RESUMO
Genome characterization of California poppy (Eschscholzia californica cv. "Hitoezaki"), which produces pharmaceutically important benzylisoquinoline alkaloids (BIAs), was carried out using the draft genome sequence. The numbers of tRNA and rRNA genes were close to those of the other plant species tested, whereas the frequency of repetitive sequences was distinct from those species. Comparison of the predicted genes with those of Amborella trichopoda, Nelumbo nucifera, Solanum lycopersicum, and Arabidopsis thaliana, and analyses of gene ontology and Kyoto Encyclopedia of Genes and Genomes pathway indicated that the enzyme genes involved in BIA biosynthesis were highly enriched in the California poppy genome. Further comparative analysis using the genome information of Papaver somniferum and Aquilegia coerulea, both BIA-producing plants, revealed that many genes encoding BIA biosynthetic enzymes, transcription factors, transporters, and candidate proteins, possibly related to BIA biosynthesis, were specifically distributed in these plant species.
Assuntos
Alcaloides/biossíntese , Benzilisoquinolinas/metabolismo , Eschscholzia/genética , Genoma de Planta , Regulação da Expressão Gênica de Plantas , RNA Ribossômico/genética , RNA de Transferência/genéticaRESUMO
With respect to the biosynthesis of plant alkaloids, that of benzylisoquinoline alkaloids (BIAs) has been the most investigated at the molecular level. Previous investigations have shown that the biosynthesis of BIAs is comprehensively regulated by WRKY and bHLH transcription factors, while promoter analyses of biosynthesis enzyme-encoding genes have also implicated the involvement of members of the APETALA2/ethylene responsive factor (AP2/ERF) superfamily. To investigate the physiological roles of AP2/ERF transcription factors in BIA biosynthesis, 134 AP2/ERF genes were annotated using the draft genome sequence data of Eschscholzia californica (California poppy) together with transcriptomic data. Phylogenetic analysis revealed that these genes could be classified into 20 AP2, 5 RAV, 47 DREB, 60 ERF and 2 Soloist family members. Gene structure, conserved motif and orthologous analyses were also carried out. Gene expression profiling via RNA sequencing in response to methyl jasmonate (MeJA) indicated that approximately 20 EcAP2/ERF genes, including 10 group IX genes, were upregulated by MeJA, with an increase in the expression of the transcription factor-encoding gene EcbHLH1 and the biosynthesis enzyme-encoding genes Ec6OMT and EcCYP719A5. Further quantitative RT-PCR confirmed the MeJA responsiveness of the EcAP2/ERF genes, i.e., the increased expression of 9 group IX, 2 group X and 2 group III ERF subfamily genes. Transactivation activity of group IX EcAP2/ERFs was also confirmed by a luciferase reporter assay in conjunction with the promoters of the Ec6OMT and EcCYP719A5 genes. The physiological roles of AP2/ERF genes in BIA biosynthesis and their evolution in the regulation of alkaloid biosynthesis are discussed.
Assuntos
Acetatos/farmacologia , Proteínas de Arabidopsis/genética , Ciclopentanos/farmacologia , Eschscholzia/genética , Eschscholzia/metabolismo , Estudo de Associação Genômica Ampla , Proteínas de Homeodomínio/genética , Oxilipinas/farmacologia , Fatores de Transcrição/genética , Transcriptoma/efeitos dos fármacos , Transcriptoma/genética , Proteínas de Arabidopsis/fisiologia , Benzilisoquinolinas/metabolismo , Proteínas de Homeodomínio/fisiologia , Fatores de Transcrição/fisiologiaRESUMO
Macarpine is a minor benzophenanthridine alkaloid with interesting biological activities, which is produced in only a few species of the Papaveraceae family, including Eschscholzia californica. Our present study was focused on the enhancement of macarpine production in E. californica suspension cultures using three elicitation models: salicylic acid (SA) (4; 6; 8 mg/L) elicitation, and simultaneous or sequential combinations of SA and L-tyrosine (1 mmol/L). Sanguinarine production was assessed along with macarpine formation in elicited suspension cultures. Alkaloid production was evaluated after 24, 48 and 72 h of elicitation. Among the tested elicitation models, the SA (4 mg/L), supported by L-tyrosine, stimulated sanguinarine and macarpine production the most efficiently. While sequential treatment led to a peak accumulation of sanguinarine at 24 h and macarpine at 48 h, simultaneous treatment resulted in maximum sanguinarine accumulation at 48 h and macarpine at 72 h. The effect of SA elicitation and precursor supplementation was evaluated also based on the gene expression of 4'-OMT, CYP719A2, and CYP719A3. The gene expression of investigated enzymes was increased at all used elicitation models and their changes correlated with sanguinarine but not macarpine accumulation.
