Cloning and molecular analysis of structural genes involved in flavonoid and stilbene biosynthesis in grape (Vitis vinifera L.)

Genes involved in flavonoid and stilbene biosynthesis were isolated from grape (Vitis vinifera L.). Clones coding for phenylalanine ammonia-lyase (PAL), chalcone synthase (CHS), chalcone isomerase (CHI), flavanone 3-hydoxylase (F3H), dihydroflavonol 4-reductase (DFR), leucoanthocyanidin dioxygenase (LDOX) and UDP glucose:flavonoid 3-O-glucosyl transferase (UFGT), were isolated by screening a cDNA library, obtained from mRNA from seedlings grown in light for 48 h using snapdragon (Antirrhinum majus) and maize heterologous probes. A cDNA clone coding for stilbene synthase (StSy) was isolated by probing the library with a specific oligonucleotide. These clones were sequenced and when the putative products were compared to the published amino acid sequence for corresponding enzymes, the percentages of similarity ranged from 65% (UFGT) to 90% (CHS and PAL). The analysis of the genomic organization and expression of these genes in response to light shows that PAL and StSy genes belong to large multigene families, while the others are present in one to four copies per haploid genome. The steady-state level of mRNAs encoded by the flavonoid biosynthetic genes as determined in young seedlings is coordinately induced by light, except for PAL and StSy, which appear to be constitutively expressed.     

Flavonoid synthesis in Petunia hybrida: partial characterization of dihydroflavonol-4-reductase genes

In this paper we describe the organization and expression of the genes encoding the flavonoid-biosynthetic enzyme dihydroflavonol-4-reductase (DFR) in Petunia hybrida. A nearly full-size DFR cDNA clone (1.5kb), isolated from a corolla-specific cDNA library was compared at the nucleotide level with the pallida gene from Antirrhinum majus and at the amino acid level with enzymes encoded by the pallida gene and the A1 gene from Zea mays.The P. hybrida and A. majus DFR genes transcribed in flowers contain 5 introns, at identical positions; the three introns of the A1 gene from Z. mays coincide with first three introns of the other two species. P. hybrida line V30 harbours three DFR genes (A, B, C) which were mapped by RFLP analysis on three different chromosomes (IV, II and VI respectively).Steady-state levels of DFR mRNA in the line V30 follow the same pattern during development as chalcone synthase (CHS) and chalcone flavanone isomerase (CHI) mRNA. Six mutants that accumulate dihydroflavonols in mature flowers were subjected to Northern blot analysis for the presence of DFR mRNA. Five of these mutants lack detectable levels of DFR mRNA. Four of these five also show drastically reduced levels of activity for the enzyme UDPG: flavonoid-3-O-glucosyltransferase (UFGT), which carries out the next step in flavonoid biosynthesis; these mutants might be considered as containing lesions in regulatory genes, controlling the expression of the structural genes in this part of the flavonoid biosynthetic pathway. Only the an6 mutant shows no detectable DFR mRNA but a wild-type level for UFGT activity. Since both an6 and DFR-A are located on chromosome IV and DFR-A is transcribed in floral tissues, it is postulated that the An6 locus contains the DFR structural gene. The an9 mutant shows a wild-type level of DFR mRNA and a wild-type UFGT activity.     

利用cDNA微阵列分离津田芜菁花青素生物合成相关基因

花色素苷是植物的重要次生代谢产物, 在植物体内行使多种生理功能。利用UV-A处理48 h后津田芜菁块根变红, 以黑暗处理条件下的白色块根为对照, 与削减文库特异基因片段制备的cDNA微阵列进行杂交。UV-A处理条件下津田芜菁中表达上调的基因为81个, 表达下调的基因为47个, 表达上调的基因中包括与花青素生物合成直接相关的基因片段cytochrome P450, PAL, F3H, ANS, CHS, DFR和GST等。Northern杂交结果显示, UV-A处理48 h的津田芜菁试材中, PAL、CHS、F3H、DFR和ANS基因的表达量明显高于黑暗条件下白色块根中这些基因的表达量, 进一步验证了芯片杂交结果的可靠性。     

