Biochemical complementation of the betalain biosynthetic pathway in Portulaca grandiflora by a fungal 3,4-dihydroxyphenylalanine dioxygenase

3,4-Dihydroxyphenylalanine (DOPA) dioxygenase from Amanitamuscaria catalyses the key reaction of betalain biosynthesis, namely the conversion of DOPA to betalamic acid by a 4,5-ring-opening reaction. In addition, it catalyses a 2,3 opening which yields the fungal pigment muscaflavin, a compound that has never been found in plants. In this work, a cDNA clone (DodA) encoding A. muscaria DOPA-dioxygenase was expressed in white Portulacagrandiflora petals, using the particle bombardment technique. Transformation resulted in the formation of yellow and violet spots that contained betalain pigments and muscaflavin, indicating that the fungal enzyme was expressed and active in plants, and could complement the plant betalain biosynthetic pathway. The presence of muscaflavin in transformed plants indicates a difference in the specificity of the plant and A.muscaria enzymes. Received: 24 February 1997 / Accepted: 20 May 1997     

Additional betalain accumulation by genetic engineering leads to a novel flower color in lisianthus (Eustoma grandiflorum)

Betalains, comprising violet betacyanins and yellow betaxanthins, are pigments found in plants belonging to the order Caryophyllales. In this study, we induced the accumulation of betalains in ornamental lisianthus (Eustoma grandiflorum) by genetic engineering. Three betalain biosynthetic genes encoding CYP76AD1, dihydroxyphenylalanine (DOPA) 4,5-dioxygenase (DOD), and cyclo-DOPA 5-O-glucosyltransferase (5GT) were expressed under the control of the cauliflower mosaic virus (CaMV) 35S promoter in lisianthus, in which anthocyanin pigments are responsible for the pink flower color. During the selection process on hygromycin-containing media, some shoots with red leaves were obtained. However, most red-colored shoots were suppressed root induction and incapable of further growth. Only clone #1 successfully acclimatized and bloomed, producing pinkish-red flowers, with a slightly greater intensity of red color than that in wild-type flowers. T₁ plants derived from clone #1 segregated into five typical flower color phenotypes: wine red, bright pink, pale pink, pale yellow, and salmon pink. Among these, line #1-1 showed high expression levels of all three transgenes and exhibited a novel wine-red flower color. In the flower petals of line #1-1, abundant betacyanins and low-level betaxanthins were coexistent with anthocyanins. In other lines, differences in the relative accumulation of betalain and anthocyanin pigments resulted in flower color variations, as described above. Thus, this study is the first to successfully produce novel flower color varieties in ornamental plants by controlling betalain accumulation through genetic engineering.     

A dopamine-based biosynthetic pathway produces decarboxylated betalains in Chenopodium quinoa

Betalains are the nitrogenous pigments that replace anthocyanins in the plant order Caryophyllales. Here, we describe unconventional decarboxylated betalains in quinoa (Chenopodium quinoa) grains. Decarboxylated betalains are derived from a previously unconsidered activity of the 4,5-DOPA-extradiol-dioxygenase enzyme (DODA), which has been identified as the key enzymatic step in the established biosynthetic pathway of betalains. Here, dopamine is fully characterized as an alternative substrate of the DODA enzyme able to yield an intermediate and structural unit of plant pigments: 6-decarboxy-betalamic acid, which is proposed and described. To characterize this activity, quinoa grains of different colors were analyzed in depth by chromatography, time-of-flight mass spectrometry, and reactions were performed in enzymatic assays and bioreactors. The enzymatic-chemical scheme proposed leads to an uncharacterized family of 6-decarboxylated betalains produced by a hitherto unknown enzymatic activity. All intermediate compounds as well as the final products of the dopamine-based biosynthetic pathway of pigments have been unambiguously determined and the reactions have been characterized from the enzymatic and functional perspectives. Results evidence a palette of molecules in quinoa grains of physiological relevance and which explain minor betalains described in plants of the Caryophyllales order. An entire family of betalains is anticipated.

