Phenylalanine treatment induces tomato resistance to Tuta absoluta via increased accumulation of benzenoid/phenylpropanoid volatiles serving as defense signals.

Tuta absoluta ("leafminer"), is a major pest of tomato crops worldwide. Controlling this insect is difficult due to its efficient infestation, rapid proliferation, and resilience to changing weather conditions. Furthermore, chemical pesticides have only a short-term effect due to rapid development of T. absoluta strains. Here, we show that a variety of tomato cultivars, treated with external phenylalanine solutions exhibit high resistance to T. absoluta, under both greenhouse and open field conditions, at different locations. A large-scale metabolomic study revealed that tomato leaves absorb and metabolize externally given Phe efficiently, resulting in a change in their volatile profile, and repellence of T. absoluta moths. The change in the volatile profile is due to an increase in three phenylalanine-derived benzenoid phenylpropanoid volatiles (BPVs), benzaldehyde, phenylacetaldehyde, and 2-phenylethanol. This treatment had no effect on terpenes and green leaf volatiles, known to contribute to the fight against insects. Phe-treated plants also increased the resistance of neighboring non-treated plants. RNAseq analysis of the neighboring non-treated plants revealed an exclusive upregulation of genes, with enrichment of genes related to the plant immune response system. Exposure of tomato plants to either benzaldehyde, phenylacetaldehyde, or 2-phenylethanol, resulted in induction of genes related to the plant immune system that were also induced due to neighboring Phe-treated plants. We suggest a novel role of phenylalanine-derived BPVs as mediators of plant-insect interactions, acting as inducers of the plant defense mechanisms.

Phenylalanine in motion: A tale of an essential molecule with many faces.

Phenylalanine has a unique role in plants as a source of a wide range of specialized metabolites, named phenylpropanoids that contribute to the adjustment of plants to changing developmental and environmental conditions. The profile of these metabolites differs between plants and plant organs. Some of the prominent phenylpropanoids include anthocyanins, phenolic acids, flavonoids, tannins, stilbenes, lignins, glucosinolates and benzenoid phenylpropanoid volatiles. Phenylalanine biosynthesis, leading to increased phenylpropanoid levels, is induced under stress. However, high availability of phenylalanine in plants under non-stressed conditions can be achieved either by genetically engineering plants to overproduce phenylalanine, or by external treatment of whole plants or detached plant organs with phenylalanine solutions. The objective of this review is to portray the many effects that increased phenylalanine availability has in plants under non-stressed conditions, focusing mainly on external applications. These applications include spraying and drenching whole plants with phenylalanine solutions, postharvest treatments by dipping fruit and cut flower stems, and addition of phenylalanine to cell suspensions. The results of these treatments include increased fragrance in flowers, increased aroma and pigmentation in fruit, increased production of health promoting metabolites in plant cell cultures, and increased resistance of plants, pre- and post-harvest, to a wide variety of pathogens. These effects suggest that plants can very efficiently uptake phenylalanine from their roots, leaves, flowers and fruits, translocate it from one organ to the other and between cell compartments, and metabolize it into phenylpropanoids. The mechanisms by which Phe treatment increases plant resistance to pathogens reveal new roles of phenylpropanoids in induction of genes related to the plant immune system. The simplicity of treatments with phenylalanine open many possibilities for industrial use. Many of the phenylalanine-treatment effects on increased resistance to plant pathogens have also been successful in commercial field trials.

Phenylalanine induces mango fruit resistance against chilling injuries during storage at suboptimal temperature.

Cold is the best means of prolonging fruit storage. However, tropical fruit are susceptible to cold storage. The mode of action of mango fruit tolerance to suboptimal cold temperature of 7 or 10 °C after postharvest application of 8 mM phenylalanine was investigated using transcriptomic and metabolomic analyses of mango fruit during suboptimal cold storage. Phenylalanine-treated fruit had less chilling injuries-black spot and pitting electrolyte leakage,-and reduced decay after suboptimal cold storage. Phenylalanine treatment induced genes related to plant-pathogen interactions, plant hormone signal transduction, and the phenylpropanoid pathway, increasing the levels of the flavonoids quercetin and kaempferol glycosides and anthocyanins, and antioxidant content. Reduced oxidation led to lower lipid peroxidation, and a reduction in fatty acid-degradation products, e.g., volatile aldehydes. Treatment with phenylalanine, therefore, enhances chilling tolerance of mango fruit through regulation of metabolic and defense-related pathways, maintaining high levels of flavonoids, and antioxidants enzyme activity, and reducing H2O2 content, lipid peroxidation, and volatile aldehydes.