Regulation of Flavonoid Biosynthetic Genes in Germinating Arabidopsis Seedlings

Many higher plants, including Arabidopsis, transiently display purple anthocyanin pigments just after seed germination. We observed that steady state levels of mRNAs encoded by four flavonoid biosynthetic genes, PAL1 (encoding phenylalanine ammonia-lyase 1), CHS (encoding chalcone synthase), CHI (encoding chalcone isomerase), and DFR (encoding dihydroflavonol reductase), were temporally regulated, peaking in 3-day-old seedlings grown in continuous white light. Except for the case of PAL1 mRNA, mRNA levels for these flavonoid genes were very low in seedlings grown in darkness. Light induction studies using seedlings grown in darkness showed that PAL1 mRNA began to accumulate before CHS and CHI mRNAs, which, in turn, began to accumulate before DFR mRNA. This order of induction is the same as the order of the biosynthetic steps in flavonoid biosynthesis. Our results suggest that the flavonoid biosynthetic pathway is coordinately regulated by a developmental timing mechanism during germination. Blue light and UVB light induction experiments using red light- and dark-grown seedlings showed that the flavonoid biosynthetic genes are induced most effectively by UVB light and that blue light induction is mediated by a specific blue light receptor.     

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.     

Cis-acting elements of the CHS1 gene from white mustard controlling promoter activity and spatial patterns of expression

Chalcone synthase (CHS) catalyses the first regulatory step in the branch pathway of phenylpropanoid biosynthesis specific for synthesis of ubiquitous flavonoid pigments and UV protectants. External stimuli such as stress, light and wounding induce CHS expression that is both tissue-specific and under developmental control. In order to identify cis-acting elements involved in organ and tissue specifity, we fused varying parts of the CHS1 promoter of white mustard (Sinapis alba L.) to the GUS-coding region and analysed the expression of these constructs in stably transformed Arabidopsis plants. Two different stages of development were examined, seedlings as an early stage and flowers as the final stage of development. In seedlings, the full-length promoter showed expression in all organs except the hypocotyl; in flowers expression could be observed in all whorls. Unit 1 of the mustard CHS1 promoter, an element conserved in several CHS genes, which has been recently identified as a light responsive element, is able to mediate a tissue-specific expression pattern similar to that obtained with the full-length promoter in seedlings as well as in flowers. Other elements enhance or repress expression in combination with Unit 1, or mediate defined spatial expression independently of Unit 1. One such element, located between-907 and -655, directs expression similar to that of the full-length promoter in flowers but not in seedlings and differs therefore in function to Unit 1. Our data suggest a dominant regulation of CHS1 expression by Unit 1. Other elements within this promoter might interact with Unit 1 or confer a subset of spatial expression patterns when Unit 1 is deleted.Abbreviations ADH alcohol dehydrogenase - CaMV cauliflower mosaic virus - CHS chalcone synthase - GUS -glucuronidase     

Gene expression profile of zeitlupe/lov kelch protein1 T-DNA insertion mutants in Arabidopsis thaliana: Downregulation of auxin-inducible genes in hypocotyls

Elongation of hypocotyl cells has been studied as a model for elucidating the contribution of cellular expansion to plant organ growth. ZEITLUPE (ZTL) or LOV KELCH PROTEIN1 (LKP1) is a positive regulator of warmth-induced hypocotyl elongation under white light in Arabidopsis, although the molecular mechanisms by which it promotes hypocotyl cell elongation remain unknown. Microarray analysis showed that 134 genes were upregulated and 204 genes including 15 auxin-inducible genes were downregulated in the seedlings of 2 ztl T-DNA insertion mutants grown under warm conditions with continuous white light. Application of a polar auxin transport inhibitor, an auxin antagonist or an auxin biosynthesis inhibitor inhibited hypocotyl elongation of control seedlings to the level observed with the ztl mutant. Our data suggest the involvement of auxin and auxin-inducible genes in ZTL-mediated hypocotyl elongation.     