A biosynthetic pathway produces unconventional plant pigments betalains derived from dopamine in quinoa.     

植物中甜菜色素的研究进展

甜菜色素是一类主要存在于石竹目植物中的天然植物色素，分为甜菜红素和甜菜黄素。甜菜色素不仅具有吸引昆虫授粉的作用，还可以作为一种重要的渗透调节物质和非酶促抗氧化剂来帮助植物抵抗逆境，维持植物体内正常的生理活动。甜菜色素生物合成途径独特，具有重要的医疗保健价值，目前已被广泛用于食品、药物和化妆品中。本文结合国内外最新研究进展，从理化性质、合成途径、生物学功能和应用价值等方面对甜菜色素进行了介绍，重点阐述了甜菜色素和花青素的关系以及光照对甜菜色素生物合成的影响，以期为进一步深入了解甜菜色素和开发利用甜菜色素提供参考依据。     

Ectopic Expression of the Pttkn1 Gene Induces Alterations in the Morphology of the Leaves and Flowers in Petunia hybrida Vilm

A novel knottedl-like homeobox (knox) gene, Pttknl (Populus tremula×tremuloides knotted1), isolated from the cambial region of hybrid aspen, was introduced into Petunia hybrida Vilm. using the leafdisc method mediated by Agrobacterium. A series of novel phenotypes was observed in transgenic petunia plants, including the formation of ectopic spikes on the adaxial surface of corollas and small petals on theabaxial surface of corollas, fusion of floral organs, shortening of corolla midribs, the formation of tumor-like knots along the midrib on the abaxial surface and serrated lobs of corolla margins, and alterations in petal color; except for changes in the leaves and plant architecture, RT-PCR showed that the Pttknl gene was expressed in the leaves of different petunia transgenic plants, whereas no signal was detected in wild-type plants. The possible function of Pttknl in leaf and flower development is discussed.     

Ectopic Expression of the Pttkn1 Gene Induces Alterations in the Morphology of the Leaves and Flowers in Petunia hybrida Vilm

A novel knottedl-like homeobox (knox) gene, Pttknl (Populus tremula×tremuloides knotted1), isolated from the cambial region of hybrid aspen, was introduced into Petunia hybrida Vilm. using the leafdisc method mediated by Agrobacterium. A series of novel phenotypes was observed in transgenic petunia plants, including the formation of ectopic spikes on the adaxial surface of corollas and small petals on theabaxial surface of corollas, fusion of floral organs, shortening of corolla midribs, the formation of tumor-like knots along the midrib on the abaxial surface and serrated lobs of corolla margins, and alterations in petal color; except for changes in the leaves and plant architecture, RT-PCR showed that the Pttknl gene was expressed in the leaves of different petunia transgenic plants, whereas no signal was detected in wild-type plants. The possible function of Pttknl in leaf and flower development is discussed.     

Metabolic engineering of ketocarotenoid biosynthesis in leaves and flowers of tobacco species

Ketocarotenoids and especially astaxanthin are high-valued pigments used as feed additives. Conventionally, they are provided by chemical synthesis. Their biological production is a promising alternative. For the development of a plant production system, Nicotiana glauca, a species with carotenoid-containing yellow pigmented flower petals, was transformed with a cyanobacterial ketolase gene. The resulting plants accumulated 4-ketozeaxantin (adinoxanthin), which is the first ketocarotenoid synthesized in flower petals by genetic modification. Due to the very late flowering in this tobacco species, N. tabacum was used to optimize the yield and ketocarotenoid product pattern by metabolic engineering of the ketolation steps of carotenogenesis. The highly carotenogenic nectary tissue in the flowers represents a model of a flower chromoplast system. By expression of a ketolase gene, it was possible to engineer the biosynthetic pathway towards the formation of 3'-hydroxyechinenone, 3-hydroxyechinenone, 4-ketozeaxanthin, 4-ketozeaxanthin esters, 4-ketolutein and 4-ketolutein esters. Some of these ketocarotenoids were also formed in the leaves of the trangenic plants. In particular, by co-expression of the ketolase gene in combination with a hydroxylase gene under an ubiquitous promoter, the formation of total carotenoids in nectaries increased by more than 2.5-fold. In the nectaries of this type of transformants, more than 50% of the accumulating carotenoids were keto derivatives. In addition, the levels of ketocarotenoid esters were much lower and a higher percentage of the free ketocarotenoids accumulated. These results open new promising perspectives for a successful metabolic engineering of keto-hydroxy carotenoid production in carotenogenic flowers.     

