Flavonoid hydroxylase from Catharanthus roseus: cDNA, heterologous expression, enzyme properties and cell-type specific expression in plants

We investigated the P450 dependent flavonoid hydroxylase from the ornamental plant Catharanthus roseus. cDNAs were obtained by heterologous screening with the CYP75 Hf1 cDNA from Petunia hybrida. The C. roseus protein shared 68-78% identity with other CYP75s, and genomic blots suggested one or two genes. The protein was expressed in Escherichia coli as translational fusion with the P450 reductase from C. roseus. Enzyme assays showed that it was a flavonoid 3', 5'-hydroxylase, but 3'-hydroxylated products were also detected. The substrate specificity was investigated with the C. roseus enzyme and a fusion protein of the Petunia hybrida CYP75 with the C. roseus P450 reductase. Both enzymes accepted flavanones as well as flavones, dihydroflavonols and flavonols, and both performed 3'- as well as 3'5'-hydroxylation. Kinetics with C. roseus cultures on the level of enzyme activity, protein and RNA showed that the F3'5'H was present in dark-grown cells and was induced by irradiation. The same results were obtained for cinnamic acid 4-hydroxylase and flavanone 3beta-hydroxylase. In contrast, CHS expression was strictly dependent on light, although CHS is necessary in the synthesis of the F3'5'H substrates. Immunohistochemical localization of F3'5'H had not been performed before. A comparison of CHS and F3'5'H in cotyledons and flower buds from C. roseus identified CHS expression preferentially in the epidermis, while F3'5'H was only detected in the phloem. The cell-type specific expression suggests that intercellular transport may play an important role in the compartmentation of the pathways to the different flavonoids.     

Identification and characterisation of CYP75A31, a new flavonoid 3'5'-hydroxylase,isolated from <Emphasis Type="Italic">Solanum lycopersicum</Emphasis>

Background  

Understanding the regulation of the flavonoid pathway is important for maximising the nutritional value of crop plants and possibly enhancing their resistance towards pathogens. The flavonoid 3'5'-hydroxylase (F3'5'H) enzyme functions at an important branch point between flavonol and anthocyanin synthesis, as is evident from studies in petunia (Petunia hybrida), and potato (Solanum tuberosum). The present work involves the identification and characterisation of a F3'5'H gene from tomato (Solanum lycopersicum), and the examination of its putative role in flavonoid metabolism.     

Identification of the molecular basis for the functional difference between flavonoid 3'-hydroxylase and flavonoid 3',5'-hydroxylase

Flavonoid 3'-hydroxylase (F3'H) and flavonoid 3',5'-hydroxylase (F3'5'H) are cytochrome P450 enzymes and determine the B-ring hydroxylation pattern of flavonoids by introducing hydroxyl groups at the 3'- or the 3'- and 5'-position, respectively. Sequence identity between F3'H and F3'5'H is generally low since their divergence took place early in the evolution of higher plants. However, in the Asteraceae the family-specific evolution of an F3'5'H from an F3'H precursor occurred, and consequently sequence identity is substantially higher. We used this phenomenon for alignment studies, in order to identify regions which could be involved in determining substrate specificity and functionality. Subsequent construction and expression of chimeric genes indicated that substrate specificity of F3'H and F3'5'H is determined near the N-terminal end and the functional difference between these two enzymes near the C-terminal end. The impact on function of individual amino acids located in substrate recognition site 6 (SRS6) was further tested by site-directed mutagenesis. Most interestingly, a conservative Thr to Ser exchange at position 487 conferred additional 5'-hydroxylation activity to recombinant Gerbera hybrida F3'H, whereas the reverse substitution transformed recombinant Osteospermum hybrida F3'5'H into an F3'H with low remaining 5'-hydroxylation activity. Since the physicochemical properties of Thr and Ser are highly similar, the difference in size appears to be the main factor contributing to functional difference. The results further suggest that relatively few amino acids exchanges were required for the evolutionary extension of 3'- to 3',5'-hydroxylation activity.     

Genetic engineering of the anthocyanin biosynthetic pathway with flavonoid-3′,5′-hydroxylase: specific switching of the pathway in petunia

Flavonoid-3',5'-hydroxylase (F3'5'H) is the key enzyme in the synthesis of 3',5'-hydroxylated anthocyanins, which are generally required for the expression of blue or purple flower color. It has been predicted that the introduction of this enzyme into a plant species that lacks it would enable the production of blue or purple flowers by altering the anthocyanin composition. We present here the results of the genetic engineering of petunia flower color, pigmentation patterns and anthocyanin composition with sense or antisense constructs of the F3'5'H gene under the control of the CaMV 35S promoter. When sense constructs were introduced into pink flower varieties that are deficient in the enzyme, transgenic plants showed flower color changes from pink to magenta along with changes in anthocyanin composition. Some transgenic plants showed novel pigmentation patterns, e.g. a star-shaped pattern. When sense constructs were introduced into blue flower petunia varieties, the flower color of the transgenic plants changed from deep blue to pale blue or even pale pink. Pigment composition analysis of the transgenic plants suggested that the F3'5'H transgene not only created or inhibited the biosynthetic pathway to 3',5'-hydroxylated anthocyanins but switched the pathway to 3',5'-hydroxylated or 3'-hydroxylated anthocyanins.     