Over 1000-Fold Synergistic Boost in Viniferin Levels by Elicitation of Vitis vinifera cv. Gamay Red Cell Cultures over Accumulating Phenylalanine.

Elicitation treatments of grape cell cultures with methyl jasmonate (MeJA), ultraviolet-C (UV-C) irradiation, and sucrose induce mild production of stilbenes and flavonoids due to limited substrate availability. However, these treatments cause a synergistic boost of stilbenes production when applied to two phenylalanine (Phe)-enriched transgenic grape cell lines, AroG* + STS and AroG* + FLS. The combined treatment of UV-C elicitation on the Phe-fed AroG* + STS line resulted in the highest content of stilbenes (37.8-fold increase, 17.39 mg/g dry weight (DW)) mainly due to resveratrol (64-fold, 3.23 mg/g DW) and viniferin (1343-fold, 13.43 mg/g DW). The synergistic increase following either UV-C or MeJA elicitation was due to the induction of stilbene-related genes, while sucrose treatment had no effect on gene expression levels and served as an additional carbon source for phenylpropanoids. The combined strategy presented may enable future usage of grape cell cultures for the production of stilbenes and in particular viniferin.

Preharvest Application of Phenylalanine Induces Red Color in Mango and Apple Fruit's Skin.

Anthocyanins are secondary metabolites responsible for the red coloration of mango and apple. The red color of the peel is essential for the fruit's marketability. Anthocyanins and flavonols are synthesized via the flavonoid pathway initiated from phenylalanine (Phe). Anthocyanins and flavonols have antioxidant, antifungal, and health-promoting properties. To determine if the external treatment of apple and mango trees with Phe can induce the red color of the fruit peel, the orchards were sprayed 1 to 4 weeks before the harvest of mango (cv. Kent, Shelly, and Tommy Atkins) and apple fruit (cv. Cripps pink, Gala and Starking Delicious). Preharvest Phe treatment increased the red coloring intensity and red surface area of both mango and apple fruit that was exposed to sunlight at the orchard. The best application of Phe was 2-4 weeks preharvest at a concentration of 0.12%, while a higher concentration did not have an additive effect. A combination of Phe and the positive control of prohydrojasmon (PDJ) or several applications of Phe did not have a significant added value on the increase in red color. Phe treatment increased total flavonoid, anthocyanin contents, and antioxidant activity in treated fruit compared to control fruits. High Performance Liquid Chromatography analysis of the peel of Phe treated 'Cripps pink' apples showed an increase in total flavonols and anthocyanins with no effect on the compound composition. HPLC analysis of 'Kent' mango fruit peel showed that Phe treatment had almost no effect on total flavonols content while significantly increasing the level of anthocyanins was observed. Thus preharvest application of Phe combined with sunlight exposure offers an eco-friendly, alternative treatment to improve one of the most essential quality traits-fruit color.

A Synchronized Increase of Stilbenes and Flavonoids in Metabolically Engineered Vitis vinifera cv. Gamay Red Cell Culture.

Stilbenes and flavonoids are two major health-promoting phenylpropanoid groups in grapes. Attempts to promote the accumulation of one group usually resulted in a decrease in the other. This study presents a unique strategy for simultaneously increasing metabolites in both groups in V. vinifera cv. Gamay Red grape cell culture, by overexpression of flavonol synthase (FLS) and increasing Phe availability. Increased Phe availability was achieved by transforming the cell culture with a second gene, the feedback-insensitive E. coli DAHP synthase (AroG*), and feeding them with Phe. A combined metabolomic and transcriptomic analysis reveals that the increase in both phenylpropanoid groups is accompanied by an induction of many of the flavonoid biosynthetic genes and no change in the expression levels of stilbene synthase. Furthermore, FLS overexpression with increased Phe availability resulted in higher anthocyanin levels, mainly those derived from delphinidin, due to the induction of F3'5'H. These insights may contribute to the development of grape berries with increased health benefits.