Assuntos
Benzofenantridinas/biossíntese , Eschscholzia/efeitos dos fármacos , Reguladores de Crescimento de Plantas/farmacologia , Ácido Salicílico/farmacologia , Tirosina/farmacologia , Sistema Enzimático do Citocromo P-450/biossíntese , Sistema Enzimático do Citocromo P-450/genética , Relação Dose-Resposta a Droga , Eschscholzia/genética , Eschscholzia/crescimento & desenvolvimento , Eschscholzia/metabolismo , Regulação da Expressão Gênica de Plantas , Hidroponia/métodos , Isoquinolinas , Metiltransferases/biossíntese , Metiltransferases/genética , Proteínas de Plantas/agonistas , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Tirosina/metabolismoRESUMO
The California poppy (Eschscholzia californica) is renowned for its brilliant golden-orange flowers, though white petal variants have been described. By whole-transcriptome sequencing, we have discovered in multiple white petal varieties a single deletion leading to altered splicing and C-terminal truncation of phytoene synthase (PSY), a key enzyme in carotenoid biosynthesis. Our findings underscore the diverse roles of phytoene synthase in shaping horticultural traits, and resolve a longstanding mystery of the regaled golden poppy.
Assuntos
Eschscholzia/genética , Geranil-Geranildifosfato Geranil-Geraniltransferase/genética , Mutação , Sequência de Bases , DNA Complementar/genética , Genes de Plantas , Filogenia , Análise de Sequência de DNA , TranscriptomaRESUMO
KEY MESSAGE: Viral-induced gene silencing of selected biosynthetic genes decreased overall carotenoid accumulation in California poppy. Regulation of carotenogenesis was linked with pigment sequestration, not changes in biosynthetic gene expression. Genes of carotenogenesis are well described, but understanding how they affect carotenoid accumulation has proven difficult because of plant lethality when the pigments are lacking. Here, we used a Tobacco Rattle Virus-based virus-induced-gene-silencing (VIGS) approach in California poppy (Eschscholzia californica) to investigate how silencing of the carotenoid biosynthetic pathway genes affects carotenoid metabolite accumulation and RNA transcript abundance of the carotenoid biosynthetic pathway genes. VIGS of upstream (PDS and ZDS) and downstream (ßOH and ZEP) genes reduced transcript abundance of the targeted genes in the poppy petals while having no effect on abundance of the other carotenogenesis genes. Silencing of PDS, ZDS, ßOH and ZEP genes reduced total pigment concentration by 75-90% and altered petal colour. HPLC and LC-MS measurements suggested that petal colour changes were caused by substantially altered pigment profiles and quantity. Carotenoid metabolites were different to those normally detected in wild-type petals accumulated but overall carotenoid concentration was less, suggesting the chemical form of carotenoid was important for whether it could be stored at high amounts. In poppy petals, eschscholtzxanthin and retro-carotene-triol were the predominant carotenoids, present mainly as esters. Specific esterification enzymes for specific carotenoids and/or fatty acids appear key for enabling petal carotenoids to accumulate to high amounts. Our findings argue against a direct role for carotenoid metabolites regulating carotenogenesis genes in the petals of California poppy as transcript abundance of carotenogenesis genes studied was unchanged, while the petal carotenoid metabolite profile changed substantially.
