RESUMO
Dahlia (Dahlia variabilis) is a widely cultivated ornamental and medicinal plant in China. Recently, dahlia plants with symptoms of leaf mottling and distortion were collected in Hohhot, Inner Mongolia, China. The presence of dahlia common mosaic virus (DCMV), an unassigned species in the genus Caulimovirus, was confirmed by high-throughput sequencing. Three fragments of DCMV Inner Mongolia isolate (DCMV-IN) were PCR-amplified with specific primers, sequenced and assembled into the complete genome sequence with a GenBank accession number of OR494328. The double-stranded circular DNA genome of DCMV-IN consists of 7949 bp and contains six open reading frames (ORFs). Sequence analysis showed that DCMV-IN shared high sequence identities with other DCMV isolates available in the GenBank database. Phylogenetic analysis of DCMV isolates and other representative caulimoviruses based on genome sequence clustered four DCMV isolates to a single branch which was closest to dahlia mosaic virus (DMV). No recombination event was detected among the four DCMV isolates.
Assuntos
Caulimovirus , Dahlia , Caulimovirus/genética , Dahlia/genética , Filogenia , Genoma Viral , Reação em Cadeia da PolimeraseRESUMO
MAIN CONCLUSION: A novel gene belonging to the aldo-keto reductase 13 family is involved in isoliquiritigenin biosynthesis in dahlia. The yellow pigments of dahlia flowers are derived from 6'-deoxychalcones, which are synthesized via a two-step process, involving the conversion of 3-malonyl-CoA and 4-coumaloyl-CoA into isoliquiritigenin in the first step, and the subsequent generation of butein from isoliquiritigenin. The first step reaction is catalyzed by chalcone synthase (CHS) and aldo-keto reductase (AKR). AKR has been implicated in the isoflavone biosynthesis in legumes, however, isolation of butein biosynthesis related AKR members are yet to be reported. A comparative RNA-seq analysis between two dahlia cultivars, 'Shukuhai' and its butein-deficient lateral mutant 'Rinka', was used in this study to identify a novel AKR gene involved in 6'-deoxychalcone biosynthesis. DvAKR1 encoded a AKR 13 sub-family protein with significant differential expression levels, and was phylogenetically distinct from the chalcone reductases, which belongs to the AKR 4A sub-family in legumes. DNA sequence variation and expression profiles of DvAKR1 gene were correlated with 6'-deoxychalcone accumulation in the tested dahlia cultivars. A single over-expression analysis of DvAKR1 was not sufficient to initiate the accumulation of isoliquiritigenin in tobacco, in contrast, its co-overexpression with a chalcone 4'-O-glucosyltransferase (Am4'CGT) from Antirrhinum majus and a MYB transcription factor, CaMYBA from Capsicum annuum successfully induced isoliquiritigenin accumulation. In addition, DvAKR1 homologous gene expression was detected in Coreopsideae species accumulating 6'-deoxychalcone, but not in Asteraceae species lacking 6'-deoxychalcone production. These results not only demonstrate the involvement of DvAKR1 in the biosynthesis of 6'-deoxychalcone in dahlia, but also show that 6'-deoxychalcone occurrence in Coreopsideae species developed evolutionarily independent from legume species.
