新規花色のためのカロテノイド代謝の遺伝子工学に関する研究

この博士論文は内容の要約のみの公開（または一部非公開）になっています筑波大学 (University of Tsukuba)201

Assessing Anthocyanin Biosynthesis in Solanaceae as a Model Pathway for Secondary Metabolism

Solanaceae have played an important role in elucidating how flower color is specified by the flavonoid biosynthesis pathway (FBP), which produces anthocyanins and other secondary metabolites. With well-established reverse genetics tools and rich genomic resources, Solanaceae provide a robust framework to examine the diversification of this well-studied pathway over short evolutionary timescales and to evaluate the predictability of genetic perturbation on pathway flux. Genomes of eight Solanaceae species, nine related asterids, and four rosids were mined to evaluate variation in copy number of the suite of FBP enzymes involved in anthocyanin biosynthesis. Comparison of annotation sources indicated that the NCBI annotation pipeline generated more and longer FBP annotations on average than genome-specific annotation pipelines. The pattern of diversification of each enzyme among asterids was assessed by phylogenetic analysis, showing that the CHS superfamily encompasses a large paralogous family of ancient and recent duplicates, whereas other FBP enzymes have diversified via recent duplications in particular lineages. Heterologous expression of a pansy F3050H gene in tobacco changed flower color from pink to dark purple, demonstrating that anthocyanin production can be predictably modified using reverse genetics. These results suggest that the Solanaceae FBP could be an ideal system to model genotype-to-phenotype interactions for secondary metabolism

Genetic control of anthocyanin pigmentation in Antirrhinum flowers

The genus Antirrhinum (commonly known as snapdragons) contains more than twentyfive recognised species. The genus has been divided into three morphological subsections: Antirrhinum, Streptosepalum and Kickxiella (Rothmaler, 1956). One of the major characteristics distinguishing the three subsections is flower colour. Most species in subsection Antirrhinum have dark pink or yellow flowers, Kickxiella species are white or pale pink and Streptosepalum species have yellow or pale pink flowers. All Antirrhinum species can be crossed to produce fertile hybrids which allow the genes that underlie their differences to be identified. I used quantitative trait locus (QTL) analysis on hybrids of A. majus (dark magenta flowers) and A. charidemi (pale-pink flowers) to map genomic regions underlying differences in flower colour. This identified two major-effect loci, in Linkage Group 3 (LG3) and LG7, that explained most of the differences between these species. I used near-isogenic lines (NILs) to further test involvement of two candidate genes - Rosea (Ros) in LG3, which encodes a regulator of the anthocyanin biosynthesis pathway (ABP) and Incolorata (Inc) in LG7 which encodes a rate-limiting enzyme of the ABP. In both cases, the A. majus allele increased pigmentation. Sequence differences between Ros alleles of A. majus, A. charidemi and A. molle (a Kickxiella species with white flowers) suggest that A. molle carries a ros loss-of-function mutation and that a transposon insertion in the ROS promoter might contribute to differences in expression between A. majus and A. charidemi. Ros genotypes were found to be strongly correlated with pigmentation in the corolla tube in A. majus x A. charidemi hybrids, and to a lesser extent with corolla lobe pigmentation, although NILs suggested that ROS did not correspond to the major-effect QTL indentified in LG3. I also mapped a minor-effect QTL for tube pigmentation to a region of LG4 containing the ABP structural gene Candica. Analysis of NILs revealed that Inc was not the second major-effect QTL mapped to LG7, although sequence differences were detected between Inc alleles of A. majus and A. charidemi. I was further able to narrow down the region containing the second LG7 major-effect QTL to an interval of 11 cM, between two molecular markers, which could be used to determine the likely QTL genotypes of segregating NILs. Surprisingly, several ABP genes, particularly Nivea, Inc and Pallida, were expressed at higher levels in pale flowers that were homozygous for the A. chardemi QTL allele than in their dark flowered siblings that carried an A. majus allele. This suggests that ABP genes might be up-regulated in pale flowers as part of a negative feedback mechanism. Two potential roles of the LG7 QTL are considered 1) its requirement for anthocyanin modification or transport to the vacuole, so that a build-up of cytosolic anthocyanins or their break-down products in pale flowers increases structural gene expression but cannot compensate for the overall reduction in anthocyanin, or 2) a role in promoting production of flavonols at the expense of anthocyanins

