Fruit Metabolome Research Articles

Metabolic engineering of tomato fruit enriched in L-DOPA

L-DOPA, also known as Levodopa or L-3,4-dihydroxyphenylalanine, is a non-standard amino acid, and the gold standard drug for the treatment for Parkinson's Disease (PD). Recently, a gene encoding the enzyme that is responsible for its synthesis, as a precursor of the coloured pigment group betalains, was identified in beetroot, BvCYP76AD6. We have engineered tomato fruit enriched in L-DOPA through overexpression of BvCYP76AD6 in a fruit specific manner. Analysis of the transgenic fruit revealed the feasibility of accumulating L-DOPA in a non-naturally betalain-producing plant. Fruit accumulating L-DOPA also showed major effects on the fruit metabolome. Some of these changes included elevation of amino acids levels, changes in the levels of intermediates of the TCA and glycolysis pathways and reductions in the levels of phenolic compounds and nitrogen-containing specialised metabolites. Furthermore, we were able to increase the L-DOPA levels further by elevating the expression of the metabolic master regulator, MYB12, specifically in tomato fruit, together with BvCYP76AD6. Our study elucidated new roles for L-DOPA in plants, because it impacted fruit quality parameters including antioxidant capacity and firmness. The L-DOPA levels achieved in tomato fruit were comparable to the levels in other non-seed organs of L-DOPA - accumulating plants, offering an opportunity to develop new biological sources of L-DOPA by widening the repertoire of L-DOPA-accumulating plants. These tomato fruit could be used as an alternative source of this important pharmaceutical.

Early metabolic priming under differing carbon sufficiency conditions influences peach fruit quality development

Crop load management is an important preharvest factor to balance yield, quality, and maturation in peach. However, few studies have addressed how preharvest factors impact metabolism on fruit of equal maturity. An experiment was conducted to understand how carbon competition impacts fruit internal quality and metabolism in ‘Cresthaven’ peach trees by imposing distinct thinning severities. Fruit quality was evaluated at three developmental stages (S2, S3, S4), while controlling for equal maturity using non-destructive visual to near-infrared spectroscopy. Non-targeted metabolite profiling was used to characterize fruit at each developmental stage from trees that were unthinned (carbon starvation) or thinned (carbon sufficiency). Carbon sufficiency resulted in significantly higher fruit dry matter content and soluble solids concentration at harvest when compared to the carbon starved, underscoring the true impact of carbon manipulation on fruit quality. Significant differences in the fruit metabolome between treatments were observed at S2 when phenotypes were similar, while less differences were observed at S4 when the carbon sufficient fruit exhibited a superior phenotype. This suggests a potential metabolic priming effect on fruit quality when carbon is sufficiently supplied during early fruit growth and development. In particular, elevated levels of catechin may suggest a link between secondary/primary metabolism and fruit quality development.

Metabolome classification via GC/MS and UHPLC/MS of olive fruit varieties grown in Egypt reveal pickling process impact on their composition

The role of variety and effects of pickling on Egyptian olive fruit metabolome was determined using mass spectrometry-based metabolomics targeting nutrients and bioactive metabolities. The analyzed fresh olive fruit varieties included Manzanilo, Picual, Koroneiki, and Coratina, while the pickled samples included the Manzanilo and Picual varieties. Profiling of primary and secondary metabolites resulted in the detection of 201 metabolites. Variation between varieties was mostly observed among sugars, sugar alcohols, secoiridoids, and flavonoids. An abundance of carbohydrates and O-glycosides in Picual and Manzanilo versus enrichment of secoiridoids in Picual and Coratina olives viz. dehyro-oleuropein could account for the difference in palatability and health benefits among varieties. Herein, 13 new compounds are reported in the tested varieties, of which 10 appeared exclusively in pickled samples. Generally, pickled samples were characterized by the relative abundance of secoiridoids regarded as important markers for the pickling process. Metabolites profiling provided greater insight into the pickling process as a preservation method and accounted for the improved organoleptic characters in pickled fruits.

Modulation of the Tomato Fruit Metabolome by LED Light.