Metabolic Engineering Strategy Enables a Hundred-Fold Increase in Viniferin Levels in Vitis vinifera cv. Gamay Red Cell Culture.

Stilbenes are phytoalexins with health-promoting benefits for humans. Here, we boost stilbenes' production, and in particular the resveratrol dehydrodimer viniferin, with significant pharmacological properties, by overexpressing stilbene synthase (STS) under unlimited phenylalanine (Phe) supply. Vitis vinifera cell cultures were co-transformed with a feedback-insensitive E. coli DAHP synthase (AroG*) and STS genes, under constitutive promoters. All transgenic lines had increased levels of Phe and stilbenes (74-fold higher viniferin reaching 0.74 mg/g DW). External Phe feeding of AroG* + STS lines caused a synergistic effect on resveratrol and viniferin accumulation, achieving a 26-fold (1.33 mg/g DW) increase in resveratrol and a 620-fold increase (6.2 mg/g DW) in viniferin, which to date is the highest viniferin accumulation reported in plant cultures. We suggest that this strategy of combining higher Phe availability and STS expression generates grape cell cultures as potential factories for sustainable production of stilbenes with a minor effect on the levels of flavonoids.

Induced defense response in red mango fruit against Colletotrichum gloeosporioides.

Mango fruit exposed to sunlight develops red skin and are more resistant to biotic and abiotic stresses. Here we show that harvested red mango fruit that was exposed to sunlight at the orchard is more resistant than green fruit to Colletotrichum gloeosporioides. LCMS analysis showed high amounts of antifungal compounds, as glycosylated flavonols, glycosylated anthocyanins, and mangiferin in red vs. green mango skin, correlated with higher antioxidant and lower ROS. However, also the green side of red mango fruit that has low levels of flavonoids was resistant, indicated induced resistance. Transcriptomes of red and green fruit inoculated on their red and green sides with C. gloeosporioides were analyzed. Overall, in red fruit skin, 2,187 genes were upregulated in response to C. gloeosporioides. On the green side of red mango, upregulation of 22 transcription factors and 33 signaling-related transcripts indicated induced resistance. The RNA-Seq analysis suggests that resistance of the whole red fruit involved upregulation of ethylene, brassinosteroid, and phenylpropanoid pathways. To conclude, red fruit resistance to fungal pathogen was related to both flavonoid toxicity and primed resistance of fruit that was exposed to light at the orchard.

Increased substrate availability reveals the potential of scentless lisianthus flowers in producing fragrant benzenoid-phenylpropanoids.

Lisianthus (Eustoma grandiflorum), a leading plant in the cut flower industry, is scentless. Here we show that lisianthus flowers have potential to produce several fragrant benzenoid-phenylpropanoids when substrate availability is not limited. To enable hyperaccumulation of substrates for the production of volatile benzenoid-phenylpropanoids, lisianthus commercial hybrid "Excalibur Pink" was transformed via floral dipping with a feedback-insensitive Escherichia coli DAHP synthase (AroG*) and Clarkia breweri benzyl alcohol acetyltransferase (BEAT), under constitutive promoters. The T1 progeny of "Excalibur Pink" plants segregated into four visual phenotypes, with pink or white colored petals and multiple or single petal layers. Interestingly, transformation with AroG* and BEAT caused no significant effect in the pigment composition among phenotypes, but did increase the levels of down-stream fragrant volatile benzenoids. All the transgenic lines exclusively accumulated methyl benzoate, a fragrant benzenoid, either in their petals or leaves. Furthermore, feeding with benzyl alcohol resulted in the accumulation of two novel benzenoids, benzyl acetate (the product of BEAT) and benzoate, as well as a dramatic increase in the concentrations of additional benzenoid-phenylpropanoid volatiles. Presumably, the degree of benzaldehyde overproduction after benzyl alcohol feeding in both leaves and flowers revealed their reverse conversion in lisianthus plants. These findings demonstrate the concealed capability of lisianthus plants to produce a wide array of fragrant benzenoid-phenylpropanoids, given high substrate concentrations, which could in turn open opportunities for future scent engineering.

Phenylalanine increases chrysanthemum flower immunity against Botrytis cinerea attack.

