flavonoid compounds in higher plants ( Bueno et al., 2012 ; Tripathi et al., 2018 ). Their biosynthesis is catalyzed by chalcone synthase (CHS), chalcone isomerase (CHI), flavanone 3-hydroxylase (F3H), flavonoid 3'-hydroxylase (F3'H), dihydroflavonol 4
Ying Fang, Ting Lei, Yanmei Wu, and Xuehua Jin
Ki-Ho Son, Jin-Hui Lee, Youngjae Oh, Daeil Kim, Myung-Min Oh, and Byung-Chun In
treatments were collected every week and frozen with liquid nitrogen. Differences in the transcript levels of phenylalanine ammonia-lyase ( PAL ) and chalcone synthase ( CHS ), which are the key genes for the biosynthesis of phenolic compounds and flavonoids
Tomomi Tsuda, Masami Yamaguchi, Chikako Honda, and Takaya Moriguchi
We used RNA blot analysis to examine the expression of six genes of the anthocyanin biosynthesis pathway in the flowers and fruit skins at three developmental stages of white and red peaches and a deep-red nectarine [Prunus persica (L.) Batch]. In the red peach `Akatsuki' and the deep-red nectarine `Flavortop', expression levels of anthocyanin biosynthesis genes were related to anthocyanin accumulation in the fruit skin; expression of all six genes dramatically increased at Stage III of fruit development, and anthocyanin concentration also increased at this stage. In the white peach `Mochizuki', however, expression of the chalcone synthase gene (CHS) and the dihydroflavonol 4-reductase gene (DFR) was undetectable in Stage III, although the chalcone isomerase gene (CHI), the flavanone 3-hydroxylase gene (F3H), the anthocyanidin synthase gene (ANS), and the UDP-glucose-flavonoid 3-O-glucosyltransferase gene (UFGT) were expressed. We occasionally found red pigment in the skin of `Mochizuki' peach. In these red skin areas, both CHS and DFR were clearly expressed in Stage III. These results suggest that CHS and DFR are the key regulatory genes in the process of anthocyanin biosynthesis in mature red peach and nectarine.
John R. Stommel* and Robert J. Griesbach
Anthocyanins contribute to color development in economically important vegetables, fruits and floral crops. Their expression is critical to product sensory quality attributes, potential nutritive value, and stress response. Anthocyanins are synthesized in response to numerous environmental factors including temperature and light stress and pathogen attack. We have developed several Capsicum lines, including `02C27', expressing anthocyanin pigmentation differentially in various tissues (leaf, stem, fruit and flower). HPLC analysis demonstrated that the anthocyanins within the fruit, flower and leaves of Capsicum `02C27' were identical and that the major anthocyanidin was a delphinidin glycoside. Line `02C27' exhibits anthocyanin foliar pigmentation that is accumulated differentially in response to temperature stress. Under unfavorable low temperature (20 °C day/18 °C night), mature Capsicum leaves contained 4.6 times less anthocyanin per gram fresh weight than under high (30 °C day/28 °C; day/night) temperatures. Besides containing less anthocyanin in mature leaves, young immature leaves did not develop color as quickly under the lower temperature. Utilizing cloned and sequenced gene fragments of pepper chalcone synthase (CHS), dihydroflavonol 4-reductase (DFR), and anthocyanidin synthase (ANS), we evaluated the role of transcription in regulation of flavonol biosynthesis. Analysis of anthocyanin composition and gene expression data indicated that the block in anthocyanin formation in less pigmented leaves occurred at anthocyanin synthase. In contrast to wild tupe plants, this mutant also exhibited reduced flowering and failed to set fruit under high temperature, long day conditions.