Molecular characterization of an anthocyanin-related glutathione S-transferase gene in cyclamen

Anthocyanins are a subclass of flavonoids and are a major contributor to flower colors ranging from red to blue and purple. Previous studies in model and ornamental plants indicate a member of the glutathione S-transferase (GST) gene family is involved in vacuolar accumulation of anthocyanins. In order to identify the anthocyanin-related GST in cyclamen, degenerate PCR was performed using total RNA from immature young petals. Four candidates of GSTs (CkmGST1 to CkmGST4) were isolated. Phylogenetic analysis indicated that CkmGST3 was closely related to PhAN9, an anthocyanin-related GST of petunia, and this clade was clustered with other known anthocyanin-related GSTs. Expression analysis at different developmental stages of petals revealed that CkmGST3 was strongly expressed in paler pigmented petals than in fully pigmented petals, in contrast to the constitutive expression of the other three candidates during petal development. This expression pattern of CkmGST3 was correlated with those of other anthocyanin biosynthetic genes such as CkmF3'5'H and CkmDFR2. Molecular complementation of Arabidopsis tt19, a knockout mutant of an anthocyanin-related GST gene, demonstrated that CkmGST3 could complement the anthocyanin-less phenotype of tt19. Transgenic plants that expressed the other three CkmGSTs did not show anthocyanin accumulation. These results indicate CkmGST3 functions in anthocyanin accumulation in cyclamen.     

A chalcone isomerase‐like protein enhances flavonoid production and flower pigmentation

Flavonoids are major pigments in plants, and their biosynthetic pathway is one of the best‐studied metabolic pathways. Here we have identified three mutations within a gene that result in pale‐colored flowers in the Japanese morning glory (Ipomoea nil). As the mutations lead to a reduction of the colorless flavonoid compound flavonol as well as of anthocyanins in the flower petal, the identified gene was designated enhancer of flavonoid production (EFP). EFP encodes a chalcone isomerase (CHI)‐related protein classified as a type IV CHI protein. CHI is the second committed enzyme of the flavonoid biosynthetic pathway, but type IV CHI proteins are thought to lack CHI enzymatic activity, and their functions remain unknown. The spatio‐temporal expression of EFP and structural genes encoding enzymes that produce flavonoids is very similar. Expression of both EFP and the structural genes is coordinately promoted by genes encoding R2R3‐MYB and WD40 family proteins. The EFP gene is widely distributed in land plants, and RNAi knockdown mutants of the EFP homologs in petunia (Petunia hybrida) and torenia (Torenia hybrida) had pale‐colored flowers and low amounts of anthocyanins. The flavonol and flavone contents in the knockdown petunia and torenia flowers, respectively, were also significantly decreased, suggesting that the EFP protein contributes in early step(s) of the flavonoid biosynthetic pathway to ensure production of flavonoid compounds. From these results, we conclude that EFP is an enhancer of flavonoid production and flower pigmentation, and its function is conserved among diverse land plant species.     

Isolation and characterization of the fragrant cyclamen O-methyltransferase involved in flower coloration

