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Takashi Nakatsuka,
Eri Yamada,
Hideyuki Takahashi,
Tomohiro Imamura,
Mariko Suzuki, Yoshihiro Ozeki,
Ikuko Tsujimura,
Misa Saito,
Yuichi Sakamoto,
Nobuhiro Sasaki,
Masahiro Nishihara
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ABSTRACT: Betalains are one of the major plant pigment groups found in some higher plants and higher fungi. They are not produced naturally in any plant species outside of the order Caryophyllales, nor are they produced by anthocyanin-accumulating Caryophyllales. Here, we attempted to reconstruct the betalain biosynthetic pathway as a self-contained system in an anthocyanin-producing plant species. The combined expressions of a tyrosinase gene from shiitake mushroom and a DOPA 4,5-dioxygenase gene from the four-o'clock plant resulted in successful betalain production in cultured cells of tobacco BY2 and Arabidopsis T87. Transgenic tobacco BY2 cells were bright yellow because of the accumulation of betaxanthins. LC-TOF-MS analyses showed that proline-betaxanthin (Pro-Bx) accumulated as the major betaxanthin in these transgenic BY2 cells. Transgenic Arabidopsis T87 cells also produced betaxanthins, but produced lower levels than transgenic BY2 cells. These results illustrate the success of a novel genetic engineering strategy for betalain biosynthesis.
Scientific Reports 06/2013; 3:1970.
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ABSTRACT: The molecular mechanisms underlying spontaneous bud mutations, which provide an important breeding tool in carnation, are poorly understood. Here we describe a new active hAT type transposable element, designated Tdic101, the movement of which caused a bud mutation in carnation that led to a change of flower color from purple to deep pink. The color change was attributed to Tdic101 insertion into the second intron of F3'H, the gene for flavonoid 3'-hydroxylase responsible for purple pigment production. Regions on the deep pink flowers of the mutant can revert to purple, a visible phenotype of, as we show, excision of the transposable element. Sequence analysis revealed that Tdic101 has the characteristics of an autonomous element encoding a transposase. A related, but non-autonomous element dTdic102 was found to move in the genome of the bud mutant as well. Its mobilization might be the result of transposase activities provided by other elements such as Tdic101. In carnation, therefore, the movement of transposable elements plays an important role in the emergence of a bud mutation.
MGG Molecular & General Genetics 03/2013; · 2.58 Impact Factor
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ABSTRACT: The biosynthetic pathways that produce anthocyanins, the principal pigments for flower and leaf coloration in plants, have been extensively investigated. As a result, many of the enzymes involved in these pathways have been identified. Here, we make use of an inducible Arabidopsis thaliana system and demonstrate that the final step in the formation of the major anthocyanin molecule occurs via a glucosylation step catalyzed by acyl-glucose-dependent anthocyanin glucosyltransferase (AAGT). The glucosylation occurs at the 4-coumarate moiety of the anthocyanin molecule cyanidin 3-O-[2″-O-(2'″-O-(sinapoyl) xylosyl) 6″-O-(p-coumaroyl) glucoside] 5-O-[6″″-O-(malonyl) glucoside] leading to completion of the main anthocyanin structure, a reaction that has not previously been identified in studies of Arabidopsis anthocyanins. Earlier studies on flower AAGTs showed that they conjugate a glucose directly to the basic skeleton of anthocyanin. The present study provides the first evidence that an AAGT of Arabidopsis can conjugate a glucose to an acyl moiety of an anthocyanin modified with sugars and organic acids. The results from analyses of gene expression and of anthocyanin composition in a knock-out (KO) mutant and from a complementation test indicate that AtBGLU10 might encode this AAGT.