Sunggil Kim*, Marla Binzel, Sunghun Park, Kil-Sun Yoo, and Leonard Pike
Anthocyanin, one of the flavonoids, is a primary determinant of red color in onions. Inheritance studies indicate that a single gene determines the color difference between yellow and red onions. In order to establish which gene might be responsible for this color difference, full-length cDNAs of five structural genes: chalcone synthase (CHS), flavanone 3-hydroxylase (F3H), dihydroflavonol 4-reductase (DFR), anthocyanidin synthase (ANS), and flavonol synthase (FLS) were cloned using degenerate PCR and RACE (Rapid Amplification of cDNA Ends). RT-PCR was carried out for these five genes to examine differential expression between yellow and red colored bulbs. Accumulation of the DFR gene transcript only occurred in red onions. In F3 populations which originated from the cross between yellow and red parents, DFR transcript was detected only in red F3 lines, not in yellow F3 lines. To design molecular markers for selection of yellow and red DFR alleles, the DFR gene was sequenced from genomic DNA isolated from both types of onions. The genomic DNA sequence revealed the DFR gene consists of six exons and five introns. A PCR-RFLP marker was designed based on 2% polymorphic nucleotide sequence of the DFR gene between yellow and red onions. The co-segregation of markers and red color were observed in F2 segregating populations, supporting the conclusion that color difference in red and yellow onions is likely to be due to the lack of an active DFR gene.
The biosynthetic pathway for anthocyanins has been studied using genetic, biochemical and molecular biological tools. In the past decade, the core pathway genes have been cloned; a number of genes which act to modify anthocyanin structure have been cloned more recently. The first results in color modification have been reduced flower color intensity using gene suppression methods. In particular, we have utilized chalcone synthase (CHS) and dihydroflavonol reductase (DFR) genes and sense suppression in our experimental system, Petunia hybrida, and in the commercial crops, chrysan-themum (Dendranthema morifolium) and rose (Rosa hybrida). In petunia a range of new phenotypes was obtained; genetic stability of suppressed pheno-types will be described. In chrysanthemum a white-flowering derivative of a pink-flowering variety will be described. In rose uniform, partial reduction in pigment intensity throughout the flower was observed in over a dozen trans-genie derivatives of a red-flowering variety.
Zhiguo Ju, Chenglian Liu, Yongbing Yuan, Yongzhang Wang, and Gongshi Liu
Crosses between red cultivars produced high frequency of less-colored progeny, while hybridization between non-red cultivars yielded some red-fruited F1 trees. When harvest was delayed and light intensity increased, both green and yellow cultivars accumulated some anthocyanin with higher UDPGal:flavonoid-3-o-glycosyltransferase (UFGalT) activity in colored areas. Overall, anthocyanin accumulation and UFGalT activity were highly correlated (r = 0.8921, P = 0.0001) in fruit from both parental trees and their F1 progeny, but UFGalT activity always was relatively high in fruit peel, whether anthocyanin accumulated or not. There were no significant differences in phenylalanine ammonia-lyase or chalcone synthase activities among the cultivars, and they did not change much after hybridization.
Paola S. Cotroneo, Maria P. Russo, Manuela Ciuni, Giuseppe Reforgiato Recupero, and Angela R. Lo Piero
Genes encoding chalcone synthase (CHS), anthocyanidin synthase (ANS), and UDP-glucose-flavonoid 3-O-glucosyltransferase (UFGT), some of the enzymes of anthocyanin biosynthetic pathway, were assayed in two different experiments using quantitative real-time reverse transcriptase (RT)-PCR, in order to test their transcription levels in the flesh of blood and common orange [Citrus sinensis (L.) Osbeck] fruit, and to investigate their role in anthocyanin accumulation in the same tissue. The first experiment compared a blood orange and a common orange cultivar during seven different fruit maturation stages. This was followed by the test of 11 different genotypes at the end of the winter season. Data collected from the first experiment, over the blood orange cultivar, were statistically analyzed using the Pearson correlation coefficient. Results show that CHS, ANS, and UFGT mRNA transcripts are up- and co-regulated in the blood orange cultivar, whereas they are down-regulated in the common orange cultivar. There is evidence of correspondence between the target genes expression level and the content of the pigment assessed. The second test confirms this correlation and shows that enzyme synthesis levels and pigment accumulation, in plants grown under the same environmental conditions, are dependent on the differences occurring among the genotypes tested. These results suggest that the absence of pigment in the common orange cultivars may be caused by the lack of induction on the structural genes expression. This is the first report on the characterization of the relationships between biosynthetic genes expression and fruit flesh anthocyanin content in blood oranges.