Assuntos
Vias Biossintéticas , Carotenoides/metabolismo , Eschscholzia/metabolismo , Eschscholzia/virologia , Flores/metabolismo , Flores/virologia , Inativação Gênica , Vírus de Plantas/fisiologia , Vias Biossintéticas/genética , Eschscholzia/genética , Regulação da Expressão Gênica de Plantas , Genes de Plantas , Pigmentação , RNA Mensageiro/genética , RNA Mensageiro/metabolismoRESUMO
The basal production of secondary metabolites in medicinal plants is limited. One of the effective approaches that encourages plants to produce a remarkable amount of precious compounds is an application of elicitors. Our work was focused on the elicitation of Eschscholzia californica Cham. suspension cultures using various concentrations of MnCl2 (5; 10; 15 mg/L) with the aim of evaluating its effect on sanguinarine, chelerythrine, and macarpine production and gene expression of enzymes involved in the biosynthesis of mentioned secondary metabolites (BBE, 4'-OMT, CYP80B1) or in defense processes (LOX). Suspension cultures were exposed to elicitor for 24, 48, and 72 h. The content of alkaloids in phytomass was determined on the basis of their fluorescence properties. The relative mRNA expression of selected genes was analyzed using the ΔΔCt value method. PCR products were evaluated by melting curve analysis to confirm the specific amplification. Our results demonstrated that Eschscholzia californica Cham. cell suspension cultures evince sensitivity to the presence of MnCl2 in growth media resulting in the increased production of benzophenanthridine alkaloids and gene expression of selected enzymes. Manganese chloride seems to be a potential elicitor supporting natural biosynthetic properties in plant cell cultures and can be applied for the sustained production of valuable secondary metabolites.
Assuntos
Cloretos/metabolismo , Eschscholzia/metabolismo , Compostos de Manganês/metabolismo , Alcaloides/biossíntese , Vias Biossintéticas/efeitos dos fármacos , Cloretos/farmacologia , Eschscholzia/efeitos dos fármacos , Eschscholzia/genética , Compostos de Manganês/farmacologiaRESUMO
Land plants produce specialized low molecular weight metabolites to adapt to various environmental stressors, such as UV radiation, pathogen infection, wounding and animal feeding damage. Due to the large variety of stresses, plants produce various chemicals, particularly plant species-specific alkaloids, through specialized biosynthetic pathways. In this study, using a draft genome sequence and querying known biosynthetic cytochrome P450 (P450) enzyme-encoding genes, we characterized the P450 genes involved in benzylisoquinoline alkaloid (BIA) biosynthesis in California poppy (Eschscholzia californica), as P450s are key enzymes involved in the diversification of specialized metabolism. Our in silico studies showed that all identified enzyme-encoding genes involved in BIA biosynthesis were found in the draft genome sequence of approximately 489 Mb, which covered approximately 97% of the whole genome (502 Mb). Further analyses showed that some P450 families involved in BIA biosynthesis, i.e. the CYP80, CYP82 and CYP719 families, were more enriched in the genome of E. californica than in the genome of Arabidopsis thaliana, a plant that does not produce BIAs. CYP82 family genes were highly abundant, so we measured the expression of CYP82 genes with respect to alkaloid accumulation in different plant tissues and two cell lines whose BIA production differs to estimate the functions of the genes. Further characterization revealed two highly homologous P450s (CYP82P2 and CYP82P3) that exhibited 10-hydroxylase activities with different substrate specificities. Here, we discuss the evolution of the P450 genes and the potential for further genome mining of the genes encoding the enzymes involved in BIA biosynthesis.
Assuntos
Alcaloides/biossíntese , Vias Biossintéticas/genética , Sistema Enzimático do Citocromo P-450/genética , Mineração de Dados , Eschscholzia/enzimologia , Eschscholzia/genética , Genes de Plantas , Alcaloides/química , Sequência de Bases , Sistema Enzimático do Citocromo P-450/metabolismo , Regulação Enzimológica da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Família Multigênica , Pichia , Análise de Sequência de DNARESUMO
Transcription factors control many processes in plants and have high potentials to manipulate specialized metabolic pathways. Transcriptional regulation of the biosynthesis of monoterpenoid indole alkaloids (MIAs), nicotine alkaloids, and benzylisoquinoline alkaloids (BIAs) has been characterized using Catharanthus roseus, Nicotiana and Coptis plants. However, metabolic engineering in which specific transcription factors are used in alkaloid biosynthesis is limited. In this study, we characterized the effects of ectopic expression of CjWRKY1, which is a transcriptional activator with many targets in BIA biosynthesis in Coptis japonica (Ranunculaceae) and Eschscholzia californica (California poppy, Papaveraceae). Heterologous expression of CjWRKY1 in cultured California poppy cells induced increases in transcripts of several genes encoding BIA biosynthetic enzymes. Metabolite analyses indicated that the overexpression of the CjWRKY1 gene also induced increases in the accumulation of BIAs such as sanguinarine, chelerythrine, chelirubine, protopine, allocryptopine, and 10-hydroxychelerythrine in the culture medium. Previous characterization of EcbHLH1 and current results indicated that both transcription factors, WRKY1 and bHLH1, are substantially involved in the regulation of BIA biosynthesis. We discuss the function of CjWRKY1 in E. californica cells and its potential for metabolic engineering in BIA biosynthesis.