Journal of plant physiology 01/2013; · 2.50 Impact Factor
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ABSTRACT: A cDNA encoding an acyl-glucose-dependent anthocyanin 7-O-glucosyltransferase (AaAA7GT) was isolated from Agapanthus africanus petals; this is the first AAGT identified in a monocot. Peak expression of AaAA7GT in developing A. africanus petals occurred before the flowering stage, and was later than found previously for other anthocyanin biosynthetic genes. Analysis of recombinant proteins showed AaAA7GT had strict substrate preference for anthocyanidin 3-O-glycosides. The AaAA7GT amino acid had high sequence similarity to glycoside hydrolase family 1 (GH1) proteins, which typically act as β-glycosidases. A phylogenetic analysis of amino acid sequences suggested that AAGTs were derived from glycosidase early in the angiosperm lineage.
Journal of plant physiology 06/2012; 169(13):1321-6. · 2.50 Impact Factor
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ABSTRACT: The natural bicolor floral traits of the horticultural petunia (Petunia hybrida) cultivars Picotee and Star are caused by the spatial repression of the chalcone synthase A (CHS-A) gene, which encodes an anthocyanin biosynthetic enzyme. Here we show that Picotee and Star petunias carry the same short interfering RNA (siRNA)-producing locus, consisting of two intact CHS-A copies, PhCHS-A1 and PhCHS-A2, in a tandem head-to-tail orientation. The precursor CHS mRNAs are transcribed from the two CHS-A copies throughout the bicolored petals, but the mature CHS mRNAs are not found in the white tissues. An analysis of small RNAs revealed the accumulation of siRNAs of 21 nucleotides that originated from the exon 2 region of both CHS-A copies. This accumulation is closely correlated with the disappearance of the CHS mRNAs, indicating that the bicolor floral phenotype is caused by the spatially regulated post-transcriptional silencing of both CHS-A genes. Linkage between the tandemly arranged CHS-A allele and the bicolor floral trait indicates that the CHS-A allele is a necessary factor to confer the trait. We suppose that the spatially regulated production of siRNAs in Picotee and Star flowers is triggered by another putative regulatory locus, and that the silencing mechanism in this case may be different from other known mechanisms of post-transcriptional gene silencing in plants. A sequence analysis of wild Petunia species indicated that these tandem CHS-A genes originated from Petunia integrifolia and/or Petunia inflata, the parental species of P. hybrida, as a result of a chromosomal rearrangement rather than a gene duplication event.
The Plant Journal 01/2012; 70(5):739-49. · 6.16 Impact Factor
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ABSTRACT: The components of commercial sodium stearoyl lactylate (SSL), purchased in Japan, were determined and identified using thin layer chromatography (TLC) and liquid chromatography with mass spectroscopy (LC-MS). Stearoyl lactate (SL) and stearoyl-2-lactylate (SLL) were purified using TLC and silica gel chromatography to obtain standards. The results show that SSL consisted of lactic acid (8.4%), stearic acid (15%), SL (57%), and SLL (13%). The total amounts of free lactic acid, lactic acid derived from SL and lactic acid derived from SLL were determined using LC-MS. The mean value was approximately equal to that determined using the JECFA method. This is the first study to determine and identify the components of SSL purchased in Japan, using TLC and LC-MS.
Journal of the Food Hygienic Society of Japan (Shokuhin Eiseigaku Zasshi) 01/2012; 53(1):14-8. · 0.43 Impact Factor
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ABSTRACT: The pink, red and crimson petal colors of carnations (Dianthus caryophyllus) are produced by anthocyanins. The anthocyanins, pelargonidin and cyanidin can be modified by two glucoses at the 3 and 5 positions, and by a single malic acid. Petal color variation can result from failure of such modification, for example, the lack of a glucose at the 5 position is responsible for the color variants of some commercial varieties. With respect to this variation, modification by 5-O-glucosyltransferase plays the most important role in glucosylation at the 5 position. Recently, we identified a novel acyl-glucose-dependent anthocyanin 5-O-glucosyltransferase (AA5GT), that uses acyl-glucoses, but not UDP-glucose, as the glucose donor. Although we showed that loss of AA5GT expression was responsible for loss of glucosylation at the 5 position of anthocyanin in some varieties, the cause of this repression of AA5GT expression could not be determined. Here, we have succeeded in isolating the AA5GT gene and found that it consists of 12 exons and 11 introns. In carnation varieties lacking a glucose at the 5 position, we identified the insertion of two different retrotransposons, Ty1dic1 and Retdic1, into AA5GT. Ty1dic1, which belongs to the class I long terminal repeat (LTR)-retrotransposons of Ty1/copia families, was inserted into exon 10. Retdic1, which includes a long interspersed nuclear element (LINE)-like sequence, was inserted into intron 5. Thus, insertion of either Ty1dic1 or Retdic1 can disrupt AA5GT and result in the lack of glucosylation at the 5 position in anthocyanins.
