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.

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.

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.

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.

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.

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.

Enhanced formation of aromatic amino acids increases fragrance without affecting flower longevity or pigmentation in Petunia × hybrida.

Purple Petunia × hybrida V26 plants accumulate fragrant benzenoid-phenylpropanoid molecules and anthocyanin pigments in their petals. These specialized metabolites are synthesized mainly from the aromatic amino acids phenylalanine. Here, we studied the profile of secondary metabolites of petunia plants, expressing a feedback-insensitive bacterial form of 3-deoxy-di-arabino-heptulosonate 7-phosphate synthase enzyme (AroG*) of the shikimate pathway, as a tool to stimulate the conversion of primary to secondary metabolism via the aromatic amino acids. We focused on specialized metabolites contributing to flower showy traits. The presence of AroG* protein led to increased aromatic amino acid levels in the leaves and high phenylalanine levels in the petals. In addition, the AroG* petals accumulated significantly higher levels of fragrant benzenoid-phenylpropanoid volatiles, without affecting the flowers' lifetime. In contrast, AroG* abundance had no effect on flavonoids and anthocyanins levels. The metabolic profile of all five AroG* lines was comparable, even though two lines produced the transgene in the leaves, but not in the petals. This implies that phenylalanine produced in leaves can be transported through the stem to the flowers and serve as a precursor for formation of fragrant metabolites. Dipping cut petunia stems in labelled phenylalanine solution resulted in production of labelled fragrant volatiles in the flowers. This study emphasizes further the potential of this metabolic engineering approach to stimulate the production of specialized metabolites and enhance the quality of various plant organs. Furthermore, transformation of vegetative tissues with AroG* is sufficient for induced production of specialized metabolites in organs such as the flowers.

In planta anthocyanin degradation by a vacuolar class III peroxidase in Brunfelsia calycina flowers.

In contrast to detailed knowledge regarding the biosynthesis of anthocyanins, the largest group of plant pigments, little is known about their in planta degradation. It has been suggested that anthocyanin degradation is enzymatically controlled and induced when beneficial to the plant. Here we investigated the enzymatic process in Brunfelsia calycina flowers, as they changed color from purple to white. We characterized the enzymatic process by which B. calycina protein extracts degrade anthocyanins. A candidate peroxidase was partially purified and characterized and its intracellular localization was determined. The transcript sequence of this peroxidase was fully identified. A basic peroxidase, BcPrx01, is responsible for the in planta degradation of anthocyanins in B. calycina flowers. BcPrx01 has the ability to degrade complex anthocyanins, it co-localizes with these pigments in the vacuoles of petals, and both the mRNA and protein levels of BcPrx01 are greatly induced parallel to the degradation of anthocyanins. Both isoelectric focusing (IEF) gel analysis and 3D structure prediction indicated that BcPrx01 is cationic. Identification of BcPrx01 is a significant breakthrough both in the understanding of anthocyanin catabolism in plants and in the field of peroxidases, where such a consistent relationship between expression levels, in planta subcellular localization and activity has seldom been demonstrated.

ANTHOCYANIN1 from Solanum chilense is more efficient in accumulating anthocyanin metabolites than its Solanum lycopersicum counterpart in association with the ANTHOCYANIN FRUIT phenotype of tomato.

Anthocyanins are flavonoid metabolites contributing attractive colors and antioxidant qualities to the human diet. Accordingly, there is a growing interest in developing crops enriched with these compounds. Fruits of the cultivated tomato, Solanum (S.) lycopersicum, do not normally produce high levels of anthocyanins. However, several wild tomato species yield anthocyanin-pigmented fruits, and this trait has been introgressed into the cultivated tomato. Two genes encoding homologous R2R3 MYB transcription factors, termed ANT1 and AN2, were previously genetically implicated in anthocyanin accumulation in tomato fruit peels of the ANTHOCYANIN FRUIT (AFT) genotype originating from S. chilense. Here we compared transgenic tomato plants constitutively over-expressing the S. lycopersicum (35S::ANT1 ( L ) ) or the S. chilense (35S::ANT1 ( C )) allele, and show that each displayed variable levels of purple pigmentation in vegetative as well as reproductive tissues. However, 35S::ANT1 ( C ) was significantly more efficient in producing anthocyanin pigments, attributed to its gene coding-sequence rather than to its transcript levels. These results expand the potential of enhancing anthocyanin levels through engineering coding-sequence polymorphisms in addition to the transcriptional alterations commonly used. In addition, a segregating population obtained from a recombinant genotype revealed that the native ANT1, and not AN2, is fully associated with the AFT phenotype and that ANT1 alone can generate the characteristic phenotype of anthocyanin accumulation in AFT fruits. Our results therefore provide further support to the hypothesis that ANT1 is the gene responsible for anthocyanin accumulation in fruits of the AFT genotype.