Assuntos
Alcaloides/biossíntese , Benzilisoquinolinas/metabolismo , Eschscholzia/metabolismo , Proteínas de Plantas/genética , Eschscholzia/genética , Genes de Plantas , Ativação TranscricionalRESUMO
California poppy (Eschscholzia californica), a member of the Papaveraceae family, produces many biologically active benzylisoquinoline alkaloids (BIAs), such as sanguinarine, macarpine and chelerythrine. Sanguinarine biosynthesis has been elucidated at the molecular level, and its biosynthetic genes have been isolated and used in synthetic biology approaches to produce BIAs in vitro. However, several genes involved in the biosynthesis of macarpine and chelerythrine have not yet been characterized. In this study, we report the isolation and characterization of a novel O-methyltransferase (OMT) involved in the biosynthesis of partially characterized BIAs, especially chelerythrine. A search of the RNA sequence database from NCBI and PhytoMetaSyn for the conserved OMT domain identified 68 new OMT-like sequences, of which the longest 22 sequences were selected based on sequence similarity. Based on their expression in cell lines with different macarpine/chelerythrine profiles, we selected three OMTs (G2, G3 and G11) for further characterization. G3 expression in Escherichia coli indicated O-methylation activity of the simple benzylisoquinolines, including reticuline and norreticuline, and the protoberberine scoulerine with dual regio-reactivities. G3 produced 7-O-methylated, 3'-O-methylated and dual O-methylated products from reticuline and norreticuline, and 9-O-methylated tetrahydrocolumbamine, 2-O-methylscoulerine and tetrahydropalmatine from scoulerine. Further enzymatic analyses suggested that G3 is a scoulerine-9-O-methyltransferase for the biosynthesis of chelerythrine in California poppy. In the present study, we discuss the physiological role of G3 in BIA biosynthesis.
Assuntos
Alcaloides/biossíntese , Benzilisoquinolinas/metabolismo , Eschscholzia/metabolismo , Sequência de Aminoácidos , Benzilisoquinolinas/química , Cromatografia Líquida , Eschscholzia/enzimologia , Eschscholzia/genética , Regulação da Expressão Gênica de Plantas , Estudos de Associação Genética , Cinética , Espectrometria de Massas , Metilação , Metiltransferases/química , Metiltransferases/metabolismo , Filogenia , Proteínas Recombinantes/metabolismoRESUMO
Cell cultures of Eschscholzia californica react to a fungal elicitor by the overproduction of antimicrobial benzophenanthridine alkaloids. The signal cascade toward the expression of biosynthetic enzymes includes (1) the activation of phospholipase A2 at the plasma membrane, resulting in a peak of lysophosphatidylcholine, and (2) a subsequent, transient efflux of vacuolar protons, resulting in a peak of cytosolic H(+). This study demonstrates that one of the Na(+)/H(+) antiporters acting at the tonoplast of E. californica cells mediates this proton flux. Four antiporter-encoding genes were isolated and cloned from complementary DNA (EcNHX1-EcNHX4). RNA interference-based, simultaneous silencing of EcNHX1, EcNHX3, and EcNHX4 resulted in stable cell lines with largely diminished capacities of (1) sodium-dependent efflux of vacuolar protons and (2) elicitor-triggered overproduction of alkaloids. Each of the four EcNHX genes of E. californica reconstituted the lack of Na(+)-dependent H(+) efflux in a Δnhx null mutant of Saccharomyces cerevisiae. Only the yeast strain transformed with and expressing the EcNHX1 gene displayed Na(+)-dependent proton fluxes that were stimulated by lysophosphatidylcholine, thus giving rise to a net efflux of vacuolar H(+). This finding was supported by three-dimensional protein homology models that predict a plausible recognition site for lysophosphatidylcholine only in EcNHX1. We conclude that the EcNHX1 antiporter functions in the elicitor-initiated expression of alkaloid biosynthetic genes by recruiting the vacuolar proton pool for the signaling process.