MGG Molecular & General Genetics 11/2011; 286(5-6):383-94. · 2.58 Impact Factor
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03/2011: pages 321 - 342; , ISBN: 9781119991311
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ABSTRACT: The herbicide-tolerant genetically modified Roundup Ready canola (Brassica napus) line RT73 has been approved worldwide for use in animal feed and human food. However, RT73 Brassica rapa lines derived from interspecific crosses with RT73 B. napus have not been approved in Japan. Here, we report on a novel system using individual kernel analyses for the qualitative detection of RT73 B. rapa in canola grain samples. We developed a duplex real-time polymerase chain reaction (PCR) method to discriminate B. napus and B. rapa DNA using scatter plots of the end-point analyses; this method was able to discriminate a group comprising B. rapa and Brassica juncea from a group comprising B. napus, Brassica carinata, and Brassica oleracea. We also developed a duplex real-time PCR method for the simultaneous detection of an RT73-specific sequence and an endogenous FatA gene. Additionally, a DNA-extraction method using 96-well silica-membrane plates was developed and optimized for use with individual canola kernels. Our detection system could identify RT73 B. rapa kernels in canola grain samples enabling the accurate and reliable monitoring of RT73 B. rapa contamination in canola, thus playing a role in its governmental regulation in Japan.
Analytical Chemistry 11/2010; 82(23):9909-16. · 5.86 Impact Factor
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ABSTRACT: Betalamic acid, the chromophore of betaxanthins, was enzymatically synthesized on a large scale from l-dihydroxyphenylalanine (L-DOPA) using recombinant Mirabilis jalapa DOPA 4,5-dioxygenase. After synthesis, proline was directly added to the concentrated reaction mixture to generate proline-betaxanthin. The molecular mass and nuclear magnetic resonance spectrum of the purified product were identical to those previously reported for proline-betaxanthin. Twenty-four betaxanthin species were synthesized by the condensation reaction of purified betalamic acid and amino acids or amines. An HPLC protocol was established for identifying the different betaxanthin species. Proline-, dopamine-, and γ-aminobutyric acid (GABA)-betaxanthins were prepared as representative betaxanthins under large-scale conditions, and their 1,1-diphenyl-2-picrylhydrazyl radical-scavenging activities were compared against those of known antioxidants. GABA-betaxanthin showed comparatively low activity, whereas dopamine-betaxanthin had similar activity to the red pigment betanin and the anthocyanin cyanidin 3-glucoside. Proline-betaxanthin had the highest activity of the three synthesized compounds and was similar to the flavonoid quercetin.