Anthocyanin O-methyltransferase (OMT) is one of the key enzymes for anthocyanin modification and flower pigmentation. We previously bred a novel red-purple-flowered fragrant cyclamen (KMrp) from the purple-flowered fragrant cyclamen 'Kaori-no-mai' (KM) by ion-beam irradiation. Since the major anthocyanins in KMrp and KM petals were delphinidin 3,5-diglucoside and malvidin 3,5-diglucoside, respectively, inactivation of a methylation step in the anthocyanin biosynthetic pathway was indicated in KMrp. We isolated and compared OMT genes expressed in KM and KMrp petals. RT-PCR analysis revealed that CkmOMT2 was expressed in the petals of KM but not in KMrp. Three additional CkmOMTs with identical sequences were expressed in petals of both KM and KMrp. Genomic PCR analysis revealed that CkmOMT2 was not amplified from the KMrp genome, indicating that ion-beam irradiation caused a loss of the entire CkmOMT2 region in KMrp. In vitro enzyme assay demonstrated that CkmOMT2 catalyzes the 3' or 3',5' O-methylation of the B-ring of anthocyanin substrates. These results suggest that CkmOMT2 is functional for anthocyanin methylation, and defective expression of CkmOMT2 is responsible for changes in anthocyanin composition and flower coloration in KMrp.     

高等植物二氢黄酮醇4-还原酶基因研究进展

花青素苷是影响植物花瓣呈色的重要色素,而花色是决定花卉观赏价值和商业价值的一个重要因素。在花青素苷的生物合成过程中,二氢黄酮醇4-还原酶（DFR）是花青素苷生物合成下游途径中的第一个关键的酶。因此,DFR在高等植物花色的形成过程中发挥极其重要的作用,是形成花青素苷的一个非常重要的调控点。DFR对3种二氢黄酮醇底物具有选择特异性,但决定DFR底物特异性的分子机制目前仍不十分清楚。该文简单概述了花青素苷生物合成途径及其转录调控机制,并结合作者的工作重点综述了DFR的底物特异性以及克隆的DFR基因在植物基因工程中的应用。     

查尔酮合酶基因对转基因植物花色和育性的影响

查尔酮合酶 ( chalcone synthase,CHS)是花色素合成途径中的一个关键酶 ,它在植物中表达量的改变可能影响花的颜色。从矮牵牛 ( Petunia hybrida)特定发育阶段的花瓣的 c DNA中 ,克隆到查尔酮合酶基因 ,并正向插入到原核表达载体和含有花椰菜花叶病毒 Ca MV 35 S启动子的真核表达载体中 ,在原核中得到高效表达 ,并通过土壤农杆菌介导的方法转化矮牵牛。转基因植物的花色不但发生了明显的变异 ,其育性也受到了影响 ,不能产生正常花粉粒 ,成为雄性不育植株。 Northern杂交表明 ,转基因植物花瓣中 ,内源及外源查尔酮合酶基因转录均受到抑制     

Transient Expression of the Cytochrome p450 CYP78A2 Enhances Anthocyanin Production in Flowers

Transient expression of a cytochrome P450 gene (CYP78A2) cloned from Phalaenopsis was shown to enhance the anthocyanin contents in the petals of transformed Phalaenopsis. In this study, it was characterized further to understand the relationship between this P450 and the anthocyanin biosynthesis in flowers. The enhancement effect exerted by the P450 gene exhibits the following characteristics. First, its product seems to be able to effectively boost the existing pathway of biosynthesis without causing synthesis of any new anthocyanin. Second, the effect is not limited to Phalaenopsis, a monocotyledon, but also occurs in dicotyledons such as carnation and rose, indicating its wide range of action in heterologous plants. Third, the gene is not expressed in petals at any stage of flower development of Phalaenopsis, thus ruling out its direct participation in anthocyanin biosynthesis. It is possible that this P450 gene is associated with the biosynthesis of plant hormones or second metabolites, and through which to positively and indirectly influence the existing biosynthesis pathway of anthocyanins in petals.     

植物甜菜素研究进展

植物花色素的研究已经非常广泛和深入, 但是甜菜素作为类似花色素的一种重要的植物次生代谢物质还不被人们所熟悉。为了增加人们对甜菜素的了解, 本文结合国内外研究的最新进展, 从种类与化学结构、提纯与鉴定、稳定性及影响因素、合成途径、生物功能和应用价值等方面对甜菜素进行了介绍, 重点比较分析了甜菜素和花色素结构和合成途径的不同与联系, 并对当前甜菜素研究的热点领域和亟待解决的问题进行了展望。