Assuntos
Eschscholzia/metabolismo , Proteínas de Plantas/metabolismo , Prótons , Metabolismo Secundário , Trocadores de Sódio-Hidrogênio/metabolismo , Vacúolos/metabolismo , Sequência de Aminoácidos , Cátions , Permeabilidade da Membrana Celular , Eschscholzia/genética , Fluorescência , Teste de Complementação Genética , Concentração de Íons de Hidrogênio , Lisofosfatidilcolinas/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Mutação/genética , Proteínas de Plantas/química , Plantas Geneticamente Modificadas , Interferência de RNA , Alinhamento de Sequência , Transdução de Sinais , Trocadores de Sódio-Hidrogênio/química , Homologia Estrutural de ProteínaRESUMO
Isoquinoline alkaloids (IQAs), terpenoid indole alkaloid and nicotine are some of the most studied alkaloids. Recently, several groups have reported that the biosynthesis of these alkaloids is regulated by basic helix-loop-helix (bHLH) transcription factors. Whereas the biosyntheses of nicotine and terpenoid indole alkaloid in Nicotiana plants and Catharanthus roseus are directly or indirectly regulated by Arabidopsis thaliana MYC2 homologs, a non-MYC2-type bHLH transcription factor, CjbHLH1, comprehensively regulates berberine biosynthesis in Coptis japonica. Interestingly, CjbHLH1 homologous genes were found in many IQA-producing plant species, which suggests that non-MYC2-type CjbHLH homologs are specifically associated with IQA biosynthesis. To test whether CjbHLH1 homologs are involved in the biosynthesis of IQA in a plant other than C. japonica, we isolated two genes homologous to CjbHLH1, i.e. EcbHLH1-1 and EcbHLH1-2, from Eschscholzia californica (California poppy). Stable transformants in which the expression levels of EcbHLH1 genes were constitutively suppressed by RNA interference (RNAi) showed a reduced expression of some IQA biosynthetic enzyme genes. A metabolite analysis confirmed that the suppression of EcbHLH1, particularly EcbHLH1-2, caused a decrease in sanguinarine accumulation in transgenic cultured cells. These results indicate that non-MYC2-type EcbHLH1s regulate IQA biosynthesis in California poppy like CjbHLH1 in C. japonica.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Benzofenantridinas/biossíntese , Coptis/metabolismo , Eschscholzia/metabolismo , Proteínas de Plantas/metabolismo , Acetatos/farmacologia , Benzofenantridinas/química , Berberina/química , Vias Biossintéticas/efeitos dos fármacos , Vias Biossintéticas/genética , Ciclopentanos/farmacologia , Regulação para Baixo/efeitos dos fármacos , Eschscholzia/efeitos dos fármacos , Eschscholzia/genética , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Inativação Gênica/efeitos dos fármacos , Isoquinolinas/química , Especificidade de Órgãos/efeitos dos fármacos , Oxilipinas/farmacologia , Plantas Geneticamente Modificadas , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Plântula/efeitos dos fármacos , Plântula/genética , Homologia de Sequência de AminoácidosRESUMO
Carpel development and evolution are central issues for plant biology. The conservation of genetic functions conferring carpel identity has been widely studied in higher plants. However, although genetic networks directing the development of characteristic features of angiosperm carpels such as stigma and style are increasingly known in Arabidopsis thaliana, little information is available on the conservation and diversification of these networks in other species. Here, we have studied the functional conservation of NGATHA transcription factors in widely divergent species within the eudicots. We determined by in situ hybridization the expression patterns of NGATHA orthologs in Eschscholzia californica and Nicotiana benthamiana. Virus-induced gene silencing (VIGS)-mediated inactivation of NGATHA genes in both species was performed and different microscopy techniques were used for phenotypic characterization. We found the expression patterns of EcNGA and NbNGA genes during flower development to be highly similar to each other, as well as to those reported for Arabidopsis NGATHA genes. Inactivation of EcNGA and NbNGA also caused severe defects in style and stigma development in both species. These results demonstrate the widely conserved essential role of NGATHA genes in style and stigma specification and suggest that the angiosperm-specific NGATHA genes were likely recruited to direct a carpel-specific developmental program.
