Erratum: Engineering of flower color in forsythia by expression of two independently-transformed dihydroflavonol 4-reductase and anthocyanidin synthase genes of flavonoid pathway

Molecular Breeding -     

Production of red-flowered plants by genetic engineering of multiple flavonoid biosynthetic genes

Orange- to red-colored flowers are difficult to produce by conventional breeding techniques in some floricultural plants. This is due to the deficiency in the formation of pelargonidin, which confers orange to red colors, in their flowers. Previous researchers have reported that brick-red colored flowers can be produced by introducing a foreign dihydroflavonol 4-reductase (DFR) with different substrate specificity in Petunia hybrida, which does not accumulate pelargonidin pigments naturally. However, because these experiments used dihydrokaempferol (DHK)-accumulated mutants as transformation hosts, this strategy cannot be applied directly to other floricultural plants. Thus in this study, we attempted to produce red-flowered plants by suppressing two endogenous genes and expressing one foreign gene using tobacco as a model plant. We used a chimeric RNAi construct for suppression of two genes (flavonol synthase [FLS] and flavonoid 3′-hydroxylase [F3′H]) and expression of the gerbera DFR gene in order to accumulate pelargonidin pigments in tobacco flowers. We successfully produced red-flowered tobacco plants containing high amounts of additional pelargonidin as confirmed by HPLC analysis. The flavonol content was reduced in the transgenic plants as expected, although complete inhibition was not achieved. Expression analysis also showed that reduction of the two-targeted genes and expression of the foreign gene occurred simultaneously. These results demonstrate that flower color modification can be achieved by multiple gene regulation without use of mutants if the vector constructs are designed resourcefully. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Identification and expression analysis of chalcone synthase and dihydroflavonol 4-reductase in Clivia miniata

Clivia miniata is a popular breeding variety. The production of anthocyanin has been studied in Clivia species and the presence of key genes in anthocyanin production, chalcone synthase (CHS) and dihydroflavonol 4-reductase (DFR) confirmed. However, it is currently unknown to what extent these genes are expressed in different flower tissue during flower development. Thus the aim of this study was to determine the expression of CHS and DFR in C. miniata var. miniata, an orange flowered variety, and C. miniata var. citrina, a yellow flowered variety, in tepal, carpel and stamen at flower developmental stage two to six. As expected, the anthocyanin content in orange flowers was higher than that of yellow flowers. The expression of CHS and DFR correlated to anthocyanin content. Anthocyanin gene expression and production was found primarily in the tepal. There was a high correlation between CHS and DFR expression suggesting that these genes are subject to coordinate regulation in C. miniata.     

Engineering of the rose flavonoid biosynthetic pathway successfully generated blue-hued flowers accumulating delphinidin

Flower color is mainly determined by anthocyanins. Rosa hybrida lacks violet to blue flower varieties due to the absence of delphinidin-based anthocyanins, usually the major constituents of violet and blue flowers, because roses do not possess flavonoid 3',5'-hydoxylase (F3'5'H), a key enzyme for delphinidin biosynthesis. Other factors such as the presence of co-pigments and the vacuolar pH also affect flower color. We analyzed the flavonoid composition of hundreds of rose cultivars and measured the pH of their petal juice in order to select hosts of genetic transformation that would be suitable for the exclusive accumulation of delphinidin and the resulting color change toward blue. Expression of the viola F3'5'H gene in some of the selected cultivars resulted in the accumulation of a high percentage of delphinidin (up to 95%) and a novel bluish flower color. For more exclusive and dominant accumulation of delphinidin irrespective of the hosts, we down-regulated the endogenous dihydroflavonol 4-reductase (DFR) gene and overexpressed the Irisxhollandica DFR gene in addition to the viola F3'5'H gene in a rose cultivar. The resultant roses exclusively accumulated delphinidin in the petals, and the flowers had blue hues not achieved by hybridization breeding. Moreover, the ability for exclusive accumulation of delphinidin was inherited by the next generations.     

Modification of flower color and fragrance by antisense suppression of the flavanone 3-hydroxylase gene

Anthocyanins are the major pigments contributing to carnation flowercoloration. Most carnation varieties are sterile and hence molecular breedingis an attractive approach to creating novel colors in this commercially importantcrop. Characterization of anthocyanins in the flowers of the modern carnationcv. Eilat revealed that only the orange pelargonidin accumulates, due to a lackof both flavonoid 3,5-hydroxylase and flavonoid3-hydroxylase activities. To modify flower color in cv. Eilat, we usedantisense suppression to block the expression of a gene encoding flavanone3-hydroxylase, a key step in the anthocyanin pathway. The transgenic plantsexhibited flower color modifications ranging from attenuation to complete lossof their original orange/reddish color. In the latter, only traces ofpelargonidin were detected. Dramatic suppression of flavanone 3-hydroxylaselevel/activity in these transgenes was confirmed by northern blot, RT-PCR andenzymatic assays. The new phenotype has been stable for over 4 years ofvegetative propagation. Moreover, transgenic plants with severe colormodification were more fragrant than control plants. GC-MS headspace analysesrevealed that transgenic anti-f3h flowers emit higherlevels of methyl benzoate. The possible interrelation between pathways leadingto anthocyanin and fragrance production is discussed.     