Assuntos
Chalconas , Dahlia , Aldeído Redutase/metabolismo , Aldo-Ceto Redutases/genética , Aldo-Ceto Redutases/metabolismo , Chalconas/metabolismo , Coenzima A/metabolismo , Dahlia/genéticaRESUMO
We investigated the bi-colored dahlia cultivar 'Seattle', which exhibits bright yellow petals with white tips, for its potential use as a model system for studies of the anthochlor biosynthesis. The yellow base contained high amounts of the 6'-deoxychalcones and the structurally related 4-deoxyaurones, as well as flavones. In contrast, only traces of anthochlors and flavones were detected in the white tips. No anthocyanins, flavonols, flavanones or dihydroflavonols were found in the petals. Gene expression studies indicated that the absence of anthocyanins in the petals is caused by a lack of flavanone 3-hydroxylase (FHT) expression, which is accompanied by a lack of expression of the bHLH transcription factor IVS. Expression of other genes involved in anthocyanidin biosynthesis such as dihydroflavonol 4-reductase (DFR) and anthocyanidin synthase (ANS) was not affected. The yellow and white petal parts showed significant differences in the expression of chalcone synthase 2 (CHS2), which is sufficient to explain the absence of yellow pigments in the white tips. Transcriptomes of both petal parts were de novo assembled and three candidate genes for chalcone reductase (CHR) were identified. None of them showed a significantly higher expression in the yellow base compared to the white tips. In summary, it was shown that the bicolouration is most likely caused by a bottleneck in chalcone formation in the white tip. The relative prevalence of flavones compared to the anthochlors in the white tips could be an indication for the presence of a so far unknown differentially expressed CHR.
Assuntos
Dahlia , Regulação da Expressão Gênica de Plantas , Modelos Biológicos , Pigmentos Biológicos , Antocianinas/genética , Dahlia/genética , Dahlia/metabolismo , Flores/química , Flores/genética , Flores/metabolismo , Genes de Plantas/genética , Pigmentos Biológicos/biossínteseRESUMO
Wheat (Triticum aestivum L.) is an important staple crop. Sharp eyespot and common root rot are destructive diseases of wheat. Antimicrobial peptides (AMPs) are small peptides with broad-spectrum antimicrobial activity. In this study, we synthesized the DmAMP1W gene, encoding Dahlia merckii DmAMP1, and investigated the antifungal role of DmAMP1W in vitro and in transgenic wheat. Protein electrophoresis analysis and in vitro inhibition results demonstrated that the synthesized DmAMP1W correctly translated to the expected peptide DmAMP1W, and the purified peptide inhibited growths of the fungi Rhizoctonia cerealis and Bipolaris sorokiniana, the pathogenic causes of wheat sharp eyespot and common root rot. DmAMP1W was introduced into a wheat variety Zhoumai18 via Agrobacterium-mediated transformation. The molecular characteristics indicated that DmAMP1W could be heritable and expressed in five transgenic wheat lines in T1-T2 generations. Average sharp eyespot infection types of these five DmAMP1W transgenic wheat lines in T1-T2 generations decreased 0.69-1.54 and 0.40-0.82 compared with non-transformed Zhoumai18, respectively. Average common root rot infection types of these transgenic lines and non-transformed Zhoumai18 were 1.23-1.48 and 2.27, respectively. These results indicated that DmAMP1W-expressing transgenic wheat lines displayed enhanced-resistance to both sharp eyespot and common root rot. This study provides new broad-spectrum antifungal resources for wheat breeding.
Assuntos
Peptídeos Catiônicos Antimicrobianos/metabolismo , Resistência à Doença , Plantas Geneticamente Modificadas/crescimento & desenvolvimento , Triticum/genética , Peptídeos Catiônicos Antimicrobianos/genética , Peptídeos Catiônicos Antimicrobianos/farmacologia , Dahlia/genética , Dahlia/metabolismo , Fungos/efeitos dos fármacos , Regulação da Expressão Gênica de Plantas , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Raízes de Plantas/genética , Raízes de Plantas/crescimento & desenvolvimento , Raízes de Plantas/microbiologia , Plantas Geneticamente Modificadas/microbiologia , Característica Quantitativa Herdável , Triticum/crescimento & desenvolvimento , Triticum/microbiologiaRESUMO
