-
[show abstract]
[hide abstract]
ABSTRACT: Anthocyanins are a subclass of flavonoids and are a major contributor to flower colors ranging from red to blue and purple. Previous studies in model and ornamental plants indicate a member of the glutathione S-transferase (GST) gene family is involved in vacuolar accumulation of anthocyanins. In order to identify the anthocyanin-related GST in cyclamen, degenerate PCR was performed using total RNA from immature young petals. Four candidates of GSTs (CkmGST1 to CkmGST4) were isolated. Phylogenetic analysis indicated that CkmGST3 was closely related to PhAN9, an anthocyanin-related GST of petunia, and this clade was clustered with other known anthocyanin-related GSTs. Expression analysis at different developmental stages of petals revealed that CkmGST3 was strongly expressed in paler pigmented petals than in fully pigmented petals, in contrast to the constitutive expression of the other three candidates during petal development. This expression pattern of CkmGST3 was correlated with those of other anthocyanin biosynthetic genes such as CkmF3'5'H and CkmDFR2. Molecular complementation of Arabidopsis tt19, a knockout mutant of an anthocyanin-related GST gene, demonstrated that CkmGST3 could complement the anthocyanin-less phenotype of tt19. Transgenic plants that expressed the other three CkmGSTs did not show anthocyanin accumulation. These results indicate CkmGST3 functions in anthocyanin accumulation in cyclamen.
Journal of plant physiology 04/2012; 169(6):636-42. · 2.50 Impact Factor
-
Yusuke Akita, Satoshi Kitamura,
Yoshihiro Hase,
Issay Narumi,
Hiroshi Ishizaka,
Emiko Kondo,
Naoko Kameari,
Masayoshi Nakayama,
Natsu Tanikawa,
Yasumasa Morita,
Atsushi Tanaka
[show abstract]
[hide abstract]
ABSTRACT: Anthocyanin O-methyltransferase (OMT) is one of the key enzymes for anthocyanin modification and flower pigmentation. We previously bred a novel red-purple-flowered fragrant cyclamen (KMrp) from the purple-flowered fragrant cyclamen 'Kaori-no-mai' (KM) by ion-beam irradiation. Since the major anthocyanins in KMrp and KM petals were delphinidin 3,5-diglucoside and malvidin 3,5-diglucoside, respectively, inactivation of a methylation step in the anthocyanin biosynthetic pathway was indicated in KMrp. We isolated and compared OMT genes expressed in KM and KMrp petals. RT-PCR analysis revealed that CkmOMT2 was expressed in the petals of KM but not in KMrp. Three additional CkmOMTs with identical sequences were expressed in petals of both KM and KMrp. Genomic PCR analysis revealed that CkmOMT2 was not amplified from the KMrp genome, indicating that ion-beam irradiation caused a loss of the entire CkmOMT2 region in KMrp. In vitro enzyme assay demonstrated that CkmOMT2 catalyzes the 3' or 3',5' O-methylation of the B-ring of anthocyanin substrates. These results suggest that CkmOMT2 is functional for anthocyanin methylation, and defective expression of CkmOMT2 is responsible for changes in anthocyanin composition and flower coloration in KMrp.
Planta 07/2011; 234(6):1127-36. · 3.00 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: The flower colour of Cyclamen graecum gra6 (wild-type) is pink-purple in the main part of the petal, referred to as the ‘slip’, and deep purple at the petal base, referred to as the ‘eye’. On the other hand, flowers of C. graecum gra50 (a white-flowered variant) exhibit a white colour in both the ‘slip’ and ‘eye’ regions. In this study, the relationship between floral pigmentation and the expression of several anthocyanin biosynthesis genes was investigated in C. graecum gra6 and gra50.The pigments in the ‘slip’ and ‘eye’ regions consist mainly of malvidin 3,5-diglucoside in gra6, suggesting that the difference between the colour of the ‘slip’ and ‘eye’ regions is related to the amount of anthocyanin present.White-flowered C. graecum gra50 possessed lower amounts of anthocyanins, but higher amounts of flavonols compared to gra6, suggesting a change in metabolism caused by a disruption of anthocyanin biosynthesis. Gene expression analysis demonstrated that expression of the dihydroflavonol 4-reductase gene 2 (CgraDFR2) was lower in gra50 compared with gra6, whereas expression of the three other key genes (dihydroflavonol 4-reductase gene 1, flavonoid 3’,5’-hydroxylase, and anthocyanidin synthase) did not differ greatly. These results suggest that the white-flowered variant (gra50) may result from a defect in expression of the CgraDFR2 gene.