植物花色呈现的生物化学、分子生物学机制及其基因工程改良

文章综述了植物花色基因工程改良的前提、策略和特殊性。植物主要花色色素类黄酮、类胡萝卜素的合成途径和相关酶的基因表达已基本研究清楚,关键酶的基因已被克隆。花色的基因工程改良遵循一般植物基因工程规律,而且是系统工程,要求花器官特异性启动子,基本策略为抑制关键酶的基因表达、导入调节基因或新的外源基因。花色基因工程可能成为获得雄性不育的方式之一,应与传统育种技术有机结合,当然也存在安全性问题。     

DNA rearrangements at the region of the dihydroflavonol 4-reductase gene for flower pigmentation and incomplete dominance in morning glory carrying the mutable flaked mutation

 The a-3 ^flecked [J] variegated line of Japanese morning glory bearing white flowers with normal-colored flecks and sectors has been shown to carry a 6.4-kb transposable element, Tpn1, inserted within the DFR-B gene, one of the anthocyanin biosynthesis genes encoding dihydroflavonol 4-reductase (DFR). The a ^flaked [M] variegated line of morning glory also bears white flowers with normal-colored flakes and sectors, and it was shown to carry multiple DNA rearrangements, including insertions of mobile element-like sequences, MELSIP1 and MELSIP2, in its DFR gene region. Unlike the a-3 ^flecked [J] mutation, the mutable a ^flaked [M] allele exhibited incomplete dominance. Interestingly, not only intensely colored flakes but also white spots and sectors were often observed in lightly colored flowers of morning glory in the heterozygous state A[M]/a ^flaked [M]. The interspecific F₁ hybrids between Japanese morning glory and morning glory carrying both a-3 ^flecked [J]/A-3[M] and A[J]/ a ^flaked [M] in the heterozygous condition bear lightly colored flowers with intensely colored sectors as well as white flakes. The results clearly demonstrated that the DFR gene in the a ^flaked [M] line of morning glory is active and complements the DFR-B gene carrying Tpn1 in the a-3 ^flecked [J] line of Japanese morning glory. Interspecific allelic interactions between the mutable a ^flaked [M] gene of morning glory and the corresponding wild-type A[J] gene of Japanese morning glory resulted in incomplete dominance and the formation of white flakes and sectors. The appearance of the white flakes may be due to a somatic mutation of the A[J] gene. Received: 4 November 1996/Accepted: 13 December 1996     

Flower color diversity revealed by differential expression of flavonoid biosynthetic genes and flavonoid accumulation in herbaceous peony (Paeonia lactiflora Pall.)

Herbaceous peony (Paeonia lactiflora Pall.) is an important ornamental plant which contains different flower colors. In this paper, eight genes encoding phenylalanine ammonialyase (PAL), chalcone synthase (CHS), chalcone isomerase (CHI), flavanone 3-hydroxylase (F3H), flavonoid 3′-hydroxylase (F3′H), dihydroflavonol 4-reductase (DFR), anthocyanidin synthase (ANS), UDP-glucose: flavonoid 3-o-glucosyltransferase (UF3GT) were isolated. Moreover, the expression patterns of these eight genes and UF5GT in the flowers were investigated in three cultivars, that is, ‘Hongyanzhenghui’, ‘Yulouhongxing’ and ‘Huangjinlun’ with purplish-red, white and yellow flower respectively. Furthermore, flavonoid accumulation in the flowers was also analyzed. The results showed that in different organs, most of genes expressed higher in flowers than in other organs. During the development of flowers, all genes could be divided into four groups. The first group (PlPAL) was highly expressed in S1 and S4. The second group (PlCHS and PlCHI) was at a high expression level throughout the whole developmental stages. The third group (PlF3H, PlF3′H, PlDFR, PlANS and PlUF5GT) gradually decreased with the development of flowers. The fourth group (PlUF3GT) gradually increased during the flower development. In addition, anthoxanthins and anthocyanins were detected in ‘Hongyanzhenghui’ and ‘Yulouhongxing’, chalcones and anthoxanthins were found in ‘Huangjinlun’. When different color flowers were concerned, low expression level of PlCHI induced most of the substrate accumulation in the form of chalcones and displaying yellow, changing a small part of substrates to anthoxanthins, and there was no anthocyanin synthesis in ‘Huangjinlun’ because of low expression level of DFR. In ‘Yulouhongxing’, massive expressions of upstream genes and low expression of DFR caused synthesis of a great deal of anthoxanthins and a small amount of colorless anthocyanins. In ‘Hongyanzhenghui’, a large number of colored anthocyanins were changed from anthoxanthins because of PlDFR, PlANS and PlUF3GT high expressions. These results would provide us a theoretical basis to understand the formation of P. lactiflora flower colors.     