MAIN CONCLUSION: Post-transcriptional gene silencing (PTGS) of a chalcone synthase ( DvCHS2 ) occurred in the white part of bicolor petals and flavonoid-poor leaves; however, it did not in red petals and flavonoid-rich leaves. Petal color lability is a prominent feature of bicolor dahlia cultivars, and causes plants to produce not only original bicolor petals with colored bases and pure white tips, but also frequently single-colored petals without white tips. In this study, we analysed the molecular mechanisms that are associated with petal color lability using the red-white bicolor cultivar 'Yuino'. Red single-colored petals lose their white tips as a result of recover of flavonoid biosynthesis. Among flavonoid biosynthetic genes including four chalcone synthase (CHS)-like genes (DvCHS1, DvCHS2, DvCHS3, and DvCHS4), DvCHS1 and DvCHS2 had significantly lower expression levels in the white part of bicolor petals than in red petals, while DvCHS3, DvCHS4, and other flavonoid biosynthetic genes had almost the same expression levels. Small RNAs from the white part of a bicolor petal were mapped onto DvCHS1 and DvCHS2, while small RNAs from a red single-colored petal were not mapped onto any of the four CHS genes. A relationship between petal color and leaf flavonoid accumulation has previously been demonstrated, whereby red petal-producing plants accumulate flavonoids in their leaves, while bicolor petal-producing plants tend not to. The expression level of DvCHS2 was down-regulated in flavonoid-poor leaves and small RNAs from flavonoid-poor leaves were mapped onto DvCHS2, suggesting that the down-regulation of DvCHS2 in flavonoid-poor leaves occurs post-transcriptionally. Genomic analysis also suggested that DvCHS2 is the key gene involved in bicolor formation. Together, these results suggest that post-transcriptional gene silencing of DvCHS2 plays a key role in phenotypic lability in this bicolor dahlia.
Assuntos
Aciltransferases/genética , Dahlia/enzimologia , Flavonoides/metabolismo , Interferência de RNA , Aciltransferases/metabolismo , Cor , Dahlia/genética , Dahlia/crescimento & desenvolvimento , Flavonoides/análise , Flores/enzimologia , Flores/genética , Flores/crescimento & desenvolvimento , Fenótipo , Pigmentação , Folhas de Planta/enzimologia , Folhas de Planta/genética , Folhas de Planta/crescimento & desenvolvimento , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismoRESUMO
MAIN CONCLUSION: Tobacco streak virus suppressed post-transcriptional gene silencing and caused a flower color change in black dahlias, which supported the role of cyanidin-based anthocyanins for black flower appearance. Black flower color of dahlia (Dahlia variabilis) has been attributed, in part, to the high accumulation of cyanidin-based anthocyanins that occurs when flavone synthesis is reduced because of post-transcriptional gene silencing (PTGS) of flavone synthase II (DvFNS). There are also purple-flowering plants that have emerged from a black cultivar 'Kokucho'. We report that the purple color is not caused by a mutation, as previously thought, but by infection with tobacco streak virus (TSVdahlia), which suppresses the PTGS of DvFNS. When TSVdahlia was eliminated from the purple-flowering 'Kokucho' by leaf primordia-free shoot apical meristem culture, the resulting flowers were black. TSVdahlia-infected purple flowers had lower numbers of siRNAs to DvFNS than black flowers, suggesting that TSVdahlia has a silencing suppressor. The graft inoculation of other black cultivars with TSVdahlia altered their flower color drastically except for 'Fidalgo Blacky', a very deep black cultivar with the highest amount of cyanidin-based anthocyanins. The flowers of all six TSVdahlia-infected cultivars accumulated increased amounts of flavones and reduced amounts of cyanidin-based anthocyanins. 'Fidalgo Blacky' remained black despite the change in pigment accumulation, and the amounts of cyanidin-based anthocyanins in its TSVdahlia-infected plants were still higher than those of other cultivars. We propose that black flower color in dahlia is controlled by two different mechanisms that increase the amount of cyanidin-based anthocyanins: DvFNS PTGS-dependent and -independent mechanisms. If both mechanisms occur simultaneously, the flower color will be blacker than if only a single mechanism is active.
