A new allele of flower color gene W1 encoding flavonoid 3'5'-hydroxylase is responsible for light purple flowers in wild soybean Glycine soja.
ABSTRACT: BACKGROUND: Glycine soja is a wild relative of soybean that has purple flowers. No flower color variant of Glycine soja has been found in the natural habitat. RESULTS: B09121, an accession with light purple flowers, was discovered in southern Japan. Genetic analysis revealed that the gene responsible for the light purple flowers was allelic to the W1 locus encoding flavonoid 3'5'-hydroxylase (F3'5'H). The new allele was designated as w1-lp. The dominance relationship of the locus was W1 >w1-lp >w1. One F2 plant and four F3 plants with purple flowers were generated in the cross between B09121 and a Clark near-isogenic line with w1 allele. Flower petals of B09121 contained lower amounts of four major anthocyanins (malvidin 3,5-di-O-glucoside, petunidin 3,5-di-O-glucoside, delphinidin 3,5-di-O-glucoside and delphinidin 3-O-glucoside) common in purple flowers and contained small amounts of the 5'-unsubstituted versions of the above anthocyanins, peonidin 3,5-di-O-glucoside, cyanidin 3,5-di-O-glucoside and cyanidin 3-O-glucoside, suggesting that F3'5'H activity was reduced and flavonoid 3'-hydroxylase activity was increased. F3'5'H cDNAs were cloned from Clark and B09121 by RT-PCR. The cDNA of B09121 had a unique base substitution resulting in the substitution of valine with methionine at amino acid position 210. The base substitution was ascertained by dCAPS analysis. The polymorphism associated with the dCAPS markers co-segregated with flower color in the F2 population. F3 progeny test, and dCAPS and indel analyses suggested that the plants with purple flowers might be due to intragenic recombination and that the 65 bp insertion responsible for gene dysfunction might have been eliminated in such plants. CONCLUSIONS: B09121 may be the first example of a flower color variant found in nature. The light purple flower was controlled by a new allele of the W1 locus encoding F3'5'H. The flower petals contained unique anthocyanins not found in soybean and G. soja. B09121 may be a useful tool for studies of the structural and functional properties of F3'5'H genes as well as investigations on the role of flower color in relation to adaptation of G. soja to natural habitats.
Project description:In soybean, flavonoid 3'5'-hydroxylase (F3'5'H) and dihydroflavonol-4-reductase (DFR) play a crucial role in the production of anthocyanin pigments. Loss-of-function of the W1 locus, which encodes the former, or W3 and W4, which encode the latter, always produces white flowers. In this study, we searched for new genetic components responsible for the production of white flowers in soybean and isolated four white-flowered mutant lines, i.e., two Glycine soja accessions (CW12700 and CW13381) and two EMS-induced mutants of Glycine max (PE1837 and PE636). F3'5'H expression in CW12700, PE1837, and PE636 was normal, whereas that in CW13381 was aberrant and missing the third exon. Sequence analysis of F3'5'H of CW13381 revealed the presence of an indel (~90-bp AT-repeat) in the second intron. In addition, the F3'5'H of CW12700, PE1837, and PE636 harbored unique single-nucleotide substitutions. The single nucleotide polymorphisms resulted in substitutions of amino acid residues located in or near the SRS4 domain of F3'5'H, which is essential for substrate recognition. 3D structure modeling of F3'5'H indicated that the substitutions could interfere with an interaction between the substrate and heme group and compromise the conformation of the active site of F3'5'H. Recombination analysis revealed a tight correlation between all of the mutant alleles at the W1 locus and white flower color. On the basis of the characterization of the new mutant alleles, we discussed the biological implications of F3'5'H and DFR in the determination of flower colors in soybean.
Project description:BACKGROUND: Flower 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). RESULTS: Complementation 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. CONCLUSIONS: Complete 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.