Flowers are the most vulnerable plant organ to infection by the necrotrophic fungus Botrytis cinerea. Here we show that pre-treatment of chrysanthemum (Chrysanthemum morifolium) flowers with phenylalanine (Phe) significantly reduces their susceptibility to B. cinerea. To comprehend how Phe treatment induces resistance, we monitored the dynamics of metabolites (by GC/LC-MS) and transcriptomes (by RNAseq) in flowers after Phe treatment and B. cinerea infection. Phe treatment resulted in accumulation of 3-phenyllactate and benzaldehyde, and in particular induced the expression of genes related to Ca2+ signaling and receptor kinases, implicating an induction of the defense response. Interestingly, the main effects of Phe treatment were observed in flowers exposed to B. cinerea infection, stabilizing the global fluctuations in the levels of metabolites and transcripts while reducing susceptibility to the fungus. We suggest that Phe-induced resistance is associated to cell priming, enabling rapid and targeted reprogramming of cellular defense responses to resist disease development. After Phe pre-treatment, the levels of the anti-fungal volatiles phenylacetaldehyde and eugenol were maintained and the level of coniferin, a plausible monolignol precursor in cell wall lignification, was strongly increased. In addition, Phe pre-treatment reduced ROS generation, prevented ethylene emission, and caused changes in the expression of a minor number of genes related to cell wall biogenesis, encoding the RLK THESEUS1, or involved in Ca2+ and hormonal signaling processes. Our findings point to Phe pre-treatment as a potential orchestrator of a broad-spectrum defense response which may not only provide an ecologically friendly pest control strategy but also offers a promising way of priming plants to induce defense responses against B. cinerea.

Phenylalanine: A Promising Inducer of Fruit Resistance to Postharvest Pathogens.

More than 40% of harvested fruit is lost, largely due to decay. In parallel, restrictions on postharvest fungicides call for eco-friendly alternatives. Fruit's natural resistance depends mainly on flavonoids and anthocyanins-which have antioxidant and antifungal activity-synthesized from the phenylpropanoid pathway with phenylalanine as a precursor. We hypothesized that phenylalanine could induce fruit's natural defense response and tolerance to fungal pathogens. The postharvest application of phenylalanine to mango and avocado fruit reduced anthracnose and stem-end rot caused by Colletotrichum gloeosporioides and Lasiodiplodia theobromae, respectively. The postharvest application of phenylalanine to citrus fruit reduced green mold caused by Penicillium digitatum. The optimal phenylalanine concentrations for postharvest application were 6 mM for citrus fruits and 8 mM for mangoes and avocadoes. The preharvest application of phenylalanine to strawberries, mangoes, and citrus fruits also reduced postharvest decay. Interestingly, citrus fruit resistance to P. digitatum inoculated immediately after phenylalanine application was not improved, whereas inoculation performed 2 days after phenylalanine treatment induced the defense response. Five hours after the treatment, no phenylalanine residue was detected on/in the fruit, probably due to rapid phenylalanine metabolism. Additionally, in vitro testing showed no inhibitory effect of phenylalanine on conidial germination. Altogether, we characterized a new inducer of the fruit defense response-phenylalanine. Preharvest or postharvest application to fruit led to the inhibition of fungal pathogen-induced postharvest decay, suggesting that the application of phenylalanine could become an eco-friendly and healthy alternative to fungicides.

Glycosylated flavonoids: fruit's concealed antifungal arsenal.

Fruit defense against pathogens relies on induced and preformed mechanisms. The present contribution evaluated performed resistance of red and green mango fruit against the fungal pathogen Colletotrichum gloeosporioides and identified the main active antifungal components. High-performance liquid chromatography analysis of nonhydrolyzed mango peel extracts identified major anthocyanin peaks of glycosylated cyanidin and methylcyanidin, and flavonol peaks of glycosylated quercetin and kaempferol, which were more abundant on the 'red side' of red mango fruit. Organic extracts of red vs green mango peel were more efficient in inhibiting C. gloeosporioides. Transcriptome analysis of the mango-C. gloeosporioides interaction showed increased expression of glucosidase genes related to both fungal pathogenicity and host defense. Glucosidase treatment of organic peel extract increased its antifungal activity. Additionally, quercetin and cyanidin had significantly higher antifungal activity than their glycosylated derivatives. Peel extract volatiles treated with glucosidase had antifungal activity. GCMS analysis identified 15 volatiles after glucosidase hydrolysis, seven of them present only in red fruit. These results suggest that the fruit obtains a concealed arsenal of glycosylated flavonoids in its peel when they are hydrolyzed by ß-glucosidase that is induced in both fungus and host during infection process, become more toxic to the fungal pathogen, inhibiting decay development.