Metabolic profiles of tomatoes change during ripening and light can modulate the activity of relevant biochemical pathways. We investigated the effects of light directly supplied to the fruits on the metabolome of the fruit pericarp during ripening. Mature green tomatoes were exposed to well-controlled conditions with light as the only varying factor; control fruits were kept in darkness. In experiment 1 the fruits were exposed to either white light or darkness for 15 days. In experiment 2, fruits were exposed to different light spectra (blue, green, red, far-red, white) added to white background light for seven days. Changes in the global metabolome of the fruit pericarp were monitored using LCMS and GCMS (554 compounds in total). Health-beneficial compounds (carotenoids, flavonoids, tocopherols and phenolic acids) accumulated faster under white light compared to darkness, while alkaloids and chlorophylls decreased faster. Light also changed the levels of taste-related metabolites including glutamate and malate. The light spectrum treatments indicated that the addition of blue light was the most effective treatment in altering the fruit metabolome. We conclude that light during ripening of tomatoes can have various effects on the metabolome and may help with shaping the levels of key compounds involved in various fruit quality characteristics.

Deletion of beta-fructofuranosidase (invertase) genes is associated with sucrose content in Date Palm fruit.

The fruit of date palm trees are an important part of the diet for a large portion of the Middle East and North Africa. The fruit is consumed both fresh and dry and can be stored dry for extended periods of time. Date fruits vary significantly across hundreds of cultivars identified in the main regions of cultivation. Most dried date fruit are low in sucrose but high in glucose and fructose. However, high sucrose content is a distinctive feature of some date fruit and affects flavor as well as texture and water retention. To identify the genes controlling high sucrose content, we analyzed date fruit metabolomics for association with genotype data from 120 date fruits. We found significant association of dried date sucrose content and a genomic region that contains 3 tandem copies of the beta‐fructofuranosidase (invertase) gene in the reference Khalas genome, a low‐sucrose fruit. High‐sucrose cultivars including the popular Deglet Noor had a homozygous deletion of two of the 3 copies of the invertase gene. We show the deletion allele is derived when compared to the ancestral allele that retains all copies of the gene in 3 other species of Phoenix. The fact that 2 of the 3 tandem invertase copies are associated with dry fruit sucrose content will assist in better understanding the distinct roles of multiple date palm invertases in plant physiology. Identification of the recessive alleles associated with end‐point sucrose content in date fruit may be used in selective breeding in the future.

UHPLC-MS metabolome based classification of umbelliferous fruit taxa: a prospect for phyto-equivalency of its different accessions and in response to roasting.

Herbs of the Umbelliferae family are popular spices valued worldwide for their many nutritional and health benefits. Herein, five chief umbelliferous fruits viz., cumin, fennel, anise, coriander and caraway were assessed for its secondary metabolites diversity along with compositional changes incurring upon roasting as analyzed via ultra-high performance liquid chromatography coupled to photodiode array and electrospray ionization mass detectors UHPLC-qToF/MS. A total of 186 metabolites were annotated, according to metabolomics society guidelines, belonging mainly to flavonoids, fatty acids and phenolic acids. Multivariate models viz., PCA, HCA and OPLS-DA were further employed to assess fruits' heterogeneity in an untargeted manner and determine mechanistic changes in bioactive makeup post roasting viz., glycosidic cleavage, lipid degradation and Maillard reaction. Finally, the fruits' antioxidant activity showed decline upon roasting and in correlation with its total phenolic content. This study presents the first complete map of umbelliferous fruit metabolome, compositional differences and its roasting effect.

GC-MS and LC-MS analyses reveal the distribution of primary and secondary metabolites in mangosteen (Garcinia mangostana Linn.) fruit during ripening