Journal of Agricultural and Food Chemistry 11/2010; · 2.82 Impact Factor
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Yuki Matsuba,
Nobuhiro Sasaki,
Masayuki Tera,
Masachika Okamura,
Yutaka Abe,
Emi Okamoto,
Haruka Nakamura,
Hisakage Funabashi,
Makoto Takatsu,
Mikako Saito,
Hideaki Matsuoka,
Kazuo Nagasawa, Yoshihiro Ozeki
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ABSTRACT: Glucosylation of anthocyanin in carnations (Dianthus caryophyllus) and delphiniums (Delphinium grandiflorum) involves novel sugar donors, aromatic acyl-glucoses, in a reaction catalyzed by the enzymes acyl-glucose-dependent anthocyanin 5(7)-O-glucosyltransferase (AA5GT and AA7GT). The AA5GT enzyme was purified from carnation petals, and cDNAs encoding carnation Dc AA5GT and the delphinium homolog Dg AA7GT were isolated. Recombinant Dc AA5GT and Dg AA7GT proteins showed AA5GT and AA7GT activities in vitro. Although expression of Dc AA5GT in developing carnation petals was highest at early stages, AA5GT activity and anthocyanin accumulation continued to increase during later stages. Neither Dc AA5GT expression nor AA5GT activity was observed in the petals of mutant carnations; these petals accumulated anthocyanin lacking the glucosyl moiety at the 5 position. Transient expression of Dc AA5GT in petal cells of mutant carnations is expected to result in the transfer of a glucose moiety to the 5 position of anthocyanin. The amino acid sequences of Dc AA5GT and Dg AA7GT showed high similarity to glycoside hydrolase family 1 proteins, which typically act as β-glycosidases. A phylogenetic analysis of the amino acid sequences suggested that other plant species are likely to have similar acyl-glucose-dependent glucosyltransferases.
The Plant Cell 10/2010; 22(10):3374-89. · 8.99 Impact Factor
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ABSTRACT: Miniature inverted-repeat transposable elements (MITEs) are dispersed in large numbers within the genomes of eukaryotes although almost all are thought to be inactive. Plants have two major groups of such MITEs: Tourist and Stowaway. Mobile MITEs have been reported previously in rice but no active MITEs have been found in dicotyledons. Here, we provide evidence that Stowaway MITEs can be mobilized in the potato and that one of them causes a change of tuber skin color as an obvious phenotypic variation. In an original red-skinned potato clone, the gene encoding for a flavonoid 3',5'-hydroxylase, which is involved in purple anthocyanin synthesis, has been inactivated by the insertion of a Stowaway MITE named dTstu1 within the first exon. However, dTstu1 is absent from this gene in a purple somaclonal variant that was obtained as a regenerated plant from a protoplast culture of the red-skinned potato. The color change was attributed to reversion of flavonoid 3',5'-hydroxylase function by removal of dTstu1 from the gene. In this purple variant another specific transposition event has occurred involving a MITE closely related to dTstu1. Instead of being fossil elements, Stowaway MITEs, therefore, still have the ability to become active under particular conditions as represented by tissue culturing.
Genetics 09/2010; 186(1):59-66. · 4.01 Impact Factor
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ABSTRACT: Betalains are synthesized in flowers, fruits and other tissues of the plant order Caryophyllales. Betalamic acid is the chromophore of betalain pigments synthesized by a ring-cleaving enzyme reaction on l-dihydroxyphenylalanine (DOPA). Although reverse genetic evidence has proven that DOPA 4,5-dioxygenase (DOD) is a key enzyme of betalain biosynthesis, all attempts to detect recombinant plant DOD activity in vitro have failed. Here, we report on the formation of betalamic acid from DOPA under suitable assay conditions using recombinant MjDOD produced by Escherichia coli. This is the first report showing biochemical evidence for DOD activity in vitro.
Plant and Cell Physiology 05/2009; 50(5):1012-6. · 4.70 Impact Factor
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ABSTRACT: We investigated whether exogenously supplied precursors of bergapten, namely umbelliferone, psoralen and bergaptol, could be utilized to produce bergapten without elicitation in Glehnia littoralis cell suspension cultures. The levels of added psoralen and bergaptol in the medium soon decreased, and this was followed by the detection of bergapten in both culture fluid and cells. Umbelliferone was also incorporated but in this case no bergapten was produced; instead, skimmin, umbelliferone monoglucoside, was detected. To determine whether conversion of psoralen to bergapten was due to enzyme induction by precursor feeding, the transcript accumulations and enzyme activities of bergaptol O-methyltransferase (BMT, EC 2.1.1.69), which catalyzes the last step of bergapten synthesis, and of phenylalanine ammonia-lyase (PAL, EC 4.3.1.5), which catalyzes the initial step of the phenylpropanoid biosynthetic pathway and is known as a marker enzyme of elicitation, were examined. The results showed that both the expression and the activity of BMT were always detected in all cells, including control cells. Since PAL was slightly induced in the cells supplied with/without precursors, phenylethyl alcohol (PEA, a competitive inhibitor of PAL) was applied to suspension cells prior to the addition of psoralen. PAL activity was effectively inhibited by PEA at 1-5 mM concentrations. Under these conditions, PEA did not affect bergapten production by cell cultures fed with psoralen at all. These results demonstrate that BMT is constitutively expressed in G. littoralis cell cultures.
