Tsukasa Iwashina

The Structure and Distribution of the Flavonoids in Plants

6-C3-C6 skeleton, have been found in plants, and are divided into several classes, i.e., anthocyanins, flavones, flavonols, flavanones, dihydroflavonols, chalcones, aurones, flavan and proanthocyanidins, isoflavonoids, biflavonoids, etc. In this review, the chemical structures of the reported flavonoid classes are introduced and their distribution in nature are described. Additionally, some recent chemotaxonomical examples using the flavonoids are also given.

A single-base deletion in soybean flavonol synthase gene is associated with magenta flower color

The Wm locus of soybean [Glycine max (L.) Merr.] controls flower color. Dominant Wm and recessive wm allele of the locus produce purple and magenta flower, respectively. A putative full-length cDNA of flavonol synthase (FLS), gmfls1 was isolated by 5′ RACE and end-to-end PCR from a cultivar Harosoy with purple flower (WmWm). Sequence analysis revealed that gmfls1 consisted of 1,208 nucleotides encoding 334 amino acids. It had 59–72% homology with FLS proteins of other plant species. Conserved dioxygenase domains A and B were found in the deduced polypeptide. Sequence comparison between Harosoy and Harosoy-wm (magenta flower mutant of Harosoy; wmwm) revealed that they differed by a single G deletion in the coding region of Harosoy-wm. The deletion changed the subsequent reading frame resulting in a truncated polypeptide consisting of 37 amino acids that lacked the dioxygenase domains A and B. Extracts of E. coli cells expressing gmfls1 of Harosoy catalyzed the formation of quercetin from dihydroquercetin, whereas cell extracts expressing gmfls1 of Harosoy-wm had no FLS activity. Genomic Southern analysis suggested the existence of three to four copies of the FLS gene in the soybean genome. CAPS analysis was performed to detect the single-base deletion. Harosoy and Clark (WmWm) exhibited longer fragments, while Harosoy-wm had shorter fragments due to the single-base deletion. The CAPS marker co-segregated with genotypes at Wm locus in a F2 population segregating for the locus. Linkage mapping using SSR markers revealed that the Wm and gmfls1 were mapped at similar position in the molecular linkage group F. The above results strongly suggest that gmfls1 represents the Wm gene and that the single-base deletion may be responsible for magenta flower color.

Anthocyanin-flavone copigmentation in bluish purple flowers of Japanese garden iris (Iris ensata Thunb.)

The in vitro identification of copigmentation was carried out using anthocyanins, such as malvidin 3RGac5G, petunidin 3RGac5G and delphinidin 3RGac5G and the flavone isovitexin. These are major pigments of Iris ensata. These anthocyanins brought about copigmentation, i.e., the bathochromic shift (bluing effect) of visible λ max due to increased concentrations of isovitexin, and 32 to 35 nm were estimated as the magnitude (Δλ max) of each shift. In addition, the absorption spectrum of 0.1 mM malvidin 3RGac5G, 0.07 mM petunidin 3RGac5G and 0.7 mM isovitexin mixture closely matched those of the fresh outer perianths of the bluish purple cultivars, ‘Suiten-isshoku’, ‘Hekikai’ and ‘Yakonotama’ which belong to the malvidin 3RGac5G - petunidin 3RGac5G type of I. ensata. Therefore, these results indicated that the bluing effect on the flower color of the bluish purple cultivars of this species was caused at least in part by the copigmentation between these anthocyanins and the flavone isovitexin. For the copigment effects of isovitexin among malvidin 3RGac5G, petunidin 3RGac5G and delphinidin 3RGac5G, λ max and Δλ max of delphinidin 3RGac5G was slightly higher than those of malvidin 3RGac5G or petunidin 3RGac5G, and the copigmentation of delphinidin 3RGac5G was characterized by a higher concentration of isovitexin. Finally, the breeding for blue flowers due to copigmentation of delphinidin 3RGac5G with isovitexin in I. ensata was discussed.