Mangosteen (Garcinia mangostana Linn.) is a functional fruit rich in highly beneficial metabolites, particularly xanthones, which exhibit various bioactive properties, including antioxidant and anti-inflammatory activities. Given its pharmaceutical benefits, the market demand for mangosteen is continuing to increase; however, little is known about the metabolic changes that occur during ripening. Here we used a combination of gas chromatography-mass spectrometry (GC-MS) and liquid chromatography-MS (LC-MS) to simultaneously analyze the composition of primary and secondary metabolites in mangosteen fruit pericarp, aril, and seed at four different ripening stages (stages 0, 2, 4, and 6). A total of 57 and 98 metabolites were identified from GC-MS and LC-MS analyses, respectively. These metabolites comprised various metabolite classes, including sugars and derivatives, amino acids and derivatives, organic acids, an alcohol, aldehydes, glycosides, fatty acids, phenolics, alkaloids, terpenoids, xanthones, and a quinone. Our results showed that metabolites with variable influence on projection (VIP) ≥ 1.00 and p ≤ 0.05 included primary metabolites (e.g., L-mannopyranose, myo-inositol, arabinofuranose, galacturonic acid, l-(+)-tartaric acid and aspartic acid) and secondary metabolites (e.g., neoisostegane, epirobinetinidol-(4β,8)-catechin, α-mangostin, and gartanin). Such metabolites may contribute to the degradation of the cell wall, development of flavor, color, and aroma, and defense against frugivores, insects, and pathogens during mangosteen ripening. The metabolic pathways of galactose, ascorbate, aldarate, starch, sucrose, cysteine, and methionine, as well as glycolysis and the tricarboxylic acid (TCA) cycle, were differentially regulated during mangosteen ripening. Profiling the fruit metabolome during the ripening provided a snapshot for understanding the intricate biological and molecular regulation of fruit development. Our results will facilitate further improvements in post-harvest technologies that will help increase the shelf life of mangosteen fruit, thus assisting farmers and producers in the global commercialization of this superfruit.

Comparative Metabolomics Unravel the Effect of Magnesium Oversupply on Tomato Fruit Quality and Associated Plant Metabolism.

In general, greenhouse cultivation involves the rampant application of chemical fertilizers, with the aim of achieving high yields. Oversaturation with mineral nutrients that aid plant growth, development, and yield may lead to abiotic stress conditions. We explore the effects of excess magnesium on tomato plant metabolism, as well as tomato fruit quality using non-targeted mass spectrometry (MS)-based metabolomic approaches. Tomato plants were subjected to three different experiments, including high magnesium stress (MgH), extremely high magnesium stress (MgEH), and a control with optimal nutrient levels. Leaves, roots, and fruits were harvested at 16 weeks following the treatment. A metabolic pathway analysis showed that the metabolism induced by Mg oversupply was remarkably different between the leaf and root. Tomato plants allocated more resources to roots by upregulating carbohydrate and polyamine metabolism, while these pathways were downregulated in leaves. Mg oversupply affects the fruit metabolome in plants. In particular, the relative abundance of threonic acid, xylose, fucose, glucose, fumaric acid, malic acid, citric acid, oxoglutaric acid, threonine, glutamic acid, phenylalanine, and asparagine responsible for the flavor of tomato fruits was significantly decreased in the presence of Mg oversupply. Altogether, we concluded that Mg oversupply leads to drastically higher metabolite transport from sources (fully expanded leaves) to sinks (young leaves and roots), and thus, produces unfavorable outcomes in fruit quality and development.

Evaluating the metabolic perturbations in Mangifera indica (mango) ripened with various ripening agents/practices through gas chromatography - mass spectrometry based metabolomics.

Mangifera indica L. (mango) is said to be the king of fruits due to its rich nutritional properties and mainly originates from the Indian sub-continent. The consumption pattern of the mangoes has increased drastically, due to which, many ripening practices/agents were used to make it ready-to-eat fruit or juice for the consumers. The fruit quality and metabolic composition are said to be altered due to different ripening agents/practices. The present communication mainly deals to understand the metabolic perturbations in mango fruits due to different ripening practices/agents (room temperature ripening, ethylene, and calcium carbide) using gas chromatography - mass spectrometry based metabolomics. The partial least square-discriminant analysis has found 16 differential metabolites for different ripening agents/practices which are belong to the classes of amino acids, fatty acids, sugars, and polyols. Four metabolic pathways were found to alter in the fruit metabolome due to different ripening agents/practices. Fructose, glucose, and galactose were found to be significantly up-regulated due to calcium carbide ripening in comparison to other ripening agents/practices. Overall findings from the present study advocates that mass spectrometry based metabolomics can be valuable tool to understand the fruit quality and safety with respect to consumer health.

Effects of 2,4-Dichlorophenoxyacetic Acid on Cucumber Fruit Development and Metabolism.