Satoru Kondo, Kentaro Hiraoka, Shozo Kobayashi, Chikako Honda, and Norihiko Terahara
Cyanidin 3-galactoside was the primary anthocyanin in red `Tsugaru' apples [Malus sylvestris (L.) Mill. var. domestica (Borkh.) Mansf.]. The concentration of cyanidin 3-galactoside in the skin decreased from 20 to 62 days after full bloom (DAFB), then increased rapidly after 104 DAFB. Small amounts of cyanidin 3-arabinoside and cyanidin 3-glucoside were detected at 122 and 133 DAFB (harvest). The expression of five anthocyanin biosynthetic genes of chalcone synthase (MdCHS), flavanone 3-hydroxylase (MdF3H), dihydroflavonol 4-reductase (pDFR), anthocyanidin synthase (MdANS), and UDP glucose-flavonoid 3-O-glucosyltransferase (pUFGluT) was examined in the skin of red and nonred apples. In general, the expression of anthocyanin biosynthetic genes in red apples was strong in juvenile and ripening stages. The expression of MdCHS, MdF3H, pDFR, and MdANS was observed before ripening stage when anthocyanin was not detected. In contrast, the expression of pUFGluT was detected in the development stage only when anthocyanin was detected. However, the expression of all five genes was observed at 20 DAFB in fruit bagged after fertilization, and anthocyanin was not detected. The expression of MdCHS, MdF3H, pDFR, and MdANS, excluding pUFGluT, was detected at 98 DAFB in fruit bagged after 30 DAFB, and anthocyanin was not detected. These results suggest that pUFGluT may be closely related to the anthocyanin expression in apple skin at the ripening stage.
Clifford W. Beninger, George L. Hosfield, Mark J. Bassett, and Shirley Owens
Three common bean (Phaseolus vulgaris L.) seedcoat color (or glossiness) genotypes, differing from each other by a single substitution at a seedcoat locus, were analyzed for presence and concentration of three anthocyanins: delphinidin 3-O-glucoside, petunidin 3-O-glucoside, and malvidin 3-O-glucoside. The three anthocyanins were present in Florida common bean breeding line 5-593 (P C J G B V Asp), matte black (P C J G B V asp), and dark brown violet (P C J G b V Asp), but the amounts varied greatly depending on the genotype. Dark brown violet had 19% of the total anthocyanin content when compared to 5-593, whereas matte black had amounts intermediate between the two other genotypes. The B gene acts to regulate the production of precursors of anthocyanins in the seedcoat color pathway above the level of dihydrokaempferol formation, perhaps at the chalcone synthase or chalcone isomerase steps in the biosynthetic pathway. We hypothesize that B regulates simultaneously the flavonoid (color) and isoflavonoid (resistance) pathways. The I gene for resistance to bean common mosaic virus (BCMV) is known to be linked closely to B. It is therefore hypothesized that the I gene function may be to respond to BCMV infection by dramatically increasing (over a low constituitive level) production in the 5-dehydroxy isoflavonoid pathway, which leads to synthesis of the major phytoalexin, phaseollin, for resistance to BCMV. Alternatively, the B and I genes may be allelic. The Asp gene affects seedcoat glossiness by means of a structural change to the seedcoat. We demonstrate that Asp in the recessive condition (asp/asp) changes the size and shape of the palisade cells of the seedcoat epidermis, making them significantly smaller than either 5-593 or dark brown violet. Asp, therefore, limits the amounts of anthocyanins in the seedcoat by reducing the size of palisade cells.