Plant Cell Reports 11/2008; 28(2):257-65. · 2.27 Impact Factor
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ABSTRACT: The phenylalanine ammonia-lyase (PAL) gene, DcPAL3, was expressed during the synthesis of anthocyanin in suspension-cultured cells of carrot (Daucus carota). There were two putative cis-elements in the DcPAL3 promoter region: the box-L and GCC-box homologs. Both of these are committed to the upregulation of promoter activity. Although box-L is known as the conserved cis-element present in the promoter region of most PAL genes of many plant species targeted by the R2R3-MYB protein, among PAL genes, the GCC-box homolog is unique to the promoter region of the DcPAL3 gene. We have isolated two proteins belonging to the ethylene-responsive element-binding factor (ERF) family, DcERF1 and DcERF2, from two different cDNA libraries prepared from anthocyanin-synthesizing cells of different cultured cell lines of carrot. The methodology employed was yeast one-hybrid screening with the GCC-box homolog as a bait. Both DcERF1 and DcERF2 bound to the GCC-box homolog sequence in vitro. Transient expression analysis showed that, in carrot protoplasts, DcERF1 was able bind to the GCC-box homolog and act as an activator of the DcPAL3 promoter. In contrast, DcERF2 itself had no ability to activate DcPAL3 promoter activity, possibly because transiently expressed DcERF2 may not be exported into the nucleus. These results suggest that DcERF1 and DcERF2 may function in different ways in committing to the upregulation of the DcPAL3 promoter activity in anthocyanin-synthesizing cells of carrot.
Journal of Plant Research 07/2008; 121(5):499-508. · 1.75 Impact Factor
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ABSTRACT: The structures of a procyanidin tetramer and pentamer from unripe apple (Malus pumila) were elucidated by low-temperature NMR analysis at -34 °C. These structures were [epicatechin-(4[beta]-->6)-epicatechin-(4[beta]-->8)-epicatechin-(4[beta]-->8)-epicatechin (1)] and [epicatechin-(4[beta]-->8)-epicatechin-(4[beta]-->8)-epicatechin-(4[beta]-->8)-epicatechin-(4[beta]-->8)-epicatechin (2)].
Tetrahedron Letters 01/2008; 49(45):6413-6418. · 2.68 Impact Factor
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ABSTRACT: Structures and levels of anthocyanin-related compounds were analyzed during the development of marginal picotee petals in white-center and white-marginal cultivars of Petunia hybrida. In the white site of a white-center cultivar, higher concentrations of quercetin derivatives possessing 7-O-glucoside and/or 3'-O-glucoside occurred than in the colored site, suggesting that these two quercetin glycosylation steps are site-specifically regulated. The boundary areas of petal coloration were composed of cells showing various color densities, whose uniformity among adjacent cells varied between these cultivars. These results indicate diversity in spatiotemporal regulation of anthocyanin biosynthesis and flavonol glycosylations between Petunia cultivars during marginal picotee formation.