The auxin-like compound 2,4-dichlorophenoxyacetic acid (2,4-D) has been widely used as a plant growth regulator in cucumber fruit production; however, its influence on fruit development and metabolism has not been evaluated. In this study, the phenotype of cucumber fruits in both 2,4-D treatment and non-treatment control groups were recorded, and the metabolome of different segments of cucumber fruit at various sampling time points were profiled by a standardized non-targeted metabolomics method based on UPLC-qTOF-MS. The application of 2,4-D increased the early growth rate of the fruit length but had no significant effect on the final fruit length, and produced cucumber fruits with fresh flowers at the top. The 2,4-D treatment also affected the cucumber fruit metabolome, causing significant changes in the stylar end at 4 days after flowering (DAF). The significantly changed metabolites were mainly involved in methionine metabolism, the citric acid cycle and flavonoid metabolism pathways. At the harvest stage, 2,4–D treatment significantly decreased the levels of flavonoids and cinnamic acid derivatives while increased the levels of some of the amino acids. In summary, exogenous application of 2,4-D can greatly alter the phenotype and metabolism of cucumber fruit. These findings will assist in exploring the mechanisms of how 2,4-D treatment changes the fruit phenotype and evaluating the influence of 2,4-D treatment on the nutritional qualities of cucumber fruit.

Combined Analysis of the Fruit Metabolome and Transcriptome Reveals Candidate Genes Involved in Flavonoid Biosynthesis in Actinidia arguta.

To assess the interrelation between the change of metabolites and the change of fruit color, we performed a combined metabolome and transcriptome analysis of the flesh in two different Actinidia arguta cultivars: “HB” (“Hongbaoshixing”) and “YF” (“Yongfengyihao”) at two different fruit developmental stages: 70d (days after full bloom) and 100d (days after full bloom). Metabolite and transcript profiling was obtained by ultra-performance liquid chromatography quadrupole time-of-flight tandem mass spectrometer and high-throughput RNA sequencing, respectively. The identification and quantification results of metabolites showed that a total of 28,837 metabolites had been obtained, of which 13,715 were annotated. In comparison of HB100 vs. HB70, 41 metabolites were identified as being flavonoids, 7 of which, with significant difference, were identified as bracteatin, luteolin, dihydromyricetin, cyanidin, pelargonidin, delphinidin and (−)-epigallocatechin. Association analysis between metabolome and transcriptome revealed that there were two metabolic pathways presenting significant differences during fruit development, one of which was flavonoid biosynthesis, in which 14 structural genes were selected to conduct expression analysis, as well as 5 transcription factor genes obtained by transcriptome analysis. RT-qPCR results and cluster analysis revealed that AaF3H, AaLDOX, AaUFGT, AaMYB, AabHLH, and AaHB2 showed the best possibility of being candidate genes. A regulatory network of flavonoid biosynthesis was established to illustrate differentially expressed candidate genes involved in accumulation of metabolites with significant differences, inducing red coloring during fruit development. Such a regulatory network linking genes and flavonoids revealed a system involved in the pigmentation of all-red-fleshed and all-green-fleshed A. arguta, suggesting this conjunct analysis approach is not only useful in understanding the relationship between genotype and phenotype, but is also a powerful tool for providing more valuable information for breeding.

Post-harvest UV-B radiation modulates metabolite profile in peach fruit

The possibility to modify plant metabolic profile of plants and fruit to improve their healthy properties using eco-friendly tools, rather than transgenic approaches, gained interest in the last decades. Ultraviolet-B (UV-B) radiation, at low levels, thanks to its ability to influence plant secondary metabolism, could be successfully used to achieve this goal. However, few studies have been conducted so far on the effects of post-harvest UV-B treatments on fruit metabolomics. The present research, aimed to evaluate the impact of UV-B on peach metabolites profile through non-targeted metabolomics (UHPLC-ESI/QTOF-MS) coupled with multivariate chemometrics, provided evidence that 10 and 60 min of post-harvest UV-B irradiation influenced several classes of metabolites. Most phenolics were down-accumulated 24 h after both UV-B treatments, though, after 36 h, anthocyanins, flavones and dihydroflavonols increased (2.06−, 1.92−, 1.68-fold with 10 min UV-B; 6.65−, 2.53−, 2.05-fold with 60 min UV-B, respectively). UV-B reduced carotenoids and most lipids and increased some biosynthetic intermediates and degradation products, some of them known for their positive role in human health. Among alkaloids, some pteridines accumulated, likely derived from folates degradation, while indole alkaloids decreased. Despite the decrease of some bioprotective metabolites as carotenoids, the UV-B-induced up-accumulation of many antioxidant phenolics after 36 h from the exposure suggests an improvement of the healthy properties of peach fruit and reinforces the potential of UV-B controlled irradiation as a nutraceuticals-increasing tool in fruit.