Increased accumulation and decreased catabolism of anthocyanins in red grape cell suspension culture following magnesium treatment.

Anthocyanins are the largest and best studied group of plant pigments. However, not very much is known about the fate of these phenolic pigments after they have accumulated in the cell vacuoles of plant tissues. We have previously shown that magnesium treatment of ornamentals during the synthesis of anthocyanins in the flowers or foliage caused an increase in the pigment concentration. In this study, we characterized the effect of magnesium on the accumulation of anthocyanin in red cell suspension originating from Vitis vinifera cv. Gamay Red grapes. Magnesium treatment of the cells caused a 2.5- to 4.5-fold increase in anthocyanin concentration, with no substantial induction of the biosynthetic genes. This treatment inhibited the degradation of anthocyanins occurring in the cells, and changed the ratio between different anthocyanins determining cell color, with an increase in the relative concentration of the less stable pigment molecules. The process by which magnesium treatment affects anthocyanin accumulation is still not clear. However, the results presented suggest at least part of its effect on anthocyanin accumulation stems from inhibition of the pigments' catabolism. When anthocyanin biosynthesis was inhibited, magnesium treatments prevented the constant degradation of anthocyanins in the cell suspension. Future understanding of the catabolic processes undergone by anthocyanins in plants may enable more efficient inhibition of this process and increased accumulation of these pigments, and possibly of additional phenolic compounds.

Metabolic networking in Brunfelsia calycina petals after flower opening.

Brunfelsia calycina flowers change colour from purple to white due to anthocyanin degradation, parallel to an increase in fragrance and petal size. Here it was tested whether the production of the fragrant benzenoids is dependent on induction of the shikimate pathway, or if they are formed from the anthocyanin degradation products. An extensive characterization of the events taking place in Brunfelsia flowers is presented. Anthocyanin characterization was performed using ultraperfomance liquid chromatography-quadrupole time of flight-tandem mass specrometry (UPLC-QTOF-MS/MS). Volatiles emitted were identified by headspace solid phase microextraction-gas chromatography-mass spectrometry (HS-SPME-GC-MS). Accumulated proteins were identified by 2D gel electrophoresis. Transcription profiles were characterized by cross-species hybridization of Brunfelsia cDNAs to potato cDNA microarrays. Identification of accumulated metabolites was performed by UPLC-QTOF-MS non-targeted metabolite analysis. The results include characterization of the nine main anthocyanins in Brunfelsia flowers. In addition, 146 up-regulated genes, 19 volatiles, seven proteins, and 17 metabolites that increased during anthocyanin degradation were identified. A multilevel analysis suggests induction of the shikimate pathway. This pathway is the most probable source of the phenolic acids, which in turn are precursors of both the benzenoid and lignin production pathways. The knowledge obtained is valuable for future studies on degradation of anthocyanins, formation of volatiles, and the network of secondary metabolism in Brunfelsia and related species.

Characterization of the intense pigment tomato genotype emphasizing targeted fruit metabolites and chloroplast biogenesis.

The tomato INTENSE PIGMENT (IP) genotype is characterized by intense visual pigmentation of unripe and ripe fruits, not thoroughly analyzed thus far. This study was therefore designed to analyze key morphologic, metabolomic, and photomorphogenic phenotypes of this genotype in comparison to its near-isogenic normal counterpart and to evaluate its significance relative to other tomato mutants known for increased fruit pigmentation. The IP genotype produced smaller and darker red fruits, and a substantially increased chloroplast biogenesis was found in its green fruit and leaf tissues. Ripe-red fruits of the IP genotype produced 34-38% more soluble solids and up to 62.6% more carotenoids, but no differences were found in the concentration of flavonoid compounds in its peel tissue. The IP genotype was found to display a greater hypocotyl inhibition response to blue and yellow light, but a more prominent and novel response to total darkness. As a whole, the IP genotype exhibited highly desirable traits, making it a valuable genotype for tomato breeders attempting to introduce functional and taste qualities into tomato fruits.

