenylpropane-1,2-dione and (S)-cathinone are biosynthetic precursors of the ephedrine alkaloids in E. sinica stems and that the activity of (S)-cathinone reductases directs and determines the stereochemical branching of the pathway. Further methylations are likely due to N-methyltransferase activities.

Developmental patterns of phenylpropylamino alkaloids accumulation in khat (Catha edulis, Forsk.).

Khat (Catha edulis Forsk., Celastraceae) is a perennial shrub that was introduced to Israel by Yemenite immigrants. Khat young leaves are chewed as a stimulant. The main stimulating active principles in this plant are the phenylpropylamino alkaloids (-)-cathinone [(S)-alpha-aminopropiophenone], (+)-cathine [(+)-norpseudoephedrine] and (-)-norephedrine. A novel GC-MS analysis method for the quantitative determination of phenylpropylamino alkaloids and their putative precursor 1-phenyl-1,2-propanedione in khat leaves was developed. We found a marked diversity in the phenylpropylamino alkaloids content and composition in 9 different accessions originated in seedlings and in the commercial cultivar "Mahanaim". The highest 1-phenyl-1,2-propanedione and (-)-cathinone levels occur in young leaves, the part traditionally chewed for its psycho-stimulating properties. Older leaves lack (-)-cathinone but contain the less active (+)-cathine and (-)-norephedrine. Young stems and flowers also contain 1-phenyl-1,2-propanedione, (-)-cathinone, (+)-cathine and (-)-norephedrine. We report the presence of a (-)-cathinone reductase in khat leaves capable of reducing (-)-cathinone to (+)-cathine in the presence of NADPH. We propose that (-)-cathinone is a biosynthetic precursor of (+)-cathine and (-)-norephedrine in khat leaves.

O-methyltransferases involved in the biosynthesis of volatile phenolic derivatives in rose petals.

Rose (Rosa hybrida) flowers produce and emit a diverse array of volatiles, characteristic to their unique scent. One of the most prominent compounds in the floral volatiles of many rose varieties is the methoxylated phenolic derivative 3,5-dimethoxytoluene (orcinol dimethyl ether). Cell-free extracts derived from developing rose petals displayed O-methyltransferase (OMT) activities toward several phenolic substrates, including 3,5-dihydroxytoluene (orcinol), 3-methoxy,5-hydroxytoluene (orcinol monomethyl ether), 1-methoxy, 2-hydroxy benezene (guaiacol), and eugenol. The activity was most prominent in rose cv Golden Gate, a variety that produces relatively high levels of orcinol dimethyl ether, as compared with rose cv Fragrant Cloud, an otherwise scented variety but which emits almost no orcinol dimethyl ether. Using a functional genomics approach, we have identified and characterized two closely related cDNAs from a rose petal library that each encode a protein capable of methylating the penultimate and immediate precursors (orcinol and orcinol monomethyl ether, respectively) to give the final orcinol dimethyl ether product. The enzymes, designated orcinol OMTs (OOMT1 and OOMT2), are closely related to other plant methyltransferases whose substrates range from isoflavones to phenylpropenes. The peak in the levels of OOMT1 and OOMT2 transcripts in the flowers coincides with peak OMT activity and with the emission of orcinol dimethyl ether.