Chalcone synthase and flavonoid products in primary-leaf tissues of rye and maize

During cell and tissue differentiation of developing rye (Secale cereale L.) and maize (Zea mays L.) primary leaves, various flavonoids are synthesized and accumulate in both epidermal and mesophyll tissues. In order to prove either the biosynthetic autonomy of each tissue type and- or intercellular transport of flavonoids, the tissue distributions of chalcone synthase (CHS; EC 2.3.1.74), the key enzyme of the pathway, and of flavonoids have been comparatively investigated. Monoclonal antibodies raised against CHS from rye were used to relate enzyme activity in a particular tissue extract to the corresponding amount of CHS protein. A close correlation was found between CHS activities and amounts of CHS protein during leaf development and in the various tissues. The simultaneous occurrence of CHS in both epidermal layers as well as in the mesophyll correlated with the accumulation of flavonoid products in these tissues, indicating tissue autonomy of flavonoid biosynthesis. These data are in contrast to previous reports (Knogge and Weissenböck, 1986, Planta 167, 196–205) on primary leaves of oat (Avena sativa) where CHS and several subsequent enzymes were located mainly in the mesophyll whereas major flavonoid products accumulated predominantly in both epidermal cell layers, indicating that intertissue transport of flavonoids might occur.     

A light-independent developmental mechanism potentiates flavonoid gene expression in Arabidopsis seedlings

Throughout the plant kingdom expression of the flavonoid biosynthetic pathway is precisely regulated in response to developmental signals, nutrient status, and environmental stimuli such as light, heat and pathogen attack. Previously we showed that, in developing Arabidopsis seedlings, flavonoid genes are transiently expressed during germination in a light-dependent manner, with maximal mRNA levels occurring in 3-day-old seedlings. Here we describe the relationship between developmental and environmental regulation of flavonoid biosynthesis by examining phenylalanine ammonia-lyase (PAL), chalcone synthase (CHS), chalcone isomerase (CHI), and dihydroflavonol reductase (DFR) mRNA levels in germinating Arabidopsis seedlings as a function of light, developmental stage and temperature. We show that seedlings exhibit a transient potential for induction of these four genes, which is distinct from that observed for chlorophyll a/b-binding protein (CAB). The potential for flavonoid gene induction was similar in seedlings grown in darkness and red light, indicating that induction potential is not linked to cotyledon expansion or the development of photosynthetic capacity. The evidence for metabolic regulation of flavonoid genes during seedling development is discussed.     

The chalcone synthase multigene family of Petunia hybrida (V30): differential,light-regulated expression during flower development and UV light induction

We have analysed the expression of the 8–10 members of the gene family encoding the flavonoid biosynthetic enzyme chalcone synthase (CHS) from Petunia hybrida. During normal plant development only two members of the gene family (CHS-A and CHS-J) are expressed. Their expression is restricted to floral tissues mainly. About 90% of the total CHS mRNA pool is transcribed from CHS-A, wheares CHS-J delivers about 10% in flower corolla, tube and anthers. Expression of CHS-A and CHS-J during flower development is coordinated and (red) light-dependent. In young seedlings and cell suspension cultures expression of CHS-A and CHS-J can be induced with UV light. In addition to CHS-A and CHS-J, expression of another two CHS genes (CHS-B and CHS-G) is induced in young seedlings by UV light, albeit at a low level. In contrast to CHS genes from Leguminoseae, Petunia CHS genes are not inducible by phytopathogen-derived elicitors. Expression of CHS-A and CHS-J is reduced to a similar extent in a regulatory CHS mutant, Petunia hybrida Red Star, suggesting that both genes are regulated by the same trans-acting factors. Comparison of the promoter sequences of CHS-A and CHS-J reveals some striking homologies, which might represent cis-acting regulatory sequences.     

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.     