Project description:Color is one of the phenotypic markers mostly used to study soybean (Glycine max L. Merr.) genetic, molecular and biochemical processes. Two P450-dependent mono-oxygenases, flavonoid 3'-hydroxylase (F3'H; EC220.127.116.11) and flavonoid 3',5'-hydroxylase (F3'5'H, EC18.104.22.168), both catalyzing the hydroxylation of the B-ring in flavonoids, play an important role in coloration. Previous studies showed that the T locus was a gene encoding F3'H and the W1 locus co-segregated with a gene encoding F3'5'H in soybean. These two genetic loci have identified to control seed coat, flower and pubescence colors. However, the allelic distributions of both F3'H and F3'5'H genes in soybean were unknown. In this study, three novel alleles were identified (two of four alleles for GmF3'H and one of three alleles for GmF3'5'H). A set of gene-tagged markers was developed and verified based on the sequence diversity of all seven alleles. Furthermore, the markers were used to analyze soybean accessions including 170 cultivated soybeans (G. max) from a mini core collection and 102 wild soybeans (G. soja). For both F3'H and F3'5'H, the marker selection efficiencies for pubescence color and flower color were determined. The results showed that one GmF3'H allele explained 92.2 % of the variation in tawny and two gmf3'h alleles explained 63.8 % of the variation in gray pubescence colors. In addition, two GmF3'5'H alleles and one gmF3'5'h allele explained 94.0 % of the variation in purple and 75.3 % in white flowers, respectively. By the combination of the two loci, seed coat color was determined. In total, 90.9 % of accessions possessing both the gmf3'h-b and gmf3'5'h alleles had yellow seed coats. Therefore, seed coat colors are controlled by more than two loci.
Project description:The substitution pattern of anthocyanin pigments is a main determinant of flower color. Flavonoid 3',5'-hydroxylase (F3'5'H) is a cytochrome P450 enzyme (Cyt P450) that catalyzes the 3', 5'-hydroxylation of dihydroflavonols, the precursors of purple anthocyanins. Species such as rose and carnation lack F3'5'H activity and are, therefore, unable to generate purple or blue flowers. Petunia, on the other hand, contains two loci, termed hf1 and hf2, that encode a Cyt P450 with F3'5'H activity. Here we report the identification of an additional petunia gene that is required for 3',5' substitution of anthocyanins and purple flower colors. It encodes a cytochrome b5 and is expressed exclusively in the flower. Inactivation of the gene by targeted transposon mutagenesis reduced F3'5'H enzyme activity and the accumulation of 5'-substituted anthocyanins, resulting in an altered flower color. However, no phenotypic effect on the activity of other Cyt P450s, involved in the synthesis of hormones or general phenylpropanoids, was observed. These data provide in vivo evidence for the regulation of the activity of specific Cyt P450s by a cytochrome b5.
Project description:Almost all flowers of the tea plant (<i>Camellia sinensis</i>) are white, which has caused few researchers to pay attention to anthocyanin accumulation and color changing in tea flowers. A new purple-leaf cultivar, Baitang purple tea (BTP) was discovered in the Baitang Mountains of Guangdong, whose flowers are naturally pink, and can provide an opportunity to understand anthocyanin metabolic networks and flower color development in tea flowers. In the present study, twelve anthocyanin components were identified in the pink tea flowers, namely cyanidin <i>O</i>-syringic acid, petunidin 3-<i>O</i>-glucoside, pelargonidin 3-<i>O</i>-beta-d-glucoside, which marks the first time these compounds have been found in the tea flowers. The presence of these anthocyanins seem most likely to be the reason for the pink coloration of the flowers. Twenty-one differentially expressed genes (DEGs) involved in anthocyanin pathway were identified using KEGG pathway functional enrichment, and ten of these DEG's screened using venn and KEGG functional enrichment analysis during five subsequent stages of flower development. By comparing DEGs and their expression levels across multiple flower development stages, we found that anthocyanin biosynthesis and accumulation in BTP flowers mainly occurred between the third and fourth stages (BTP3 to BTP4). Particularly, during the period of peak anthocyanin synthesis 17 structural genes were upregulated, and four structural genes were downregulated only. Ultimately, eight critical genes were identified using weighted gene co-expression network analysis (WGCNA), which were found to have direct impact on biosynthesis and accumulation of three flavonoid compounds, namely cyanidin 3-<i>O</i>-glucoside, petunidin 3-<i>O</i>-glucoside and epicatechin gallate. These results provide useful information about the molecular mechanisms of coloration in rare pink tea flower of anthocyanin-rich tea, enriching the gene resource and guiding further research on anthocyanin accumulation in purple tea.