Journal of Horticultural Science and Biotechnology 05/2010; 85(5):437-43. · 0.64 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Arabidopsis TRANSPARENT TESTA19 (TT19) encodes a glutathione-S-transferase (GST)-like protein that is involved in the accumulation of proanthocyanidins (PAs) in the seed coat. PA accumulation sites in tt19 immature seeds were observed as small vacuolar-like structures, whereas those in tt12, a mutant of the tonoplast-bound transporter of PAs, and tt12 tt19 were observed at peripheral regions of small vacuoles. We found that tt19 immature seeds had small spherical structures showing unique thick morphology by differential interference contrast microscopy. The distribution pattern of the thick structures overlapped the location of PA accumulation sites, and the thick structures were outlined with GFP-TT12 proteins in tt19. PA analysis showed higher (eightfold) levels of solvent-insoluble PAs in tt19 immature seeds compared with the wild type. Metabolic profiling of the solvent-soluble fraction by LC-MS demonstrated that PA derivatives such as epicatechins and epicatechin oligomers, although highly accumulated in the wild type, were absent in tt19. We also revealed that tt12 specifically accumulated glycosylated epicatechins, the putative transport substrates for TT12. tt12 tt19 showed a similar metabolic profile to tt19. Given the cytosolic localization of functional GFP-TT19 proteins, our results suggest that TT19, which acts prior to TT12, functions in the cytosol to maintain the regular accumulation of PA precursors, such as epicatechin and glycosylated epicatechin, in the vacuole. The PA pathway in the Arabidopsis seed coat is discussed in relation to the subcellular localization of PA metabolites.
The Plant Journal 02/2010; 62(4):549-59. · 6.16 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: To investigate the nature of mutations induced by accelerated ions in higher plants, the effects of carbon-ion-irradiation were compared with those of electron-irradiation in Arabidopsis thaliana. Point-like mutations and rearrangements were induced at a similar frequency after carbon-ion-irradiation, whereas point-like mutations were more frequently induced after electron-irradiation. Sequence analysis revealed that carbon-ion-induced point-like mutations were mostly short deletions. In the case of rearrangements, deletions, inversions, insertions, and translocations were found. The estimated frequency of deletion induction was comparable to that of fast neutrons. Analysis of chromosome breakpoints revealed that carbon ions frequently deleted small regions around the breakpoints, whereas electron-irradiation often duplicated these regions. Moreover, for both types of radiation, broken ends with microhomologies were frequently rejoined. Results of the breakpoint and broken end analyses suggest that non-homologous end-joining (NHEJ) leads to the rejoining of double strand breaks (dsbs) after cells are exposed to both types of radiation, but the type of NHEJ that occurs as a result of damage is different. The results indicated that carbon-ion-induced mutations are most likely nulls and that the induced rearrangements may arise through a unique mechanism. These findings indicate that accelerated ions are a useful mutagen for both forward and reverse genetics for plants.
Journal of Experimental Botany 03/2005; 56(412):587-96. · 5.36 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Flavonoid compounds such as anthocyanins and proanthocyanidins (PAs; so-called condensed tannins) have a multitude of functions in plants. They must be transported from the site of synthesis in the cytosol to their final destination, the vacuoles. Three models have been proposed for sequestering anthocyanins in vacuoles, but the transport machinery for PAs is poorly understood. Novel Arabidopsis mutants, transparent testa 19 (tt19), which were induced by ion beam irradiation, showed a great reduction of anthocyanin pigments in the vegetative parts as well as brown pigments in the seed coat. The TT19 gene was isolated by chromosome walking and a candidate gene approach, and was shown to be a member of the Arabidopsis glutathione S-transferase (GST) gene family. Heterologous expression of a putative ortholog, petunia anthocyanin 9 (AN9), in tt19 complemented the anthocyanin accumulation but not the brown pigmentation in the seed coat. This suggests that the TT19 gene is required for vacuolar uptake of anthocyanins into vacuoles, but that it has also a function different from that of AN9. The depositional pattern of PA precursors in the mutant was different from that in the wild type. These results indicate that TT19 participates in the PA pathway as well as the anthocyanin pathway of Arabidopsis. As involvement of GST in the PA pathway was previously considered unlikely, the function of TT19 in the PA pathway is also discussed in the context of the putative transporter for PA precursors.
The Plant Journal 02/2004; 37(1):104-14. · 6.16 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Irradiation of Arabidopsis thaliana by carbon ions was carried out to investigate the mutational effect of ion particles in higher plants. Frequencies of embryonic lethals and chlorophyll-deficient mutants were found to be significantly higher after carbon-ion irradiation than after electron irradiation (11-fold and 7.8-fold per unit dose, respectively). To estimate the mutation rate of carbon ions, mutants with no pigments on leaves and stems (tt) and no trichomes on leaves (gl) were isolated at the M2 generation and subjected to analysis. Averaged segregation rate of the backcrossed mutants was 0.25, which suggested that large deletions reducing the viability of the gametophytes were not transmitted, if generated, in most cases. During the isolation of mutants, two new classes of flavonoid mutants (tt18, tt19) were isolated from carbon-ion-mutagenized M2 plants. From PCR and sequence analysis, two of the three tt18 mutant alleles were found to have a small deletion within the LDOX gene and the other was revealed to contain a rearrangement. Using the segregation rates, the mutation rate of carbon ions was estimated to be 17-fold higher than that of electrons. The isolation of novel mutants and the high mutation rate suggest that ion particles can be used as a valuable mutagen for plant genetics.
Genetics 05/2003; 163(4):1449-55. · 4.01 Impact Factor