Selective accumulation of delphinidin derivatives in tobacco using a putative flavonoid 3',5'-hydroxylase cDNA from Campanula medium

Blue flowers generally contain 3',5'-hydroxylated anthocyanins (delphinidin derivatives) as pigments, which are formed only in the presence of flavonoid 3',5'-hydroxylases (F3'5'H). Heterologous expression of a F3'5'H gene therefore provides an opportunity to produce novel blue flowers for a number of ornamental plants missing blue flowering varieties. However, our previous study indicated difficulties in obtaining good accumulation of delphinidin derivatives in plants expressing F3'5'H. Here we report the isolation of a putative F3'5'H cDNA (Ka1) from canterbury bells (Campanula medium) and its expression in tobacco. Surprisingly, compared with other F3'5'H cDNAs, Ka1 encoded a protein with a unique primary structure that conferred high competence in the accumulation of delphinidin derivatives (up to 99% of total anthocyanins) and produced novel purple flowers. These results suggest that, among F3'5' H cDNAs, Ka1 is the best genetic resource for the creation of fine blue flowers by genetic engineering.     

鲑鱼生长激素基因分泌型表达质粒的构建

生长激素(GH)是动物垂体前叶分泌的一种多肽类激素.应用分子重组及PCR等技术,构建了一种鲑鱼生长激素基因分泌型表达质粒pOsGH153,使编码鲑鱼生长激素成熟肽的序列克隆在大肠杆菌分泌型表达载体PIN-Ⅲ-ompA内,直接位于编码大肠杆菌外膜蛋白A信号肽序列的下游,在Lpp-Lac杂合启动子控制下,经IPTG诱导,分子量约23 000的鲑鱼生长激素在大肠杆菌中获得高效表达,该产物具有天然鲑鱼生长激素的免疫活性,直接分泌到细胞周质,而信号肽被自动剪除.     

The B gene of pea encodes a defective flavonoid 3',5'-hydroxylase, and confers pink flower color

The inheritance of flower color in pea (Pisum sativum) has been studied for more than a century, but many of the genes corresponding to these classical loci remain unidentified. Anthocyanins are the main flower pigments in pea. These are generated via the flavonoid biosynthetic pathway, which has been studied in detail and is well conserved among higher plants. A previous proposal that the Clariroseus (B) gene of pea controls hydroxylation at the 5' position of the B ring of flavonoid precursors of the anthocyanins suggested to us that the gene encoding flavonoid 3',5'-hydroxylase (F3'5'H), the enzyme that hydroxylates the 5' position of the B ring, was a good candidate for B. In order to test this hypothesis, we examined mutants generated by fast neutron bombardment. We found allelic pink-flowered b mutant lines that carried a variety of lesions in an F3'5'H gene, including complete gene deletions. The b mutants lacked glycosylated delphinidin and petunidin, the major pigments present in the progenitor purple-flowered wild-type pea. These results, combined with the finding that the F3'5'H gene cosegregates with b in a genetic mapping population, strongly support our hypothesis that the B gene of pea corresponds to a F3'5'H gene. The molecular characterization of genes involved in pigmentation in pea provides valuable anchor markers for comparative legume genomics and will help to identify differences in anthocyanin biosynthesis that lead to variation in pigmentation among legume species.     

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.     

Identification of the Arabidopsis thaliana flavonoid 3'-hydroxylase gene and functional expression of the encoded P450 enzyme

The phenylpropanoid pathway results in the synthesis of thousands of compounds, including flavonoids like flavonols, anthocyanidins and tannins. In Arabidopsis thaliana, the lack of tannins in the seed coat (testa) causes the transparent testa (tt) phenotype. In the present study, we identified the gene responsible for the tt7 mutation. We show that TT7 encodes the enzyme flavonoid 3'-hydroxylase (F3'H), and demonstrate that this P450-dependent monooxygenase has F3'H activity. The availability of the AtF3'H gene and promoter sequence will allow us to study the coregulation of a complete set of flavonol and anthocyanidin biosynthesis genes in A. thaliana, and makes in vitro synthesis of hydroxylated flavonoids more feasible.     