Development of anthocyanin markers: gene mapping, genomic analysis and genetic diversity studies in Ipomoea species

The anthocyanins are pigments responsible for a wide range of colours in plants, from blue, red and purple, play essential biological roles as well as their genes are evolutionarily conserved. Purple sweet potatoes have anthocyanins as the predominant colour, even though they are present in orange roots masked by carotenoids. Several studies have focused on molecular aspects of anthocyanin genes, mainly in wild Ipomoea species, although the structure and segregation analysis of those genes in sweet potato hexaploid species are still unknown. Based on an “exon-primed intron crossing” (EPIC) approach, fourteen pairs of primers were designed, on fi ve structural anthocyanin genes as candidates. The strategy exploits the Intron Length Polymorphism (ILP) from Candidate Genes (CG), resulting in 93% of successful markers giving scorable and reproducible alleles. The results allowed to defi ne partial structure and sequence of the introns and exons from the selected CG, and to determine patterns of sequence variation. The evaluation of marker dosage and allelic segregations in an Ipomoea batatas (L.) Lam mapping population identifi ed several alleles for linkage analysis. The study validated the utility of ILP-CG markers for genetic diversity and conservation applicability and a successful amplifi cation gradient across wild Ipomoea species validated their transferabilityFil: Arizio, Carla Marcela. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Recursos Biológicos; ArgentinaFil: Costa Tártara, Sabrina María. Universidad Nacional de Luján; ArgentinaFil: Zunino, Ignacio. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Pergamino. Agencia de Extensión Rural Mercedes; ArgentinaFil: Manifesto, Maria Marcela. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Recursos Biológicos; Argentin

Flavonoid Metabolomics in Gerbera hybrida and Elucidation of Complexity in the Flavonoid Biosynthetic Pathway

The flavonoid biosynthetic pathway and flavonoid metabolites have been extensively studied because of their biological roles in plant and animal kingdoms. Flavonoid metabolites are involved in plant resistance, UV-protection, pollinator attraction and have antioxidant effects. Plant species synthesise a large number of secondary metabolites, including flavonoids, carotenoids and others. Flower colour is an important feature for marketing of the ornamental model plant gerbera (Gerbera hybrida). In gerbera, flavonoid metabolites accumulate in the adaxial epidermal layer of flower petals and lead to different flower colours and patterns. These metabolites consist of three subgroups, flavones, flavonols and anthocyanins in gerbera, which have also been used to characterize different cultivars. The cultivars show variation in flower colour according to their anthocyanin type. The acyanic gerbera cultivars have flavonoid profiles comparable to the cyanic cultivars, except for the synthesis and accumulation of specific anthocyanins. Metabolite profiles indicated that all analysed acyanic cultivars have a block in a late stage of the anthocyanin pathway. Ivory, a sport of the pelargonidin-cultivar Estelle, has white flowers. Ivory has flavones and flavonols, but no anthocyanin. Gene expression of all flavonoid pathway genes was similar in Estelle and Ivory. However, both cultivars have two different alleles encoding dihydroflavonol 4-reductase and in Ivory one of them (GDFR1-2) was found to have a point mutation resulting in inactivation of the encoded enzyme. Still, Ivory expresses the second allele (GDFR1-3) and accumulates active DFR enzyme. The cyanidin cultivar President expresses only the GDFR1-3 allele, but cannot synthesize pelargonidin. Therefore, GDFR1-2 contributes specifically to pelargonidin biosynthesis and GDFR1-3 to cyanidin biosynthesis. This could be explained by a coordinated biosynthesis of anthocyanins in multi-enzyme complexes, metabolons. Gerbera chalcone synthases (GCHSs) belong to the superfamily of Type III polyketide synthase enzymes. GCHS1, 3 and 4 have different contributions to the flavonoid pathway, according to the tissue specific and post-transcriptional regulation. RNA interference of CHS encoding genes was used in different gerbera cultivars to show that GCHS1 has the main contribution to anthocyanin accumulation in petal tissues. GCHS4 was strongly expressed in petals but did not lead to anthocyanin accumulation. Still, GCHS4 is expressed and encoded a functional enzyme in the vegetative tissues.Flavonoidit ovat kasvien fenolisia yhdisteitä, jotka voivat olla värittömiä (flavonit ja flavonolit) tai oranssin, punaisen tai sinisen sävyisiä pigmenttejä (antosyaanit). Flavonodit suojaavat kasvia UV-säteilyltä ja taudinaiheuttajilta, ja toisaalta houkuttelevat hyödyllisiä pölyttäjiä. Flavonoidit ovat ravinnossa tärkeitä antioksidantteja ja niiden biosynteesiä on tutkittu paljon. Tässä työssä tutkittiin koristekasvina tunnetun gerberan kukinnon flavonoidien kemiaa, entsymologiaa ja geeniekspressiota. Materiaalina käytettiin lajikkeita, jotka sisälsivät flavoneja (apigeniinia tai luteiinia), flavonoleja (kemferolia tai kversetiiniä) ja/tai antosyaaneja (pelargonidiineja tai syanidiineja). Punaisesta Estelle-lajikkeen sivuhaarana syntyneessä valkoisessa Ivory-lajikkeessa todettiin mutaatio antosyaanibiosynteesille tärkeässä DFR-entsyymiä koodaavassa geenissä. Toinen DFR-alleeli kuitenkin koodasi toimivaa entsyymiä, joka kasvissa ei johtanut pigmentin biosynteesiin vaikka geeni ilmeni. Samalla tavalla flavonoidireitin alkupäässä olevan CHS-entsyymin kohdalla todettiin, että kahdesta kukinnossa ilmenevästä muodosta vain toinen oli tärkeä kukinnon antosyaanien biosynteesille. Toinen vastasi lehtiruotiin muodostuvasta antosyaanista. Yhteenvetona voidaan todeta, että flavonodien biosynteesiä säädellään geberalla geenitoiminnan lisäksi myös proteiinitasolla