Identification of Conserved and Diverse Metabolic Shift of the Stylar, Intermediate and Peduncular Segments of Cucumber Fruit during Development.

Cucumber (Cucumis sativus L.) is one of the most important vegetables and contains a high content of nutritionally beneficial metabolites. However, little is known about the metabolic variations among different parts of cucumber fruit and their kinetics during growth. In this study, the dynamic metabolic profiles in the stylar end, the intermediate segment and the peduncular end of cucumber fruit during the development were investigated by employing a non-targeted metabolomics approach, where 238 metabolites were identified. Statistical analyses revealed that both development time and tissue type influenced metabolic changes, while development time seemed to exert more effects than tissue type on the cucumber fruit metabolome. The levels of the most of the detected metabolites decreased gradually, while those of some amino acids, carbohydrates and flavonoids increased across development. The metabolomes of the stylar end and the intermediate segment were similar, although all three parts of the cucumber fruit were separated from each other in orthogonal partial least squares projection to latent structures-discriminant analysis (OPLS-DA) plots. Metabolites association analysis revealed that sn-1 and sn-2 lysophospholipids are synthesized via independent pathways in cucumber fruit. In sum, this study demonstrated both conserved and diverse metabolic kinetics of three parts of cucumber fruit, which will facilitate further study of the regulation of cucumber fruit development as well as their potential applications in nutritious quality improvement of cucumber fruit.

Rewiring of the Fruit Metabolome in Tomato Breeding

Humans heavily rely on dozens of domesticated plant species that have been further improved through intensive breeding. To evaluate how breeding changed the tomato fruit metabolome, we have generated and analyzed a dataset encompassing genomes, transcriptomes, and metabolomes from hundreds of tomato genotypes. The combined results illustrate how breeding globally altered fruit metabolite content. Selection for alleles of genes associated with larger fruits altered metabolite profiles as a consequence of linkage with nearby genes. Selection of five major loci reduced the accumulation of anti-nutritional steroidal glycoalkaloids in ripened fruits, rendering the fruit more edible. Breeding for pink tomatoes modified the content of over 100 metabolites. The introgression of resistance genes from wild relatives in cultivars also resulted in major and unexpected metabolic changes. The study reveals a multi-omics view of the metabolic breeding history of tomato, as well as provides insights into metabolome-assisted breeding and plant biology.

Untargeted metabolomics reveals specific withanolides and fatty acyl glycoside as tentative metabolites to differentiate organic and conventional Physalis peruviana fruits

The agronomic production systems may affect the levels of food metabolites. Metabolomics approaches have been applied as useful tool for the characterization of fruit metabolome. In this study, metabolomics techniques were used to assess the differences in phytochemical composition between goldenberry samples produced by organic and conventional systems. To verify that the organic samples were free of pesticides, individual pesticides were analyzed. Principal component analysis showed a clear separation of goldenberry samples from two different farming systems. Via targeted metabolomics assays, whereby carotenoids and ascorbic acid were analyzed, not statistical differences between both crops were found. Conversely, untargeted metabolomics allowed us to identify two withanolides and one fatty acyl glycoside as tentative metabolites to differentiate goldenberry fruits, recording organic fruits higher amounts of these compounds than conventional samples. Hence, untargeted metabolomics technology could be suitable to research differences on phytochemicals under different agricultural management practices and to authenticate organic products.

Multiwalled Carbon Nanotubes Dramatically Affect the Fruit Metabolome of Exposed Tomato Plants.

Here, we reported that multiwalled carbon nanotubes (MWCNT) added to hydroponics system can enhance fruit production of exposed tomato plants. We quantified the exact amount of MWCNT accumulated inside of fruits collected by MWCNT-exposed plants using an advanced microwave induced heating technique (MIH). We found that absorption of MWCNT by tomato fruits significantly affected total fruit metabolome as was confirmed by LC-MS. Our data highlight the importance of comprehensive toxicological risk assessment of plants contaminated with carbon nanomaterials.