Journal of Plant Research 08/2007; 120(4):563-8. · 1.75 Impact Factor
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ABSTRACT: Differentiated shoot and root cultures of Glehnia littoralis were examined for organ-specific responses to yeast extract (YE). Growth, and changes in phenylalanine ammonia-lyase (PAL, EC 4. 1. 3. 5) activity, as well as furanocoumarin and simple phenylpropanoid production were then determined. YE affected root growth positively but negatively affected the growth of both the leaf and petiole. PAL activity was induced in all organs and reached a maximum after 2days of treatment, though the activity in leaves was about three times higher than that in roots. A large amount of p-coumaric acid (p-CA) was transiently excreted into the culture medium of leaves, which has only been rarely reported to date. Subsequently, bergapten and xanthotoxin appeared in the medium. In contrast, no furanocoumarin was detected in the root cultures throughout the course of treatment. Changes in simple phenylpropanoid contents such as p-CA, caffeic acid (CafA) and ferulic acid (FA) in tissues were analyzed in three forms, i.e., free, soluble-conjugated and insoluble-conjugated forms. In leaves, little difference between control and YE-treated tissues was found except free p-CA, but every form of simple phenylpropanoid was increasingly elicited in the roots. These results indicate that YE acts bi-functionally on the root as a nutrient and an elicitor, but only as an elicitor in the leaf.
Journal of Natural Medicines 12/2006; 61(1):30-37. · 1.39 Impact Factor
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ABSTRACT: Anthocyanin synthesis and chlorophyll degradation in regenerated torenia (Torenia fournieri Linden ex Fourn.) shoots induced by osmotic stress with 7% sucrose were examined to identify the genes regulating the underlying molecular mechanism. To achieve this, suppression subtractive hybridization was performed to enrich the cDNAs of genes induced in anthocyanin-synthesizing and chlorophyll-degrading regenerated shoots. The nucleotide sequences of 1,388 random cDNAs were determined, and these were used in the preparation of cDNA microarrays for high-throughput screening. From 1,056 cDNAs analyzed in the microarrays, 116 nonredundant genes were identified, which were up regulated by 7% sucrose to induce anthocyanin synthesis and chlorophyll degradation in regenerated shoots. Of these, eight genes were selected and RNAi transformants prepared, six of which exhibited anthocyanin synthesis inhibition and/or chlorophyll degradation in their leaf discs. Notably, the RNAi transformants of the glucose 6-phosphate/phosphate translocator gene displayed inhibition both of anthocyanin synthesis and chlorophyll degradation in both leaf discs and regenerated shoots. There was also less accumulation of anthocyanin in the petals, and flowering time was shortened. The genes we identified as being up-regulated in the regenerated torenia shoots may help further elucidate the molecular mechanism underlying the induction of anthocyanin synthesis and chlorophyll degradation.
Journal of Plant Research 06/2006; 119(3):217-30. · 1.75 Impact Factor
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ABSTRACT: Changes in the amount of mRNA for phenylalanine ammonia-lyase (PAL, EC 4. 3. 1. 5) and chalcone synthase (CHS, EC 2. 3. 1. 74) induced by light were investigated in carrot (Daucus carota L. cv. Kurodagosun) cells cultured in suspension. Cells were cultured for 5 days in 2,4-dichlorophenoxy acetic acid (2,4-D)-free medium and absolute darkness. Transcription of pal and chs genes was induced by irradiation at day 5, resulting in the induction of anthocyanin synthesis. PAL mRNA transcripts were rapidly and transiently up-regulated within 6 h after onset of irradiation, after which they were immediately down-regulated. Then, PAL mRNA increased again slowly, the time course being coordinated with induction of CHS transcription and anthocyanin synthesis. The semi-quantitative polymerase chain reaction (PCR) and primer extension using gene specific oligonucleotides for pal genes revealed that two different pal genes were induced sequentially by light. The rapid and transient induction of the pal gene was not related to anthocyanin synthesis, but was identical to that induced by dilution or transfer effect. This slowly-induced pal gene, which was induced in parallel to the chs gene, corresponded to the pal gene which was induced for anthocyanin synthesis. Anthocyanin synthesis was induced by simultaneous activation of both ant-pal and chs genes, and both mRNAs remained at a high level during anthocyanin synthesis. Expression of ant-pal and chs was partially repressed by either depletion of light or addition of 2,4-D. These genes were completely repressed by addition of 2,4-D in darkness. Therefore, the two signals may act at different points. Trn-pal was not repressed by depletion of light or addition of 2,4-D.
Physiologia Plantarum 04/2006; 89(1):4 - 10. · 3.11 Impact Factor