Assuntos
Sistema Enzimático do Citocromo P-450/metabolismo , Dahlia/metabolismo , Flores/metabolismo , Ilarvirus/patogenicidade , Pigmentação/fisiologia , Proteínas de Plantas/metabolismo , Sistema Enzimático do Citocromo P-450/genética , Dahlia/genética , Dahlia/virologia , Flores/genética , Flores/virologia , Regulação da Expressão Gênica de Plantas , Pigmentação/genética , Proteínas de Plantas/genéticaRESUMO
Transgenic tobacco plants expressing the AINTEGUMENTA gene of rape under control of the 35S promoter and the promoter of dahlia mosaic virus were obtained. The transgenic plants were characterized by increase in the length of the leaves, flower sizes, stem height, and weight of seeds; at the same time, the degree of increase was greater in the case of use of the dahlia mosaic virus promoter as a regulator of transcription. Ectopic expression of the AINTEGUMENTA gene promoted prolongation of leaf growth, while sizes of epidermal cells of the leaves remained unchanged.
Assuntos
Regulação da Expressão Gênica de Plantas , Proteínas de Homeodomínio/genética , Nicotiana/genética , Proteínas de Plantas/genética , Plantas Geneticamente Modificadas , Brassica napus/genética , Caulimovirus/genética , Dahlia/genética , Dahlia/virologia , Flores/anatomia & histologia , Flores/genética , Flores/crescimento & desenvolvimento , Folhas de Planta/anatomia & histologia , Folhas de Planta/genética , Folhas de Planta/crescimento & desenvolvimento , Caules de Planta/anatomia & histologia , Caules de Planta/genética , Caules de Planta/crescimento & desenvolvimento , Reação em Cadeia da Polimerase , Regiões Promotoras Genéticas , Sementes/anatomia & histologia , Sementes/genética , Sementes/crescimento & desenvolvimento , Nicotiana/anatomia & histologia , Nicotiana/crescimento & desenvolvimento , Transcrição GênicaRESUMO
The study was aimed to identify the factors that regulate the intensity of flower color in cyanic dahlia (Dahlia variabilis), using fifteen cultivars with different color intensities in their petals. The cultivars were classified into three groups based on their flavonoid composition: ivory white cultivars with flavones; purple and pink cultivars with flavones and anthocyanins; and red cultivars with flavones, anthocyanins, and chalcones. Among the purple, pink, and ivory white cultivars, an inverse relationship was detected between lightness, which was used as an indicator for color intensity and anthocyanin content. A positive correlation was detected between anthocyanin contents and the expression of some structural genes in the anthocyanin synthesis pathway that are regulated by DvIVS, a basic helix-loop-helix transcription factor. A positive correlation between anthocyanin content and expression of DvIVS was also found. The promoter region of DvIVS was classified into three types, with cultivars carrying Type 1 promoter exhibited deep coloring, those carrying Type 2 and/or Type 3 exhibited pale coloring, and those carrying Type 1 and Type 2 and/or Type 3 exhibited medium coloring. The transcripts of the genes from these promoters encoded full-length predicted proteins. These results suggested that the genotype of the promoter region in DvIVS is one of the key factors determining the flower color intensity.
Assuntos
Antocianinas/metabolismo , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Dahlia/genética , Flavonoides/metabolismo , Regulação da Expressão Gênica de Plantas , Pigmentação/genética , Antocianinas/análise , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , DNA Complementar/genética , Dahlia/metabolismo , Flores/anatomia & histologia , Flores/genética , Flores/metabolismo , Genótipo , Concentração de Íons de Hidrogênio , Fenótipo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Polimorfismo Genético , Regiões Promotoras Genéticas/genética , RNA/genética , RNA de Plantas/genética , Análise de Sequência de DNA , Especificidade da Espécie , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
Black color in flowers is a highly attractive trait in the floricultural industry, but its underlying mechanisms are largely unknown. This study was performed to identify the bases of the high accumulation of anthocyanidins in black cultivars and to determine whether the high accumulation of total anthocyanidins alone leads to the black appearance. Our approach was to compare black dahlia (Dahlia variabilis) cultivars with purple cultivars and a purple flowering mutant of a black cultivar, using pigment and molecular analyses. Black cultivars characteristically exhibited low lightness, high petal accumulation of cyanidin and total anthocyanidins without flavones, and marked suppression of flavone synthase (DvFNS) expression. A comparative study using black and purple cultivars revealed that neither the absence of flavones nor high accumulation of total anthocyanidins is solely sufficient for black appearance, but that cyanidin content in petals is also an important factor in the phenotype. A study comparing the black cultivar 'Kokucho' and its purple mutant showed that suppression of DvFNS abolishes the competition between anthocyanidin and flavone synthesis and leads to accumulation of cyanidin and total anthocyanidins that produce a black appearance. Surprisingly, in black cultivars the suppression of DvFNS occurred in a post-transcriptional manner, as determined by small RNA mapping.