1H NMR foodomics reveals that the biodynamic and the organic cultivation managements produce different grape berries (Vitis vinifera L. cv. Sangiovese)

The increasing demand for natural foods and beverages, i.e. prepared by excluding synthetic chemicals along the whole production chain, has boosted the adoption of organic and biodynamic cultivation methods which are based on protocols avoiding use of synthetic pesticides. This trend is striking in viticulture, since wine production is largely shaped by the varying drinking attitudes of environment-friendly consumers. Using 1H NMR, the compositions of grape berries, collected at harvest in 2009 and 2011, in experimental plots cultivated either with biodynamic or organic methods, were compared. Although the analysis provides a comprehensive metabolic profile of berries, the resulting distinctive pattern consists of a few molecules. Lower content of sugars, coumaric and caffeic acids, as well as higher amount of γ-aminobutyric acid (GABA) were observed in biodynamic grapes. The 1H NMR foodomics approach evidenced a diverse fruit metabolome that could be associated to a different physiological response of plants to the agronomic environment.

Metabolomics of Lonicera caerulea fruit during ripening and its relationship with color and antioxidant activity

We performed mass spectrometry-based metabolites profiling in Lonicera caerulea fruits according to seven ripening stages. During ripening, fruit color significantly changed from green to red, with sugars, organic acids, phenolic acids, anthocyanins, and flavonoids significantly altered. In particular, the contents of cyanidin-3-glucoside, peonidin-glucoside, peonidin-3-rutinoside and cyanidin-3-rutinoside, which are closely associated with color, were elevated from stages four to seven. The changes of antioxidant activity during ripening were similar to those of total phenolic and flavonoid contents. L. caerulea fruits at stage six (pale-purple) had higher antioxidant activity and total phenolic and flavonoid contents with higher cyanidin-3,5-diglucoside contents than those at stage seven (fully purple). From this study, we revealed the changes in the contents of primary and secondary metabolites with antioxidant properties during ripening, and these results could be helpful to determine the optimal harvest stage of L. caerulea fruit.

Ethylene is all around.

Ethylene is all around.

Genetic introgression of ethylene-suppressed transgenic tomatoes with higher-polyamines trait overcomes many unintended effects due to reduced ethylene on the primary metabolome.

Ethylene regulates a myriad physiological and biochemical processes in ripening fruits and is accepted as the ripening hormone for the climacteric fruits. However, its effects on metabolome and resulting fruit quality are not yet fully understood, particularly when some of the ripening-associated biochemical changes are independent of ethylene action. We have generated a homozygous transgenic tomato genotype (2AS-AS) that exhibits reduced ethylene production as a result of impaired expression of 1-aminocyclopropane-1-carboxylate synthase 2 gene by its antisense RNA and had a longer shelf life. Double transgenic hybrid (2AS-AS × 579HO) developed through a genetic cross between 2AS-AS and 579HO (Mehta et al., 2002) lines resulted in significantly higher ethylene production than either the WT or 2AS-AS fruit. To determine the effects of reduced ethylene and introgression of higher polyamines’ trait, the metabolic profiles of ripening fruits from WT (556AZ), 2AS-AS, and 2AS-AS × 579HO lines were determined using 1H-NMR spectroscopy. The levels of Glu, Asp, AMP, Adenosine, Nucl1, and Nucl2 increased during ripening of the WT fruit. The increases in Glu, Asp, and AMP levels were attenuated in 2AS-AS fruit but recovered in the double hybrid with higher ethylene and polyamine levels. The ripening-associated decreases in Ala, Tyr, Val, Ile, Phe, malate, and myo-inositol levels in the 2AS-AS line were not reversed in the double hybrid line suggesting a developmental/ripening regulated accumulation of these metabolites independent of ethylene. Significant increases in the levels of fumarate, formate, choline, Nucl1, and Nucl2 at most stages of ripening fruit were found in the double transgenic line due to introgression with higher-polyamines trait. Taken together these results show that the ripening-associated metabolic changes are both ethylene dependent and independent, and that the fruit metabolome is under the control of multiple regulators, including ethylene and polyamines.