Increased phenylalanine levels in plant leaves reduces susceptibility to Botrytis cinerea.

Botrytis cinerea is a major plant pathogen, causing losses in crops during growth and storage. Here we show that increased accumulation of phenylalanine (Phe) and Phe-derived metabolites in plant leaves significantly reduces their susceptibility to B. cinerea. Arabidopsis, petunia and tomato plants were enriched with Phe by either overexpressing a feedback-insensitive E.coli DAHP synthase (AroG*), or by spraying or drenching detached leaves or whole plants with external Phe, prior to infection with B. cinerea. Metabolic analysis of Arabidopsis and petunia plants overexpressing AroG* as well as wt petunia plants treated externally with Phe, revealed an increase in Phe-derived phenylpropanoids accumulated in their leaves, and specifically in those inhibiting B. cinerea germination and growth, suggesting that different compounds reduce susceptibility to B. cinerea in different plants. Phe itself had no inhibitory effect on germination or growth of B. cinerea, and inhibition of Phe metabolism in petunia plants treated with external Phe prevented decreased susceptibility to the fungus. Thus, Phe metabolism into an array of metabolites, unique to each plant and plant organ, is the most probable cause for increased resistance to Botrytis. This mechanism may provide a basis for ecologically friendly control of a wide range of plant pathogens.

Ethylene Induces a Rapid Degradation of Petal Anthocyanins in Cut Vanda 'Sansai Blue' Orchid Flowers.

Ethylene plays a major role in the regulation of flower senescence, including in the ethylene-sensitive Vanda 'Sansai Blue' orchid flowers. This cut flower is popular in Thailand due to its light blue big size florets possessing a beautiful shape pattern. In the present study, we further examined the rapid ethylene-induced process of active anthocyanin degradation in cut Vanda 'Sansai Blue' flowers, which occurred much before detection of other typical senescence-related symptoms. For this purpose, the cut inflorescences were exposed to air (control), 1 or 10 µl L-1 ethylene for 24 h, or to 0.2 µl L-1 1-methylcyclopropene (1-MCP) for 6 h followed by 10 µl L-1 ethylene for 24 h at 21°C, and the effects of these treatments on various parameters were assayed. While the fading-induced effect of ethylene was not concentration-dependent in this range, the ethylene treatment significantly reduced the flower vase life in a concentration-dependent manner, further confirming the separation of the bleaching process from senescence. Exposure of the inflorescences to 1-MCP pre-treatment followed by 10 µl L-1 ethylene, recovered both inflorescence color and anthocyanin content to control levels. Quantification of total anthocyanin content, performed by HPLC analysis on the basis of cyanidin-3-glocuside equivalents, showed that ethylene reduced and 1-MCP recovered the anthocyanins profile in non-hydrolyzed anthocyanin samples of Vanda 'Sansai Blue' florets, assayed at half bloom and bloom developmental stages. The results showed that the ethylene-induced color fading, observed immediately after treatment, resulted from a significant reduction in the levels of the two main anthocyanidins, cyanidin and delphinidin, as well as of other anthocyanidins present in low abundance, but not from changes in the levels of flavonols, such as kaempferol. This anthocyanin degradation process seems to operate via ethylene-increased peroxidase activity, detected at the bud stage. Taken together, our results suggest that the ethylene-induced rapid color bleaching in petals of cut Vanda 'Sansai Blue' flowers is an outcome of in-planta anthocyanin degradation, partially mediated by increased peroxidase activity, and proceeds independently of the flower senescence process.

Nutritional value of potato (Solanum tuberosum) in hot climates: anthocyanins, carotenoids, and steroidal glycoalkaloids.