Assuntos
Padronização Corporal/genética , Sequência Conservada , Flores/crescimento & desenvolvimento , Flores/genética , Genes de Plantas , Magnoliopsida/crescimento & desenvolvimento , Magnoliopsida/genética , Sequência de Aminoácidos , Arabidopsis/genética , Arabidopsis/crescimento & desenvolvimento , Eschscholzia/genética , Eschscholzia/crescimento & desenvolvimento , Regulação da Expressão Gênica de Plantas , Inativação Gênica , Dados de Sequência Molecular , Fenótipo , Proteínas de Plantas/química , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Alinhamento de Sequência , Nicotiana/genética , Nicotiana/crescimento & desenvolvimentoRESUMO
BACKGROUND: Two-channel (or two-color) microarrays are cost-effective platforms for comparative analysis of gene expression. They are traditionally analysed in terms of the log-ratios (M-values) of the two channel intensities at each spot, but this analysis does not use all the information available in the separate channel observations. Mixed models have been proposed to analyse intensities from the two channels as separate observations, but such models can be complex to use and the gain in efficiency over the log-ratio analysis is difficult to quantify. Mixed models yield test statistics for the null distributions can be specified only approximately, and some approaches do not borrow strength between genes. RESULTS: This article reformulates the mixed model to clarify the relationship with the traditional log-ratio analysis, to facilitate information borrowing between genes, and to obtain an exact distributional theory for the resulting test statistics. The mixed model is transformed to operate on the M-values and A-values (average log-expression for each spot) instead of on the log-expression values. The log-ratio analysis is shown to ignore information contained in the A-values. The relative efficiency of the log-ratio analysis is shown to depend on the size of the intraspot correlation. A new separate channel analysis method is proposed that assumes a constant intra-spot correlation coefficient across all genes. This approach permits the mixed model to be transformed into an ordinary linear model, allowing the data analysis to use a well-understood empirical Bayes analysis pipeline for linear modeling of microarray data. This yields statistically powerful test statistics that have an exact distributional theory. The log-ratio, mixed model and common correlation methods are compared using three case studies. The results show that separate channel analyses that borrow strength between genes are more powerful than log-ratio analyses. The common correlation analysis is the most powerful of all. CONCLUSIONS: The common correlation method proposed in this article for separate-channel analysis of two-channel microarray data is no more difficult to apply in practice than the traditional log-ratio analysis. It provides an intuitive and powerful means to conduct analyses and make comparisons that might otherwise not be possible.
Assuntos
Perfilação da Expressão Gênica/métodos , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Apolipoproteína A-I/genética , Teorema de Bayes , Eschscholzia/genética , Humanos , Células Jurkat , Modelos Lineares , Células MCF-7 , Modelos EstatísticosRESUMO
In angiosperms, the shoot apical meristem is at the origin of leaves and stems and is eventually transformed into the floral meristem. Class I knotted-like homeobox (KNOX I) genes are known as crucial regulators of shoot meristem formation and maintenance. KNOX I genes maintain the undifferentiated state of the apical meristem and are locally downregulated upon leaf initiation. In Arabidopsis, KNOX I genes, especially SHOOTMERISTEMLESS (STM), have been shown to regulate flower development and the formation of carpels. We investigated the role of STM-like genes in the reproductive development of Eschscholzia californica, to learn more about the evolution of KNOX I gene function in basal eudicots. We identified two orthologs of STM in Eschscholzia, EcSTM1 and EcSTM2, which are predominantly expressed in floral tissues. In contrast, a KNAT1/BP-like and a KNAT2/6-like KNOX I gene are mainly expressed in vegetative organs. Virus-induced gene silencing (VIGS) was used to knockdown gene expression, revealing that both EcSTM genes are required for the formation of reproductive organs. Silencing of EcSTM1 resulted in the loss of the gynoecium and a reduced number of stamens. EcSTM2-VIGS flowers had reduced and defective gynoecia and a stronger reduction in the number of stamen than observed in EcSTM1-VIGS. Co-silencing of both genes led to more pronounced phenotypes. In addition, silencing of EcSTM2 alone or together with EcSTM1 resulted in altered patterns of internodal elongation and sometimes in other floral defects. Our data suggest that some aspects of STM function present in Arabidopsis evolved already before the basal eudicots diverged from core eudicots.