Project description:Tea (<i>Camellia sinensis</i>) flowers are normally white, even though the leaves could be purple. We previously discovered a specific variety with purple leaves and flowers. In the face of such a phenomenon, researchers usually focus on the mechanism of color formation but ignore the change of aroma. The purple tea flowers contain more anthocyanins, which belong to flavonoids. Meanwhile, phenylalanine (Phe), derived from the shikimate pathway, is a precursor for both flavonoids and volatile benzenoid-phenylpropanoids (BPs). Thus, it is not clear whether the BP aroma was attenuated for the appearance of purple color. In this study, we integrated metabolome and transcriptome of petals of two tea varieties, namely, Zijuan (ZJ) with white flowers and Baitang (BT) with purple flowers, to reveal the relationship between color (anthocyanins) and aroma (volatile BPs). The results indicated that in purple petals, the upstream shikimate pathway promoted for 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase (DAHPS) was elevated. Among the increased anthocyanins, delphinidin-3-<i>O</i>-glucoside (DpG) was extremely higher; volatile BPs, including benzyl aldehyde, benzyl alcohol, acetophenone (AP), 1-phenylethanol, and 2-phenylethanol, were also enhanced, and AP was largely elevated. The structural genes related to the biosynthesis of volatile BPs were induced, while the whole flavonoid biosynthesis pathway was downregulated, except for the genes <i>flavonoid 3'-hydroxylase</i> (<i>F3'H</i>) and <i>flavonoid 3',5'-hydroxylase</i> (<i>F3'5'H</i>), which were highly expressed to shift the carbon flux to delphinidin, which was then conjugated to glucoside by increased bronze-1 (BZ1) (UDP-glucose: flavonoid 3-<i>O</i>-glucosyltransferase) to form DpG. Transcription factors (TFs) highly related to AP and DpG were selected to investigate their correlation with the differentially expressed structural genes. TFs, such as MYB, AP2/ERF, bZIP, TCP, and GATA, were dramatically expressed and focused on the regulation of genes in the upstream synthesis of Phe (<i>DAHPS</i>; <i>arogenate dehydratase/prephenate</i> <i>dehydratase</i>) and the synthesis of AP (<i>phenylacetaldehyde reductase</i>; <i>short-chain dehydrogenase/reductase</i>), Dp (<i>F3'H</i>; <i>F3'5'H</i>), and DpG (<i>BZ1</i>), but inhibited the formation of flavones (<i>flavonol synthase</i>) and catechins (<i>leucoanthocyanidin reductase</i>). These results discovered an unexpected promotion of volatile BPs in purple tea flowers and extended our understanding of the relationship between the BP-type color and aroma in the tea plant.
Project description:Chrysanthemum (Chrysanthemum × morifolium) is one of the most important ornamental plants in the world. They are typically used as cut flowers or potted plants. Chrysanthemum can exhibit red, purple, pink, yellow and white flowers, but lack bright red and blue flowers. In this study, we identified two chrysanthemum cultivars, C × morifolium 'LPi' and C × morifolium 'LPu', that only accumulate flavonoids in their ligulate flowers. Next, we isolated seven anthocyanin biosynthesis genes, namely CmCHS, CmF3H, CmF3'H, CmDFR, CmANS, CmCHI and Cm3GT in these cultivars. RT-PCR and qRT-PCR analyses showed that CmF3'H was the most important enzyme required for cyanidin biosynthsis. To rebuild the delphinidin pathway, we downregulated CmF3'H using RNAi and overexpressed the Senecio cruentus F3'5'H (PCFH) gene in chrysanthemum. The resultant chrysanthemum demonstrated a significantly increased content of cyanidin and brighter red flower petals but did not accumulate delphinidin. These results indicated that CmF3'H in chrysanthemum is important for anthocyanin accumulation, and Senecio cruentus F3'5'H only exhibited F3'H activity in chrysanthemum but did not rebuild the delphinidin pathway to form blue flower chrysanthemum.