Cloning,expression in yeast,and functional characterization of CYP71D14, a root-specific cytochrome P450 from <Emphasis Type="Italic">Petunia hybrida</Emphasis>

Alkaloids, which are naturally occurring amines, are biosynthesized and accumulated in plant tissues such as roots, leaves, and stems. Many alkaloids have pharmacological effects on humans and animals. Cytochrome P450 (P450 or CYP) monooxygenases are known to play key roles in the biosynthesis of alkaloids in higher plants. A cDNA clone encoding a P450 protein consisting of 502 amino acids was isolated from Petunia hybrida. The deduced amino acid sequence of the cDNA clone showed a high level of similarity with the other P450 species in the CYP71D family; hence, this novel P450 was named CYP71D14. Among plant P450 species, CYP71D14 had 45.7% similarity in its amino acid sequence to CYP71D12, which is involved in the biosynthesis of the indole alkaloids vinblastine and vincristine. Expression of CYP71D14 mRNA in Petunia plants was examined by Northern blot analysis by using a full-length cDNA of CYP71D14 as a probe. CYP71D14 mRNA was expressed most abundantly in the roots. The nucleotide sequence of CYP71D14 has been submitted to the DDBJ, EMBL, and GenBank nucleotide databases under the accession number AB028462. An erratum to this article can be found at     

Characterization and structural features of a chalcone synthase mutation in a white-flowering line of Matthiola incanaR. Br. (Brassicaceae)

For Matthiola incana (Brassicaceae), used as a model system to study biochemical and genetical aspects of anthocyanin biosynthesis, several nearly isogenic colored wild type lines and white-flowering mutant lines are available, each with a specific defect in the genes responsible for anthocyanin production (genes e, f, and g). For gene f supposed to code for chalcone synthase (CHS; EC 2.3.1.74), the key enzyme of the flavonoid/anthocyanin biosynthesis pathway belonging to the group of type III polyketide synthases (PKS), the wild type genomic sequence of M. incana line 04 was determined in comparison to the white-flowering CHS mutant line 18. The type of mutation in the chs gene was characterized as a single nucleotide substitution in a triplet AGG coding for an evolutionary conserved arginine into AGT coding for serine (R72S). Northern blots and RT-PCR demonstrated that the mutated gene is expressed in flower petals. Heterologous expression of the wild type and mutated CHS cDNA in E. Scherichia coli, verified by Western blotting and enzyme assays with various starter molecules, revealed that the mutant protein had no detectable activity, indicating that the strictly conserved arginine residue is essential for the enzymatic reaction. This mutation, which previously was not detected by mutagenic screening, is discussed in the light of structural and functional information on alfalfa CHS and related type III PKS enzymes.     

Organ- and Tissue-specific Biosynthesis of Flavonoids in Seedlings of Oenothera odorata (Onagraceae)

In developing Oenothera odorata seedlings, phytochrome-mediated accumulation of various flavonoids (mainly glycosides of cyanidin and quercetin) is detectable, subsequent to a transient induction of the key enzymes of the general phenylpropanoid metabolism, L-phenylalanine ammonia lyase (PAL) and of flavonoid biosynthesis, chalcone synthase (CHS). Organ- and tissue-specific distribution of these enzymes and of the flavonoid end products was investigated in seedlings, irradiated with continuous far-red light. Anthocyanins and quercetin glycosides are mainly localized in both the upper and lower epidermis of the cotyledons and to a lesser extent also in the epidermal cell layer of the hypocotyl. An obvious organ-specific distribution was observed for the anthocyanins: cyanidin-3,5-O-diglucoside accumulates in the epidermal cells of the cotyledons, whereas cyanidin-3-O-glucoside is restricted to the epidermis of the hypocotyl. By contrast the pattern of quercetin glycosides is the same in the cotyledons and in the hypocotyl. The methylated flavonol aglycone 3-0-methylquercetin was found to be localized in the seed coat. According to this organ- and tissue-specific pattern of flavonoids, immunochemical and immunohistochemical detection of PAL and CHS revealed a predominant localization of theenzymes in the epidermal layers of the cotyledons and the hypocotyl but also in the cells surrounding the vascular bundles. The role of compartmentation in regulation of flavonoid biosynthesis and putative functions of flavonoid compounds are discussed.