Hispidulin and nepetin 4′-glucosides from Cirsium oligophyllum

Abstract Two flavone glycosides, hispidulin 4′-O-β- d -glucopyranoside and nepetin 4′-O-β- d -glucopyranoside, were isolated from the leaves of Cirsium oligophyllum and identified on the basis of their 1H and 13C NMR, FAB-MS, UV spectra and characterization of their hydrolysates; hispidulin 7,4′-glycoside, vicenin-2, hispidulin and nepetin were also isolated.

Difference in chilling-induced flavonoid profiles, antioxidant activity and chilling tolerance between soybean near-isogenic lines for the pubescence color gene

Chilling tolerance is an important trait of soybeans [Glycine max (L.) Merr.] produced in cool climates. We previously isolated a soybean flavonoid 3′ hydroxylase (F3′H) gene corresponding to the T locus, which controls pubescence and seed coat color. A genetic link between the T gene and chilling tolerance has been reported, although the exact underlying mechanisms remain unclear. Using the soybean near-isogenic lines (NILs) To7B (TT) and To7G (tt), we examined the relationship between chilling injury, antioxidant activity and flavonoid profiles associated with chilling treatment (15°C). Chilling injury was more severe in the second trifoliate leaves of To7G than in those of To7B. Hydrogen peroxide accumulation and lipid peroxidation were enhanced by chilling in To7G. Chilling-induced enhancement of antioxidant activity was more prominent in To7B than in To7G. High performance liquid chromatography analysis indicated that the contents of quercetin glycosides and isorhamnetin glycosides (3′,4′-dihydroxylated flavonol derivatives) increase in the second trifoliate leaves of To7B after chilling treatment, whereas the same treatment increased kaempferol glycoside (4′-monohydroxylated flavonol derivatives) content in the corresponding leaves of To7G. Histochemical staining also demonstrated chilling-induced flavonoid accumulation. Microarray analysis and real-time reverse transcription-PCR demonstrated that the transcript levels of soybean F3′H are upregulated by chilling. The differences in chilling injury, antioxidant activity and flavonoid species between the two NILs support the notion that soybean F3′H affects chilling tolerance by increasing antioxidant activity via production of 3′,4′-dihydroxylated flavonol derivatives.

NEW AND RARE FLAVONOL GLYCOSIDES FROM ASPLENIUM TRICHOMANES-RAMOSUM AS STABLE CHEMOTAXONOMIC MARKERS

Abstract Sixteen flavonol glycosides, including new and rare ones, were isolated from Asplenium trichomanes-ramosum . Major glycosides were quercetin 3-methyl ether 5- O -glucoside, kaempferol 3- O -arabinoside-7- O -rhamnoside, kaempferol 3- O -glucoside-7- O -rhamnoside, kaempferol 3,7-di- O -rhamnoside, quercetin 3- O -rhamnoside-7- O -arabinoside and quercetin 3- O -glucoside-7-rhamnoside, which were accompanied with traces of two quercetin 3-methyl ether 5- O -glycosides and three kaempferol 3,5- O -glycosides. Flavonol 5- O -glycosides were found in the genus Asplenium for the first time. The new or rare glycosides were consistently detected in all samples, which were collected at representative places in the Northern Hemisphere (the oldest sample was collected in 1879) and show that the flavonoids are extremely stable as chemotaxonomic markers for defining the species.

Allelic variation of soybean flower color gene W4 encoding dihydroflavonol 4-reductase 2.