Assuntos
Antocianinas/metabolismo , Dahlia/enzimologia , Dahlia/metabolismo , Oxigenases de Função Mista/metabolismo , Proteínas de Plantas/metabolismo , Dahlia/genética , Oxigenases de Função Mista/genética , Proteínas de Plantas/genética , Interferência de RNA/fisiologiaRESUMO
BACKGROUND: More than 20,000 cultivars of garden dahlia (Dahlia variabilis hort.) are available showing flower colour from white, yellow and orange to every imaginable hue of red and purple tones. Thereof, only a handful of cultivars are so-called black dahlias showing distinct black-red tints. Flower colour in dahlia is a result of the accumulation of red anthocyanins, yellow anthochlors (6'-deoxychalcones and 4-deoxyaurones) and colourless flavones and flavonols, which act as copigments. White and yellow coloration occurs only if the pathway leading to anthocyanins is incomplete. Not in all cultivars the same step of the anthocyanin pathway is affected, but the lack of dihydroflavonol 4-reductase activity is frequently observed and this seems to be based on the suppression of the transcription factor DvIVS. The hitherto unknown molecular background for black colour in dahlia is here presented. RESULTS: Black cultivars accumulate high amounts of anthocyanins, but show drastically reduced flavone contents. High activities were observed for all enzymes from the anthocyanin pathway whereas FNS II activity could not be detected or only to a low extent in 13 of 14 cultivars. cDNA clones and genomic clones of FNS II were isolated. Independently from the colour type, heterologous expression of the cDNA clones resulted in functionally active enzymes. FNS II possesses one intron of varying length. Quantitative Real-time PCR showed that FNS II expression in black cultivars is low compared to other cultivars. No differences between black and red cultivars were observed in the expression of transcription factors IVS and possible regulatory genes WDR1, WDR2, MYB1, MYB2, 3RMYB and DEL or the structural genes of the flavonoid pathway. Despite the suppression of FHT expression, flavanone 3-hydroxylase (FHT, synonym F3H) enzyme activity was clearly present in the yellow and white cultivars. CONCLUSIONS: An increased accumulation of anthocyanins establishes the black flowering phenotypes. In the majority of black cultivars this is due to decreased flavone accumulation and thus a lack of competition for flavanones as the common precursors of flavone formation and the anthocyanin pathway. The low FNS II activity is reflected by decreased FNS II expression.