Hybrid de novo transcriptome assembly of poinsettia (Euphorbia pulcherrima Willd. Ex Klotsch) bracts

Background Poinsettia is a popular and important ornamental crop, mostly during the Christmas season. Its bract coloration ranges from pink/red to creamy/white shades. Despite its ornamental value, there is a lack of knowledge about the genetics and molecular biology of poinsettia, especially on the mechanisms of color formation. We performed an RNA-Seq analysis in order to shed light on the transcriptome of poinsettia bracts. Moreover, we analyzed the transcriptome differences of red- and white-bracted poinsettia varieties during bract development and coloration. For the assembly of a bract transcriptome, two paired-end cDNA libraries from a red and white poinsettia pair were sequenced with the Illumina technology, and one library from a red-bracted variety was used for PacBio sequencing. Both short and long reads were assembled using a hybrid de novo strategy. Samples of red- and white-bracted poinsettias were sequenced and comparatively analyzed in three color developmental stages in order to understand the mechanisms of color formation and accumulation in the species. Results The final transcriptome contains 288,524 contigs, with 33% showing confident protein annotation against the TAIR10 database. The BUSCO pipeline, which is based on near-universal orthologous gene groups, was applied to assess the transcriptome completeness. From a total of 1440 BUSCO groups searched, 77% were categorized as complete (41% as single-copy and 36% as duplicated), 10% as fragmented and 13% as missing BUSCOs. The gene expression comparison between red and white varieties of poinsettia showed a differential regulation of the flavonoid biosynthesis pathway only at particular stages of bract development. An initial impairment of the flavonoid pathway early in the color accumulation process for the white poinsettia variety was observed, but these differences were no longer present in the subsequent stages of bract development. Nonetheless, GSTF11 and UGT79B10 showed a lower expression in the last stage of bract development for the white variety and, therefore, are potential candidates for further studies on poinsettia coloration. Conclusions In summary, this transcriptome analysis provides a valuable foundation for further studies on poinsettia, such as plant breeding and genetics, and highlights crucial information on the molecular mechanism of color formation

Developement and Application of Tobacco Rattle Virus Induced Gene Silencing in Gerbera hybrida