Assuntos
Eschscholzia/crescimento & desenvolvimento , Eschscholzia/genética , Flores/genética , Regulação da Expressão Gênica de Plantas , Proteínas de Homeodomínio/genética , Proteínas de Plantas/genética , Regulação para Baixo , Eschscholzia/metabolismo , Flores/metabolismo , Meristema/crescimento & desenvolvimento , Meristema/metabolismoRESUMO
Eschscholzia californica (California poppy), a member of the basal eudicot family of the Papaveraceae, is an important species to study alkaloid biosynthesis and the effect of alkaloids on plant metabolism. More recently, it has also been developed as a model system to study the evolution of plant morphogenesis. While progress has been made towards establishing methods for generating genetically modified cell culture lines, transcriptome data and gene expression analysis, the stable transformation and subsequent regeneration of transgenic plants has proven extremely time consuming and difficult. Here, we describe in detail a method to transiently down regulate expression of a target gene by virus-induced gene silencing (VIGS) and the subsequent analysis of the VIGS treated plants. VIGS in E. californica allows for the study of gene function within 2 to 3 weeks after inoculation, and the method proves very efficient, enabling the rapid analysis of gene functions.
Assuntos
Eschscholzia/genética , Vírus de Plantas/genética , Interferência de RNA , Agrobacterium tumefaciens/virologia , Alcaloides/biossíntese , Clonagem Molecular , Eschscholzia/virologia , Flores/genética , Flores/virologia , Perfilação da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Técnicas de Silenciamento de Genes , Genes de Plantas , Vetores Genéticos , Anotação de Sequência Molecular , RNA de Plantas/genética , RNA de Plantas/isolamento & purificação , Coloração e Rotulagem , Inclusão do Tecido , Transformação BacterianaRESUMO
The products of B class floral homeotic genes specify petal and stamen identity, and loss of B function results in homeotic conversions of petals into sepals and stamens into carpels. Here, we describe the molecular characterization of seirena-1 (sei-1), a mutant from the basal eudicot California poppy (Eschscholzia californica) that shows homeotic changes characteristic of floral homeotic B class mutants. SEI has been previously described as EScaGLO, one of four B class-related MADS box genes in California poppy. The C terminus of SEI, including the highly conserved PI motif, is truncated in sei-1 proteins. Nevertheless, like the wild-type SEI protein, the sei-1 mutant protein is able to bind CArG-boxes and can form homodimers, heterodimers, and several higher order complexes with other MADS domain proteins. However, unlike the wild type, the mutant protein is not able to mediate higher order complexes consisting of specific B, C, and putative E class related proteins likely involved in specifying stamen identity. Within the PI motif, five highly conserved N-terminal amino acids are specifically required for this interaction. Several families lack this short conserved sequence, including the Brassicaceae, and we propose an evolutionary scenario to explain these functional differences.
Assuntos
Eschscholzia/genética , Proteínas de Domínio MADS/genética , Mutação , Proteínas de Plantas/genética , Motivos de Aminoácidos , Proteínas de Arabidopsis/genética , Sequência de Bases , Sequência Conservada , Evolução Molecular , Flores/genética , Regulação da Expressão Gênica de Plantas , Genes Homeobox , Proteínas de Domínio MADS/metabolismo , Dados de Sequência Molecular , Filogenia , Proteínas de Plantas/metabolismo , Domínios e Motivos de Interação entre Proteínas , Multimerização ProteicaRESUMO
PREMISE OF THE STUDY: We isolated and characterized microsatellite markers for the California poppy, Eschscholzia californica, which is an invasive species in central Chile. METHODS AND RESULTS: A total of eight polymorphic and six monomorphic loci were developed for the species. Between one and 12 alleles were observed per locus. Polymorphic loci showed heterozygosity ranging from 0 to 0.875 in a sample of 96 individuals obtained from four populations. Only one locus showed significant departures from Hardy-Weinberg equilibrium at all sites. CONCLUSIONS: The successful microsatellite amplification makes this set of primers an important tool for understanding the recent and future patterns of invasion and adaptation of E. californica into the new Chilean geographic area. The variation detected is currently being used in a more inclusive study that assesses population expansion in central Chile.