Assuntos
Antocianinas/biossíntese , Sistema Enzimático do Citocromo P-450/metabolismo , Dahlia/enzimologia , Flavonas/biossíntese , Flores/enzimologia , Pigmentação/genética , Sequência de Aminoácidos , Sequência de Bases , Clonagem Molecular , Sistema Enzimático do Citocromo P-450/genética , DNA Complementar/genética , Dahlia/genética , Flores/genética , Regulação da Expressão Gênica de Plantas , Dados de Sequência Molecular , Fenótipo , Alinhamento de SequênciaRESUMO
Dahlias (Dahlia variabilis) exhibit a wide range of flower colours because of accumulation of anthocyanin and other flavonoids in their ray florets. Two lateral mutants were used that spontaneously occurred in 'Michael J' (MJW) which has yellow ray florets with orange variegation. MJOr, a bud mutant producing completely orange ray florets, accumulates anthocyanins, flavones, and butein, and MJY, another mutant producing completely yellow ray florets, accumulates flavones and butein. Reverse transcription-PCR analysis showed that expression of chalcone synthase 1 (DvCHS1), flavanone 3-hydroxylase (DvF3H), dihydroflavonol 4-reductase (DvDFR), anthocyanidin synthase (DvANS), and DvIVS encoding a basic helix-loop-helix transcription factor were suppressed, whereas that of chalcone isomerase (DvCHI) and DvCHS2, another CHS with 69% nucleotide identity with DvCHS1, was not suppressed in the yellow ray florets of MJY. A 5.4 kb CACTA superfamily transposable element, transposable element of Dahlia variabilis 1 (Tdv1), was found in the fourth intron of the DvIVS gene of MJW and MJY, and footprints of Tdv1 were detected in the variegated flowers of MJW. It is shown that only one type of DvIVS gene was expressed in MJOr, whereas these plants are likely to have three types of the DvIVS gene. On the basis of these results, the mechanism regulating the formation of orange and yellow ray florets in dahlia is discussed.
Assuntos
Antocianinas/biossíntese , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Dahlia/metabolismo , Regulação da Expressão Gênica de Plantas , Proteínas de Plantas/metabolismo , Sequência de Aminoácidos , Fatores de Transcrição Hélice-Alça-Hélice Básicos/química , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Vias Biossintéticas , Dahlia/química , Dahlia/classificação , Dahlia/genética , Flores/química , Flores/genética , Flores/crescimento & desenvolvimento , Flores/metabolismo , Dados de Sequência Molecular , Filogenia , Proteínas de Plantas/química , Proteínas de Plantas/genética , Alinhamento de SequênciaRESUMO
Garden dahlias (Dahlia variabilis) are autoallooctoploids with redundant genes producing wide color variations in flowers. There are no pure white dahlia cultivars, despite its long breeding history. However, the white areas of bicolor flower petals appear to be pure white. The objective of this experiment was to elucidate the mechanism by which the pure white color is expressed in the petals of some bicolor cultivars. A pigment analysis showed that no flavonoid derivatives were detected in the white areas of petals in a star-type cultivar 'Yuino' and the two seedling cultivars 'OriW1' and 'OriW2' borne from a red-white bicolor cultivar, 'Orihime', indicating that their white areas are pure white. Semi-quantitative RT-PCR showed that in the pure white areas, transcripts of two chalcone synthases (CHS), DvCHS1 and DvCHS2 which share 69% nucleotide similarity with each other, were barely detected. Premature mRNA of DvCHS1 and DvCHS2 were detected, indicating that these two CHS genes are silenced post-transcriptionally. RNA gel blot analysis revealed that small interfering RNAs (siRNAs) derived from CHSs were produced in these pure white areas. By high-throughput sequence analysis of small RNAs in the pure white areas with no mismatch acceptance, small RNAs were mapped to two alleles of DvCHS1 and two alleles of DvCHS2 expressed in 'Yuino' petals. Therefore, we concluded that simultaneous siRNA-mediated post-transcriptional gene silencing of redundant CHS genes results in the appearance of pure white color in dahlias.
Assuntos
Aciltransferases/genética , Dahlia/genética , Flores/fisiologia , Interferência de RNA , Aciltransferases/metabolismo , Antocianinas/metabolismo , Chalcona/metabolismo , Dahlia/classificação , Dahlia/enzimologia , Dahlia/fisiologia , Flavonas/metabolismo , Flores/metabolismo , Genes de Plantas , Filogenia , Pigmentação , Poliploidia , RNA Mensageiro/análise , RNA de Plantas/genética , RNA de Plantas/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Análise de Sequência de RNARESUMO
In the petals of Dahlia variabilis, hydroxylation of chalcones at position 3 can be detected, except the well-known flavonoid 3'-hydroxylation. Although the reaction is well characterized at the enzymatic level, it remained unclear whether it is catalyzed by a flavonoid 3'-hydroxylase (F3'H, EC1.14.13.21, CYP75B) with broad substrate specificity. Two novel allelic variants of F3'H were cloned from D. variabilis, which differ only in three amino acids within their 508 residues. The corresponding recombinant enzymes show significant differences in their chalcone 3-hydroxylase (CH3H) activity. A substitution of alanine at position 425 with valine enables CH3H activity, whereas the reciprocal substitution leads to a loss of CH3H activity. Interaction of the valine at position 425 with not yet identified structural properties seems to be decisive for chalcone acceptance. This is the first identification of an F3'H which is able to catalyze chalcone 3-hydroxylation to a physiologically relevant extent from any plant species.