MAIN CONCLUSION: Growth in hot climates selectively alters potato tuber secondary metabolism-such as the anthocyanins, carotenoids, and glycoalkaloids-changing its nutritive value and the composition of health-promoting components. Potato breeding for improved nutritional value focuses mainly on increasing the health-promoting carotenoids and anthocyanins, and controlling toxic steroidal glycoalkaloids (SGAs). Metabolite levels are genetically determined, but developmental, tissue-specific, and environmental cues affect their final content. Transcriptomic and metabolomic approaches were applied to monitor carotenoid, anthocyanin, and SGA metabolite levels and their biosynthetic genes' expression under heat stress. The studied cultivars differed in tuber flesh carotenoid concentration and peel anthocyanin concentration. Gene expression studies showed heat-induced downregulation of specific genes for SGA, anthocyanin, and carotenoid biosynthesis. KEGG database mapping of the heat transcriptome indicated reduced gene expression for specific metabolic pathways rather than a global heat response. Targeted metabolomics indicated reduced SGA concentration, but anthocyanin pigments concentration remained unchanged, probably due to their stabilization in the vacuole. Total carotenoid level did not change significantly in potato tuber flesh, but their composition did. Results suggest that growth in hot climates selectively alters tuber secondary metabolism, changing its nutritive value and composition of health-promoting components.

Successful floral-dipping transformation of post-anthesis lisianthus (Eustoma grandiflorum) flowers.

The adaptation of the Agrobacterium-mediated floral-dipping technique is limited, to date, to a small number of plants. In this paper, we present the efficient transformation of one of the leading plants in the cut flower industry, lisianthus (Eustoma grandiflorum). This method is approximately 18 months shorter than the known tissue culture-based transformation. The Excalibur Pink cultivar and two additional breeding lines, X-1042 and X-2541, were transformed using three different marker genes (benzyl alcohol acetyltransferase (BEAT) originating from Clarkia breweri, the feedback-insensitive bacterial gene AroG*, and the empty pART27 vector expressing a kanamycin-resistance cassette (nptII)). Genomic transformation was successful in all tested cases with transformation efficiency ranked from 0.2 to 2.9%, which is well in the range of results from Arabidopsis studies. Unlike Arabidopsis, in which floral-dipping transformation was efficient only at a pre-anthesis stage before ovary sealing, lisianthus flowers were transformed when dipping occurred 4 days pre-anthesis or 3-5 days post-anthesis with 1.5 and 3.7% efficiencies, respectively. Post-anthesis transformation occurred when the flower ovaries were sealed. Flower dipping of Excalibur Pink flowers with fluorescent Agrobacterium containing a GFP marker gene demonstrated Agrobacterium entrance into the sealed flower ovary through the open stigma and style tube. In this study, we demonstrated floral-dipping transformation of a commercial plant, lisianthus Excalibur Pink, occurring after sealing of the ovaries, probably via the stigma and wide open style tunnel.

Phenylpyruvate Contributes to the Synthesis of Fragrant Benzenoid-Phenylpropanoids in Petunia × hybrida Flowers.

Phenylalanine (Phe) is a precursor for a large group of plant specialized metabolites, including the fragrant volatile benzenoid-phenylpropanoids (BPs). In plants, the main pathway leading to production of Phe is via arogenate, while the pathway via phenylpyruvate (PPY) is considered merely an alternative route. Unlike plants, in most microorganisms the only pathway leading to the synthesis of Phe is via PPY. Here we studied the effect of increased PPY production in petunia on the formation of BPs volatiles and other specialized metabolites originating from Phe both in flowers and leaves. Stimulation of the pathway via PPY was achieved by transforming petunia with PheA∗ , a gene encoding a bacterial feedback insensitive bi-functional chorismate mutase/prephenate dehydratase enzyme. PheA∗ overexpression caused dramatic increase in the levels of flower BP volatiles such as phenylacetaldehyde, benzaldehyde, benzyl acetate, vanillin, and eugenol. All three BP pathways characterized in petunia flowers were stimulated in PheA∗ flowers. In contrast, PheA∗ overexpression had only a minor effect on the levels of amino acids and non-volatile metabolites both in the leaves and flowers. The one exception is a dramatic increase in the level of rosmarinate, a conjugate between Phe-derived caffeate and Tyr-derived 3,4-dihydroxyphenylacetate, in PheA∗ leaves. PheA∗ petunia flowers may serve as an excellent system for revealing the role of PPY in the production of BPs, including possible routes directly converting PPY to the fragrant volatiles. This study emphasizes the potential of the PPY route in achieving fragrance enhancement in flowering plants.

An Intracellular Laccase Is Responsible for Epicatechin-Mediated Anthocyanin Degradation in Litchi Fruit Pericarp.