Project description:Various colored cultivars of ornamental flowers have been bred by hybridization and mutation breeding; however, the generation of blue flowers for major cut flower plants, such as roses, chrysanthemums, and carnations, has not been achieved by conventional breeding or genetic engineering. Most blue-hued flowers contain delphinidin-based anthocyanins; therefore, delphinidin-producing carnation, rose, and chrysanthemum flowers have been generated by overexpression of the gene encoding flavonoid 3',5'-hydroxylase (F3'5'H), the key enzyme for delphinidin biosynthesis. Even so, the flowers are purple/violet rather than blue. To generate true blue flowers, blue pigments, such as polyacylated anthocyanins and metal complexes, must be introduced by metabolic engineering; however, introducing and controlling multiple transgenes in plants are complicated processes. We succeeded in generating blue chrysanthemum flowers by introduction of butterfly pea UDP (uridine diphosphate)-glucose:anthocyanin 3',5'-O-glucosyltransferase gene, in addition to the expression of the Canterbury bells F3'5'H. Newly synthesized 3',5'-diglucosylated delphinidin-based anthocyanins exhibited a violet color under the weakly acidic pH conditions of flower petal juice and showed a blue color only through intermolecular association, termed "copigmentation," with flavone glucosides in planta. Thus, we achieved the development of blue color by a two-step modification of the anthocyanin structure. This simple method is a promising approach to generate blue flowers in various ornamental plants by metabolic engineering.
Project description:<i>Nicotiana alata</i> is an ornamental horticultural plant with a variety of flower colors and a long flowering period. The genes in four different colored <i>N. alata</i> (white, purple, red, and lemon green) were analyzed to explain the differences in flower color using transcriptomes. A total of 32 differential expression genes in the chlorophyll biosynthesis pathway and 41 in the anthocyanin biosynthesis pathway were identified. The enrichment analysis showed that the chlorophyll biosynthesis pathway and anthocyanin biosynthesis pathway play critical roles in the color differences of <i>N. alata</i>. The <i>HEMA</i> of the chlorophyll biosynthesis pathway was up-regulated in lemon green flowers. Compared with white flowers, in the red and purple flowers, <i>F3H</i>, <i>F3'5'H</i> and <i>DFR</i> were significantly up-regulated, while <i>FLS</i> was significantly down-regulated. Seventeen differential expression genes homologous to transcription factor coding genes were obtained, and the homologues of <i>HY5</i>, <i>MYB12, AN1</i> and <i>AN4</i> were also involved in flower color differences. The discovery of these candidate genes related to flower color differences is significant for further research on the flower colors formation mechanism and color improvements of <i>N. alata</i>.
Project description:BACKGROUND:The objectives of this study were to reveal the anthocyanin biosynthesis metabolic pathway in white and purple flowers of Salvia miltiorrhiza using metabolomics and transcriptomics, to identify different anthocyanin metabolites, and to analyze the differentially expressed genes involved in anthocyanin biosynthesis. RESULTS:We analyzed the metabolomics and transcriptomics data of S. miltiorrhiza flowers. A total of 1994 differentially expressed genes and 84 flavonoid metabolites were identified between the white and purple flowers of S. miltiorrhiza. Integrated analysis of transcriptomics and metabolomics showed that cyanidin 3,5-O-diglucoside, malvidin 3,5-diglucoside, and cyanidin 3-O-galactoside were mainly responsible for the purple flower color of S. miltiorrhiza. A total of 100 unigenes encoding 10 enzymes were identified as candidate genes involved in anthocyanin biosynthesis in S. miltiorrhiza flowers. Low expression of the ANS gene decreased the anthocyanin content but enhanced the accumulation of flavonoids in S. miltiorrhiza flowers. CONCLUSIONS:Our results provide valuable information on the anthocyanin metabolites and the candidate genes involved in the anthocyanin biosynthesis pathways in S. miltiorrhiza.