Assuntos
Alelos , Chalconas/metabolismo , Sistema Enzimático do Citocromo P-450/genética , Dahlia/genética , Sequência de Aminoácidos , Sequência de Bases , Sistema Enzimático do Citocromo P-450/química , Primers do DNA , DNA Complementar , Dahlia/enzimologia , Cinética , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Homologia de Sequência de Aminoácidos , Especificidade por SubstratoRESUMO
Viruses in certain genera in family Caulimoviridae were shown to integrate their genomic sequences into their host genomes and exist as endogenous pararetroviral sequences (EPRV). However, members of the genus Caulimovirus remained to be the exception and are known to exist only as episomal elements in the infected cell. We present evidence that the DNA genome of a new and distinct Caulimovirus species, associated with dahlia mosaic, is integrated into its host genome, dahlia (Dahlia variabilis). Using cloned viral genes as probes, Southern blot hybridization of total plant DNA from dahlia seedlings showed the presence of viral DNA in the host DNA. Fluorescent in situ hybridization using labeled DNA probes from the D10 genome localized the viral sequences in dahlia chromosomes. The natural integration of a Caulimovirus genome into its host and its existence as an EPRV suggests the co-evolution of this plant-virus pathosystem.
Assuntos
Caulimovirus/genética , Dahlia/genética , Cromossomos de Plantas/genética , DNA de Plantas/genética , DNA Viral/genética , Dahlia/virologia , Doenças das Plantas/virologia , Plântula/genética , Plântula/virologia , Integração ViralRESUMO
Phytophthora spp., some of the more important casual agents of plant diseases, are responsible for heavy economic losses worldwide. Plant defensins have been introduced as transgenes into a range of species to increase host resistance to pathogens to which they were originally susceptible. However, the effectiveness and mechanism of interaction of the defensins with Phytophthora spp. have not been clearly characterized in planta. In this study, we expressed the Dahlia merckii defensin, DmAMP1, in papaya (Carica papaya L.), a plant highly susceptible to a root, stem, and fruit rot disease caused by Phytophthora palmivora. Extracts of total leaf proteins from transformed plants inhibited growth of Phytophthora in vitro and discs cut from the leaves of transformed plants inhibited growth of Phytophthora in a bioassay. Results from our greenhouse inoculation experiments demonstrate that expressing the DmAMP1 gene in papaya plants increased resistance against P. palmivora and that this increased resistance was associated with reduced hyphae growth of P. palmivora at the infection sites. The inhibitory effects of DmAMP1 expression in papaya suggest this approach has good potential to impart transgenic resistance against Phytophthora in papaya.