Gene characterization, analysis of expression and in vitro synthesis of dihydroflavonol 4-reductase from [Citrus sinensis (L.) Osbeck

Dihydroflavonol 4-reductase (DFR, EC 1.1.1.219) catalyzes the reduction of dihydroflavonols to leucoanthocyanins, a key "late" step in the biosynthesis of anthocyanins. In this study we showed that a strong reduction in DFR expression occurs in the non-red orange cultivar (Navel and Ovale) compared to that of the red orange (Tarocco) suggesting that the enzyme could be involved in the lack of production of anthocyanins. Therefore, we isolated and compared the cDNAs, the genomic clones, as well as the promoter regions of blood and blond orange dfrs. Our data revealed that the cDNA sequences of pigmented and non-pigmented orange DFRs were 100% homologous and contained a 1017 bp open reading frame which encodes a protein of 338 amino acid residues, corresponding to a molecular mass of 38010.76 Da, with a theoretical pI of 5.96. Moreover, we found that there were no significant differences in non-coding regions (introns and 5' upstream region) of dfr sequences. Southern blot analysis of genomic DNA indicated that dfr was present as a single copy gene in both cultivars. From these findings the low expression level of blond orange dfr, which might play a role in the phenotypic change from blood to blond orange, is thought to be the result of a likely mutation in a regulatory gene controlling the expression of dfr. In addition, here we reported the successful expression of orange DFR cDNAs leading to an active DFR enzyme which converts dihydroquercetin to leucoanthocyanidin, thus confirming the involvement of the isolated genes in the biosynthesis of anthocyanins. Moreover, as far as we know, this is the first report concerning the in vitro expression of DFR from fruit flesh whose biochemical properties might be very different from those of other plant organ DFRs.     

Flower color alteration in <Emphasis Type="Italic">Lotus japonicus</Emphasis> by modification of the carotenoid biosynthetic pathway

To establish a model system for alteration of flower color by carotenoid pigments, we modified the carotenoid biosynthesis pathway of Lotus japonicus using overexpression of the crtW gene isolated from marine bacteria Agrobacterium aurantiacum and encoding β-carotene ketolase (4,4′-β-oxygenase) for the production of pink to red color ketocarotenoids. The crtW gene with the transit peptide sequence of the pea Rubisco small subunit under the regulation of the CaMV35S promoter was introduced to L. japonicus. In most of the resulting transgenic plants, the color of flower petals changed from original light yellow to deep yellow or orange while otherwise exhibiting normal phenotype. HPLC and TLC analyses revealed that leaves and flower petals of these plants accumulated novel carotenoids, believed to be ketocarotenoids consisting of including astaxanthin, adonixanthin, canthaxanthin and echinenone. Results indicated that modification of the carotenoid biosynthesis pathway is a means of altering flower color in ornamental crops.     

Extension-growth responses and expression of flavonoid biosynthesis genes in the Arabidopsis hy4 mutant

The hy4 mutant of Arabidopsis thaliana(L.) Heynh. was previously shown to be impaired in the suppression of hypocotyl extension specifically by blue light. We report here that hy4 is altered in a range of blue-light-mediated extension-growth responses in various organs in seedlings and mature plants: it shows greater length of bolted stems, increased petiole extension and increased leaf width and area in blue light compared to the wild type. The hy4 mutant shows decreased cotyledon expansion in both red and blue light compared to the wild type. Anthocyanin formation and the expression of several flavonoid biosynthesis genes is stimulated by blue light in the wild type but to a much lower extent in hy4. The results indicate that the HY4 gene product is concerned with the perception of blue light in a range of extension-growth and gene-expression responses in Arabidopsis.Abbreviations DFR dihydroflavonol reductase - CHS chalcone synthase - CHI chalcone isomerase We thank the UK Agricultural and Food Research Council for supporting this work through the award of a research grant to G.I.J. We are grateful to Robert Brown for excellent technical assistance and Drs B.W. Shirley (Department of Biology, Virginia Polytechnic Institute and State University, Blacksburg, USA), C.D. Silflow (Department of Genetics and Cell Biology, University of Minnesota, St. Paul, USA) and I.E. Somssich (Department of Biochemistry, Max-Planck-Institut, Köln, Germany) for providing plasmid DNA.