In contrast to the detailed molecular knowledge available on anthocyanin synthesis, little is known about its catabolism in plants. Litchi (Litchi chinensis) fruit lose their attractive red color soon after harvest. The mechanism leading to quick degradation of anthocyanins in the pericarp is not well understood. An anthocyanin degradation enzyme (ADE) was purified to homogeneity by sequential column chromatography, using partially purified anthocyanins from litchi pericarp as a substrate. The purified ADE, of 116 kD by urea SDS-PAGE, was identified as a laccase (ADE/LAC). The full-length complementary DNA encoding ADE/LAC was obtained, and a polyclonal antibody raised against a deduced peptide of the gene recognized the ADE protein. The anthocyanin degradation function of the gene was confirmed by its transient expression in tobacco (Nicotiana benthamiana) leaves. The highest ADE/LAC transcript abundance was in the pericarp in comparison with other tissues, and was about 1,000-fold higher than the polyphenol oxidase gene in the pericarp. Epicatechin was found to be the favorable substrate for the ADE/LAC. The dependence of anthocyanin degradation by the enzyme on the presence of epicatechin suggests an ADE/LAC epicatechin-coupled oxidation model. This model was supported by a dramatic decrease in epicatechin content in the pericarp parallel to anthocyanin degradation. Immunogold labeling transmission electron microscopy suggested that ADE/LAC is located mainly in the vacuole, with essential phenolic substances. ADE/LAC vacuolar localization, high expression levels in the pericarp, and high epicatechin-dependent anthocyanin degradation support its central role in pigment breakdown during pericarp browning.

Phenylalanine and tyrosine levels are rate-limiting factors in production of health promoting metabolites in Vitis vinifera cv. Gamay Red cell suspension.

Environmental stresses such as high light intensity and temperature cause induction of the shikimate pathway, aromatic amino acids (AAA) pathways, and of pathways downstream from AAAs. The induction leads to production of specialized metabolites that protect the cells from oxidative damage. The regulation of the diverse AAA derived pathways is still not well understood. To gain insight on that regulation, we increased AAA production in red grape Vitis vinifera cv. Gamay Red cell suspension, without inducing external stress on the cells, and characterized the metabolic effect of this induction. Increased AAA production was achieved by expressing a feedback-insensitive bacterial form of 3-deoxy- D-arabino-heptulosonate 7-phosphate synthase enzyme (AroG (*)) of the shikimate pathway under a constitutive promoter. The presence of AroG(*) protein led to elevated levels of primary metabolites in the shikimate and AAA pathways including phenylalanine and tyrosine, and to a dramatic increase in phenylpropanoids. The AroG (*) transformed lines accumulated up to 20 and 150 fold higher levels of resveratrol and dihydroquercetin, respectively. Quercetin, formed from dihydroquercetin, and resveratrol, are health promoting metabolites that are induced due to environmental stresses. Testing the expression level of key genes along the stilbenoids, benzenoids, and phenylpropanoid pathways showed that transcription was not affected by AroG (*). This suggests that concentrations of AAAs, and of phenylalanine in particular, are rate-limiting in production of these metabolites. In contrast, increased phenylalanine production did not lead to elevated concentrations of anthocyanins, even though they are also phenylpropanoid metabolites. This suggests a control mechanism of this pathway that is independent of AAA concentration. Interestingly, total anthocyanin concentrations were slightly lower in AroG(*) cells, and the relative frequencies of the different anthocyanins changed as well.

Metabolic Engineering of the Phenylpropanoid and Its Primary, Precursor Pathway to Enhance the Flavor of Fruits and the Aroma of Flowers.

Plants produce a diverse repertoire of specialized metabolites that have multiple roles throughout their life cycle. Some of these metabolites are essential components of the aroma and flavor of flowers and fruits. Unfortunately, attempts to increase the yield and prolong the shelf life of crops have generally been associated with reduced levels of volatile specialized metabolites and hence with decreased aroma and flavor. Thus, there is a need for the development of new varieties that will retain their desired traits while gaining enhanced scent and flavor. Metabolic engineering holds great promise as a tool for improving the profile of emitted volatiles of domesticated crops. This mini review discusses recent attempts to utilize metabolic engineering of the phenylpropanoid and its primary precursor pathway to enhance the aroma and flavor of flowers and fruits.