BackgroundFlower color of soybean is primarily controlled by six genes, viz., W1, W2, W3, W4, Wm and Wp. This study was conducted to investigate the genetic and chemical basis of newly-identified flower color variants including two soybean mutant lines, 222-A-3 (near white flower) and E30-D-1 (light purple flower), a near-isogenic line (Clark-w4), flower color variants (T321 and T369) descended from the w4-mutable line and kw4 (near white flower, Glycine soja).ResultsComplementation tests revealed that the flower color of 222-A-3 and kw4 was controlled by the recessive allele (w4) of the W4 locus encoding dihydroflavonol 4-reductase 2 (DFR2). In 222-A-3, a single base was deleted in the first exon resulting in a truncated polypeptide consisting of 24 amino acids. In Clark-w4, base substitution of the first nucleotide of the fourth intron abolished the 5′ splice site, resulting in the retention of the intron. The DFR2 gene of kw4 was not expressed. The above results suggest that complete loss-of-function of DFR2 gene leads to near white flowers. Light purple flower of E30-D-1 was controlled by a new allele at the W4 locus, w4-lp. The gene symbol was approved by the Soybean Genetics Committee. In E30-D-1, a single-base substitution changed an amino acid at position 39 from arginine to histidine. Pale flowers of T369 had higher expression levels of the DFR2 gene. These flower petals contained unique dihydroflavonols that have not yet been reported to occur in soybean and G. soja.ConclusionsComplete loss-of-function of DFR2 gene leads to near white flowers. A new allele of the W4 locus, w4-lp regulates light purple flowers. Single amino acid substitution was associated with light purple flowers. Flower petals of T369 had higher levels of DFR2 gene expression and contained unique dihydroflavonols that are absent in soybean and G. soja. Thus, mutants of the DFR2 gene have unique flavonoid compositions and display a wide variety of flower color patterns in soybean, from near white, light purple, dilute purple to pale.

Three flavonol allosides from Glaucidium palmatum

Abstract Three flavonol allosides were isolated from the leaves of Glaucidium palmatum endemic to Japan. Their structures were established as quercetin, kaempferol and rhamnocitrin 3- O -β- d -allosides. The two latter glycosides were also isolated from the flowers.

On the pigmented spherical bodies and crystals in tepals of cactaceous species in reference to the nature of betalains or flavonols

Red pigmented bodies were found in tepal cells of threeRebutia species, i.e.,R. grandiflora, R. hyalacantha andR. krainziana. The pigmented body within the vacuole was spherical and normally one per cell. It contained betalains which were validated from the visible spectra of tepal extracts and microspectrophotometric survey on vacuoles. Such a particular red pigmented body has already been found in some anthocyanin-producing plants, and called “anthocyanoplast”. Now, a similar structure has also been found for the first time in betalain-producing plants. Therefore, it may be called “betalainoplast”. InR. grandiflora, yellow pigmented bodies which were not well characterized due to their minimal size were also present as a betalainoplast.On the other hand, pigment-crystals were frequently present in the cells having flavonols such as those in pale yellow tepals of fourAstrophytum species, namely,A. asterias, A. capricorne, A. myriostigma andA. ornatum, with or without a trace of betalains. Yellowish crystals were determined as the crystal mass of quercetin by the UV spectral analysis and careful comparison of chromatographic properties with an authentic specimen.

Neochilenin, a new glycoside of 3-O-methylquercetin, and other flavonols in the tepals ofNeochilenia, Neoporteria andParodia species (Cactaceae)

Abstract3-O-Methylated flavonols were isolated as crystals for the first time from the flowers ofNeochilenia, Neoporteria andParodia species belonging to the sub-family Cereoideae (Cactaceae), which are native to South America. The structures of three compounds were confirmed by chemical and spectral means. In the tepals of 7 species ofNeoporteria, 3-methyl ether of quercetin was found in the form of aglycone, whereas it was present as the 7-O-glucoside in the tepals ofParodia sanguiniflora and as the 4′-O-glucoside in the tepals of three species ofNeochilenia. Among those two glucosides of quercetin 3-methyl ether, the former has been found in a whole plant ofArtemisia transiliensis (Compositae), while the latter is new to the literature. Therefore, the term “neochilenin” may be assigned to this new pigment.