Assuntos
Carica/microbiologia , Dahlia/genética , Defensinas/genética , Phytophthora/patogenicidade , Carica/genética , Imunidade Inata , Doenças das Plantas/microbiologia , Plantas Geneticamente Modificadas/microbiologiaRESUMO
Flower color is most often conferred by colored flavonoid pigments. Aurone flavonoids confer a bright yellow color on flowers such as snapdragon (Antirrhinum majus) and dahlia (Dahlia variabilis). A. majus aureusidin synthase (AmAS1) was identified as the key enzyme that catalyzes aurone biosynthesis from chalcones, but transgenic flowers overexpressing AmAS1 gene failed to produce aurones. Here, we report that chalcone 4'-O-glucosyltransferase (4'CGT) is essential for aurone biosynthesis and yellow coloration in vivo. Coexpression of the Am4'CGT and AmAS1 genes was sufficient for the accumulation of aureusidin 6-O-glucoside in transgenic flowers (Torenia hybrida). Furthermore, their coexpression combined with down-regulation of anthocyanin biosynthesis by RNA interference (RNAi) resulted in yellow flowers. An Am4'CGT-GFP chimeric protein localized in the cytoplasm, whereas the AmAS1(N1-60)-RFP chimeric protein was localized to the vacuole. We therefore conclude that chalcones are 4'-O-glucosylated in the cytoplasm, their 4'-O-glucosides transported to the vacuole, and therein enzymatically converted to aurone 6-O-glucosides. This metabolic pathway is unique among the known examples of flavonoid, including anthocyanin biosynthesis because, for all other compounds, the carbon backbone is completed before transport to the vacuole. Our findings herein not only demonstrate the biochemical basis of aurone biosynthesis but also open the way to engineering yellow flowers for major ornamental species lacking this color variant.
Assuntos
Benzofuranos/metabolismo , Flores/metabolismo , Antirrhinum/genética , Antirrhinum/crescimento & desenvolvimento , Antirrhinum/metabolismo , Benzofuranos/química , Evolução Biológica , Chalcona/metabolismo , Cor , Dahlia/genética , Dahlia/metabolismo , Flavonoides/biossíntese , Flavonoides/química , Flavonoides/genética , Flores/genética , Regulação da Expressão Gênica de Plantas , Glucose/metabolismo , Glucosiltransferases/genética , Glucosiltransferases/metabolismo , Oxigenases de Função Mista/genética , Oxigenases de Função Mista/metabolismo , Dados de Sequência Molecular , Estrutura Molecular , Plantas Geneticamente ModificadasRESUMO
In the flowers of important ornamental Compositae plants, anthocyanins generally carry malonyl group(s) at their 3-glucosyl moiety. In this study, for the first time to our knowledge, we have identified a cDNA coding for this 3-glucoside-specific malonyltransferase for anthocyanins, i.e. malonyl-coenzyme A:anthocyanidin 3-O-glucoside-6"-O-malonyltransferase, from dahlia (Dahlia variabilis) flowers. We isolated a full-length cDNA (Dv3MaT) on the basis of amino acid sequences specifically conserved among anthocyanin acyltransferases of the versatile plant acyltransferase family. Dv3MaT coded for a protein of 460 amino acids. Quantitative real-time PCR analyses of Dv3MaT showed that the transcript was present in accordance with the distribution of 3MaT activities and the anthocyanin accumulation pattern in the dahlia plant. The Dv3MaT cDNA was expressed in Escherichia coli, and the recombinant enzyme was purified to homogeneity and characterized. The recombinant Dv3MaT catalyzed the regiospecific transfer of the malonyl group from malonyl-coenzyme A (K(m), 18.8 microM) to pelargonidin 3-O-glucoside (K(m), 46.7 microM) to produce pelargonidin 3-O-6"-O-malonylglucoside with a k(cat) value of 7.3 s(-1). The other enzymatic profiles of the recombinant Dv3MaT were closely related to those of native anthocyanin malonyltransferase activity in the extracts of dahlia flowers. Dv3MaT cDNA was introduced into petunia (Petunia hybrida) plants whose red floral color is exclusively provided by cyanidin 3-O-glucoside and 3,5-O-diglucoside. Thirteen transgenic lines of petunia were found to produce malonylated products of these anthocyanins (11-63 mol % of total anthocyanins in the flower). The spectral stability of cyanidin 3-O-6"-O-malonylglucoside at the pHs of intracellular milieus of flowers was significantly higher than that of cyanidin 3-O-glucoside. Moreover, 6"-O-malonylation of cyanidin 3-O-glucoside effectively prevented the anthocyanin from attack of beta-glucosidase. These results suggest that malonylation should serve as a strategy for pigment stabilization in the flowers.