RNA silencing is a conserved mechanism that occurs in a broad range of eukaryotes, which is regulated by small RNAs (sRNAs). RNA silencing operates to control gene expression and maintain genome integrity. Virus-induced gene silencing (VIGS) in plants is a natural antivirus mechanism that has adapted from the general RNA silencing system. To counter the antivirus RNA silencing, plant viruses have evolved to encode viral suppressors of RNA silencing (VSRs). Nowadays VIGS is usually referred to as the technology that uses recombinant viruses to knock down the expression of plant endogenous genes. Gerbera hybrida (gerbera) is a model species in the family of Asteraceae. As a highly heterozygous species, gerbera lacks efficient functional genetic approaches other than gene transfer. The aim of the present study was to develop a Tobacco rattle virus (TRV, genus Tobravirus) induced gene silencing system for gerbera, and use TRV VIGS to characterize functions of chalcone synthase (CHS) encoding genes in the plant. Preliminary VIGS experiments on the cultivar Terraregina, by syringe infiltration and applying previously developed TRV vectors, did not result in visible VIGS phenotypes due to the absent of TRV RNA2 in the up non-infiltrated leaves. Consequently, I first aimed to study the mechanism of TRV VIGS, and tried to develop new VIGS vectors based on TRV RNA1. I investigated the role of two important TRV proteins of the 16K VSR and the 29K movement protein (MP) on TRV infection and TRV VIGS, and developed TRV RNA1 based VIGS vectors. For accomplishing this, a series of TRV RNA1 mutants have been constructed to disrupt the 16K, or to replace its 29K with Tobacco mosaic virus (TMV, genus Tobamovirus) 30K MP. TRV RNA1 vector, carrying a fragment of the gene encoding Nicotiana benthamiana PDS to replace part of the 16K sequence, induced PDS gene silencing systemically in N. benthamiana. However, this has found to be less efficiently than the original TRV VIGS system when the wild-type RNA1 and RNA2:PDS were used. The infection experiments demonstrated that 16K was required for TRV long distance movement, and helped in maintaining the integrity of the TRV RNA2 genome. In addition, TRV 29K alone did not suppress RNA silencing in the co-infiltration assay, but it could suppress RNA silencing in the context of RNA1 replication. TRV 29K may be the first VSR whose silencing suppression functions are found to be directly linked to viral replication. The original TRV vector system was finally adopted for VIGS in gerbera. TRV VIGS was optimized for gerbera by screening for TRV sensitive cultivars and by improving its inoculation methods. Intensive gene silencing phenotypes were achieved both in green tissues and in floral tissues, demonstrated by knocking down genes involved in isoprenoid biosynthesis (phytoene desaturase: GPDS; H and I subunits of Mg-chelatase: GChl-H and GChl-I), flower pigmentation (chalcone synthase: GCHS1), and flower development (GLOBOSA-like MADS domain transcription factor: GGLO1). Unexpectedly, a gerbera polyketide synthase encoding gene, G2PS1, that has no apparent connections to the carotenoid or chlorophyll biosynthesis, was knocked down by the photo-bleaching that was induced by the silencing of GPDS, GChl-H and GChl-I, or by the herbicide norflurazon. We have demonstrated for the first time that the using of VIGS in an Asteraceaeous species. Our data also suggested that the selection and use of a marker gene for VIGS should be strictly evaluated. A new CHS encoding gene, GCHS4, was characterized in gerbera. Together with the two previously identified GCHS1 and GCHS3, gerbera CHSs are represented by a three-gene family. Each gerbera CHS shows a distinct expression pattern. GCHS3 is particularly expressed in gerbera pappus. In partnership with the concomitantly expressed GCHS1, they are involved in the biosynthesis of colorless flavonoids. GCHS4 is the only CHS that is naturally expressed in the leaf petiole and inflorescence scape, and it is responsible for cyanidin biosynthesis in those tissues. GCHS4 is also the only CHS that was induced by environmental stresses in the leaf blade. Both GCHS1 and GCHS4 are markedly expressed in gerbera petals, and GCHS4 mRNA actually takes the majority of CHS mRNAs in the later stages of petal development. Nonetheless, VIGS experiments, by target silencing GCHS1 or GCHS4 independently, demonstrated that GCHS1 is the predominant functional CHS in gerbera petals. Thus, GCHS4 in gerbera petals seems to be regulated post-transcriptionally. In conclusion, the results of this study shed new light on the mechanism of TRV VIGS. The established TRV VIGS system provides a valuable tool for functional genomics in gerbera