Assuntos
Eschscholzia/genética , Espécies Introduzidas , Repetições de Microssatélites/genética , Chile , Primers do DNA/metabolismo , Loci Gênicos/genética , Genética Populacional , Dados de Sequência MolecularRESUMO
YABBY genes are seed plant-specific transcriptional regulators that are involved in diverse aspects of leaf, shoot and flower development. A series of duplications gave rise to five gene groups found throughout flowering plants. In Arabidopsis and other species, expression of two gene groups, CRABS CLAW and INNER NO OUTER, is restricted to floral organs. In contrast, members of the FILAMENTOUS FLOWER, YABBY2 and YABBY5 gene groups are also expressed in leaves and have been termed 'vegetative YABBYs'. How the five paralogue groups evolved and how their expression and function diversified have remained largely unresolved, precluding a reconstruction of the natural history of this gene family. Here, we report new genes from Eschscholzia californica (Ranunculales, Papaveraceae) that we use together with currently available database sequences in a comprehensive phylogenetic re-evaluation of the YABBY gene family. Multilayered Bayesian analysis covering seed plants allowed us to locate Eschscholzia YABBY sequences within the gene family phylogeny. We established that vegetative YABBYs do not form a monophyletic clade, and that CRABS CLAW and FILAMENTOUS FLOWER arose from a common ancestor gene. INNER NO OUTER genes are sister to that ancestral gene. We identified several conserved motifs outside of known amino acid domains that define all five angiosperm YABBY gene clades. Further, we inferred the evolution of gene expression and provide evidence for release of purifying constraint in certain branches of the gene family tree. Finally, we report expression patterns for five Eschscholzia YABBY genes consistent with functional conservation between early-diverged and core eudicots.
Assuntos
Eschscholzia/genética , Sequência de Bases , Evolução Biológica , Evolução Molecular , Flores/crescimento & desenvolvimento , Regulação da Expressão Gênica de Plantas , Genes de Plantas , Dados de Sequência Molecular , Filogenia , Folhas de Planta/crescimento & desenvolvimento , Proteínas de Plantas/genética , Brotos de Planta/crescimento & desenvolvimento , Análise de Sequência de ProteínaRESUMO
BACKGROUND: Molecular genetic studies of floral development have concentrated on several core eudicots and grasses (monocots), which have canalized floral forms. Basal eudicots possess a wider range of floral morphologies than the core eudicots and grasses and can serve as an evolutionary link between core eudicots and monocots, and provide a reference for studies of other basal angiosperms. Recent advances in genomics have enabled researchers to profile gene activities during floral development, primarily in the eudicot Arabidopsis thaliana and the monocots rice and maize. However, our understanding of floral developmental processes among the basal eudicots remains limited. RESULTS: Using a recently generated expressed sequence tag (EST) set, we have designed an oligonucleotide microarray for the basal eudicot Eschscholzia californica (California poppy). We performed microarray experiments with an interwoven-loop design in order to characterize the E. californica floral transcriptome and to identify differentially expressed genes in flower buds with pre-meiotic and meiotic cells, four floral organs at preanthesis stages (sepals, petals, stamens and carpels), developing fruits, and leaves. CONCLUSIONS: Our results provide a foundation for comparative gene expression studies between eudicots and basal angiosperms. We identified whorl-specific gene expression patterns in E. californica and examined the floral expression of several gene families. Interestingly, most E. californica homologs of Arabidopsis genes important for flower development, except for genes encoding MADS-box transcription factors, show different expression patterns between the two species. Our comparative transcriptomics study highlights the unique evolutionary position of E. californica compared with basal angiosperms and core eudicots.
Assuntos
Eschscholzia/genética , Flores/crescimento & desenvolvimento , Perfilação da Expressão Gênica , Genoma de Planta , Arabidopsis/genética , Eschscholzia/crescimento & desenvolvimento , Evolução Molecular , Etiquetas de Sequências Expressas , Flores/genética , Regulação da Expressão Gênica de Plantas , Meiose , Análise de Sequência com Séries de Oligonucleotídeos , Sondas de Oligonucleotídeos/genética , Filogenia , Folhas de Planta/genética , Folhas de Planta/crescimento & desenvolvimento , RNA de Plantas/genéticaRESUMO
The model basal eudicot plant California poppy (Eschscholzia californica Cham.) typically has intense yellow to orange petals and orange pollen due to pigmentation by carotenoids. Flower color variants ranging from white to yellow and orange are common. We analyzed flower color inheritance in a diverse range of white and yellow color variants with reduced carotenoid content. The inheritance of the petal-pollen color of 24 variant flowers was investigated through complementation analysis by hybridization between different color variants and screening F(1), F(2), and BC(1) populations for segregation of petal-pollen color. All white and yellow flower color variants exhibited the pleiotropic effect with each mutation influencing both petal and pollen color, with both petal and pollen color phenotypes coinherited. A total of 5 complementation groups were identified with the color variants behaving as single recessive loci. Epistatic interactions among the loci were also identified. The white/yellow California poppy color variants described in this paper represent a unique genetic resource for analysis of carotenoid biosynthesis in this basal eudicot species.