Cloning of the pleiotropic T locus in soybean and two recessive alleles that differentially affect structure and expression of the encoded flavonoid 3' hydroxylase

Three loci (I, R, and T) control pigmentation of the seed coats in Glycine max and are genetically distinct from those controlling flower color. The T locus also controls color of the trichome hairs. We report the identification and isolation of a flavonoid 3' hydroxylase gene from G. max (GmF3'H) and the linkage of this gene to the T locus. This GmF3'H gene was highly expressed in early stages of seed coat development and was expressed at very low levels or not at all in other tissues. Evidence that the GmF3'H gene is linked to the T locus came from the occurrence of multiple RFLPs in lines with varying alleles of the T locus, as well as in a population of plants segregating at that locus. GmF3'H genomic and cDNA sequence analysis of color mutant lines with varying t alleles revealed a frameshift mutation in one of the alleles. In another line derived from a mutable genetic stock, the abundance of the mRNAs for GmF3'H was dramatically reduced. Isolation of the GmF3'H gene and its identification as the T locus will enable investigation of the pleiotropic effects of the T locus on cell wall integrity and its involvement in the regulation of the multiple branches of the flavonoid pathway in soybean.     

转基因香石竹中F3’5’H基因的克隆、表达和免疫学鉴定

在研究转基因香石竹品系月之霓裳（Moonshade）、月之伊人（Moonlite）中外源基因F3’5’H的表达中,本文克隆了F3’5’H全长基因1.5kb,构建获得工程菌株Escherichia coli BL21（DE3）（＋F3＇5＇H）。SDS-PAGE分析的结果显示,该菌株高效表达出F3’5’H重组蛋白,约占菌体总蛋白的30%。用经纯化的F3’5’H重组蛋白作为抗原,制备F3’5’H重组蛋白的抗血清,经ELISA免疫学分析表明,该抗血清的效价为1：25600。Western blot结果表明F3’5’H重组蛋白具有良好的IgG结合活性,且抗血清与转基因香石竹品系月之霓裳和月之伊人中的外源基因F3’5’H所表达的蛋白发生明显的抗原抗体反应。这样,月之霓裳和月之伊人用于评价转基因香石竹品系的环境安全性在我国也得到了验证。     

Regulation of gene expression involved in flavonol and anthocyanin biosynthesis during petal development in lisianthus (Eustoma grandiflorum)

To elucidate gene regulation of flower colour formation, the gene expressions of the enzymes involved in flavonoid biosynthesis were investigated in correlation with their product during floral development in lisianthus. Full-length cDNA clones of major responsible genes in the central flavonoid biosynthetic pathway, including chalcone synthase (CHS), chalcone isomerase (CHI), flavanone 3-hydroxylase (F3H), flavonoid 3',5'-hydroxylase (F3'5'H), dihydroflavonol 4-reductase (DFR), anthocyanidin synthase (ANS), and flavonol synthase (FLS), were isolated and characterized. In lisianthus, the stage of the accumulation of flavonols and anthocyanins was shown to be divided clearly. The flavonol content increased prior to anthocyanin accumulation during floral development and declined when anthocyanin began to accumulate. CHS, CHI, and F3H were necessary for both flavonol and anthocyanin biosynthesis and were coordinately expressed throughout all stages of floral development; their expressions were activated independently at the stages corresponding to flavonol accumulation and anthocyanin accumulation, respectively. Consistent with flavonol and anthocyanin accumulation patterns, FLS, a key enzyme in flavonol biosynthesis, was expressed prior to the expression of the genes involved in anthocyanin biosynthesis. The genes encoding F3'5'H, DFR, and ANS were expressed at later stages, just before pigmentation. The genes responsible for the flavonoid pathways branching to anthocyanins and flavonols were strictly regulated and were coordinated temporally to correspond to the biosynthetic order of their respective enzymes in the pathways, as well as in specific organs. In lisianthus, FLS and DFR, at the position of branching to flavonols and anthocyanins, were supposed to play a critical role in regulation of each biosynthesis.     

The Asc locus for resistance to Alternaria stem canker in tomato does not encode the enzyme aspartate carbamoyltransferase

The fungal disease resistance locus Alternaria stem canker (Asc) in tomato has been suggested to encode the enzyme aspartate carbamoyltransferase (AC Tase). To test this hypothesis a segment of the tomato ACTase gene was amplified by the polymerase chain reaction (PCR) using degenerate primers. The PCR product obtained was subsequently used to isolate an ACTase cDNA clone. Restriction fragment length polymorphism (RFLP) linkage analysis showed that the ACTase gene and the Asc locus do not cosegregate. RFLP mapping positioned the ACTase gene on chromosome 11, while the Asc locus is located on chromosome 3. These results exclude the possibility that the ACTase protein is encoded by the Asc locus.