Molecular aspects of Anthocyanin fruit tomato in relation to high pigment-1.

The tomato Anthocyanin fruit (Aft) genotype is characterized by purple color in skin and outer pericarp of its fruits due to higher levels of anthocyanins-flavonoid metabolites. Our objectives were to carry out metabolic and molecular characterization of this genotype, emphasizing its interaction with the high pigment-1 (hp-1) mutation, known to increase flavonoids in tomato fruits. These objectives fit the growing interest in developing tomato fruits with higher levels of functional metabolites. Our results show that 1) Aft fruits are also characterized by significantly higher levels of the flavonols quercetin and kaempferol, thus enhancing their functional value; 2) the tomato Anthocyanin1 (Ant1) gene, encoding a Myb transcription factor, displayed nucleotide and amino acid polymorphisms between the Aft genotype and cultivated genotypes; 3) a DNA marker based on Ant1 showed that the Aft trait is encoded by a single locus on chromosome 10 fully associated with Ant1; and 4) double homozygotes Aft/Aft hp-1/hp-1 plants displayed a more-than-additive effect on the production of fruit anthocyanidins and flavonols. This effect was manifested by approximately 5-, 19-, and 33-fold increase of petunidin, malvidin, and delphinidin, respectively, in the double mutants compared with the cumulative levels of their parental lines.

Active anthocyanin degradation in Brunfelsia calycina (yesterday--today--tomorrow) flowers.

Anthocyanins are the largest group of plant pigments responsible for colors ranging from red to violet and blue. The biosynthesis of anthocyanins, as part of the larger phenylpropanoid pathway, has been characterized in great detail. In contrast to the detailed molecular knowledge available on anthocyanin synthesis, very little is known about the stability and catabolism of anthocyanins in plants. In this study we present a preliminary characterization of active in planta degradation of anthocyanins, requiring novel mRNA and protein synthesis, in Brunfelsia calycina flowers. Brunfelsia is a unique system for this study, since the decrease in pigment concentration in its flowers (from dark purple to white) is extreme and rapid, and occurs at a specific and well-defined stage of flower development. Treatment of detached flowers with protein and mRNA synthesis inhibitors, at specific stages of flower development, prevented degradation. In addition, treatment of detached flowers with cytokinins delayed senescence without changing the rate of anthocyanin degradation, suggesting that degradation of anthocyanins is not part of the general senescence process of the flowers but rather a distinctive and specific pathway. Based on studies on anthocyanin degradation in wine and juices, peroxidases are reasonable candidates for the in vivo degradation. A significant increase in peroxidase activity was shown to correlate in time with the rate of anthocyanin degradation. An additional indication that oxidative enzymes are involved in the process is the fact that treatment of flowers with reducing agents, such as DTT and glutathione, caused inhibition of degradation. This study represents the first step in the elucidation of the molecular mechanism behind in vivo anthocyanin degradation in plants.

The A locus that controls anthocyanin accumulation in pepper encodes a MYB transcription factor homologous to Anthocyanin2 of Petunia.

Pepper plants containing the dominant A gene accumulate anthocyanin pigments in the foliage, flower and immature fruit. We previously mapped A to pepper chromosome 10 in the F(2) progeny of a cross between 5226 (purple-fruited) and PI 159234 (green-fruited) to a region that corresponds, in tomato, to the location of Petunia anthocyanin 2 ( An2), a regulator of anthocyanin biosynthesis. This suggested that A encodes a homologue of Petunia An2. Using the sequences of An2 and a corresponding tomato expressed sequence tag, we isolated a pepper cDNA orthologous to An2 that cosegregated with A. We subsequently determined the expression of A by Northern analysis, using RNA extracted from fruits, flowers and leaves of 5226 and PI 159234. In 5226, expression was detected in all stages of fruit development and in both flower and leaf. In contrast, A was not expressed in the sampled tissues in PI 159234. Genomic sequence comparison of A between green- and purple-fruited genotypes revealed no differences in the coding region, indicating that the lack of expression of A in the green genotypes can be attributed to variation in the promoter region. By analyzing the expression of the structural genes in the anthocyanin biosynthetic pathway in 5226 and PI 159234, it was determined that, similar to Petunia, the early genes in the pathway are regulated independently of A, while expression of the late genes is A-dependent.