RESUMO
The tagging-via-substrate approach designed for the capture of mammal prenylated proteins was adapted to Arabidopsis cell culture. In this way, proteins are in vivo tagged with an azide-modified farnesyl moiety and captured thanks to biotin alkyne Click-iT® chemistry with further streptavidin-affinity chromatography. Mass spectrometry analyses identified four small GTPases and ASG2 (ALTERED SEED GERMINATION 2), a protein previously associated to the seed germination gene network. ASG2 is a conserved protein in plants and displays a unique feature that associates WD40 domains and tetratricopeptide repeats. Additionally, we show that ASG2 has a C-terminal CaaX-box that is farnesylated in vitro. Protoplast transfections using CaaX prenyltransferase mutants show that farnesylation provokes ASG2 nucleus exclusion. Moreover, ASG2 interacts with DDB1 (DAMAGE DNA BINDING protein 1), and the subcellular localization of this complex depends on ASG2 farnesylation status. Finally, germination and root elongation experiments reveal that asg2 and the farnesyltransferase mutant era1 (ENHANCED RESPONSE TO ABSCISIC ACID (ABA) 1) behave in similar manners when exposed to ABA or salt stress. To our knowledge, ASG2 is the first farnesylated DWD (DDB1 binding WD40) protein related to ABA response in Arabidopsis that may be linked to era1 phenotypes.
Assuntos
Ácido Abscísico/metabolismo , Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , Regulação da Expressão Gênica de Plantas , Reguladores de Crescimento de Plantas/metabolismo , Transducina/metabolismo , Sequência de Aminoácidos , Arabidopsis/citologia , Arabidopsis/efeitos dos fármacos , Arabidopsis/fisiologia , Proteínas de Arabidopsis/genética , Genes Reporter , Germinação , Dados de Sequência Molecular , Mutação , Fenótipo , Raízes de Plantas/citologia , Raízes de Plantas/efeitos dos fármacos , Raízes de Plantas/genética , Raízes de Plantas/fisiologia , Plantas Geneticamente Modificadas , Prenilação de Proteína , Transporte Proteico , Proteínas Recombinantes de Fusão , Alinhamento de Sequência , Cloreto de Sódio/farmacologia , Estresse Fisiológico , Transducina/genéticaRESUMO
The Madagascar periwinkle produces monoterpenoid indole alkaloids (MIA) of high interest due to their therapeutical values. The terpenoid moiety of MIA is derived from the methyl erythritol phosphate (MEP) and seco-iridoid pathways. These pathways are regarded as the limiting branch for MIA biosynthesis in C. roseus cell and tissue cultures. In previous studies, we demonstrated a coordinated regulation at the transcriptional and spatial levels of genes from both pathways. We report here on the isolation of the 5'-flanking region (1,049 bp) of the hydroxymethylbutenyl 4-diphosphate synthase (HDS) gene from the MEP pathway. To investigate promoter transcriptional activities, the HDS promoter was fused to GUS reporter gene. Agrobacterium-mediated transformation of young tobacco leaves revealed that the cloned HDS promoter displays a tissue-specific GUS staining restricted to the vascular region of the leaves and limited to a part of the vein that encompasses the phloem in agreement with the previous localization of HDS transcripts in C. roseus aerial organs. Further functional characterizations in stably or transiently transformed C. roseus cells allowed us to identify the region that can be consider as the minimal promoter and to demonstrate the induction of HDS promoter by several hormonal signals (auxin, cytokinin, methyljasmonate and ethylene) leading to MIA production. These results, and the bioinformatic analysis of the HDS 5'-region, suggest that the HDS promoter harbours a number of cis-elements binding specific transcription factors that would regulate the flux of terpenoid precursors involved in MIA biosynthesis.
Assuntos
Catharanthus/enzimologia , Catharanthus/genética , Enzimas/genética , Eritritol/análogos & derivados , Eritritol/metabolismo , Genes de Plantas/genética , Regiões Promotoras Genéticas , Fosfatos Açúcares/metabolismo , Região 5'-Flanqueadora/genética , Acetatos/farmacologia , Sequência de Bases , Vias Biossintéticas/efeitos dos fármacos , Catharanthus/citologia , Catharanthus/efeitos dos fármacos , Clonagem Molecular , Ciclopentanos/farmacologia , Citocininas/farmacologia , Enzimas/metabolismo , Etilenos/farmacologia , Perfilação da Expressão Gênica , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Glucuronidase/metabolismo , Dados de Sequência Molecular , Motivos de Nucleotídeos/genética , Especificidade de Órgãos/efeitos dos fármacos , Especificidade de Órgãos/genética , Oxilipinas/farmacologia , Folhas de Planta/efeitos dos fármacos , Folhas de Planta/enzimologia , Folhas de Planta/genética , Plantas Geneticamente Modificadas , Alcaloides de Triptamina e Secologanina/metabolismo , Análise de Sequência de DNA , Deleção de Sequência/genética , Suspensões , Nicotiana/efeitos dos fármacos , Nicotiana/genética , Transcrição Gênica/efeitos dos fármacosRESUMO
Cytokinins (CK) have been extensively studied for their roles in plant development. Recently, they also appeared to ensure crucial functions in the pathogenicity of some bacterial and fungal plant pathogens. Thus, identifying cytokinin-producing pathogens is a prerequisite to gain a better understanding of their role in pathogenicity. Taking advantage of the cytokinin perception properties of Malus domestica CHASE Histidine Kinase receptor 2 (MdCHK2), we thereby developed a selective and highly sensitive yeast biosensor for the application of cytokinin detection in bacterial samples. The biosensor is based on the mutated sln1Δ Saccharomyces cerevisiae strain expressing MdCHK2. The biosensor does not require any extraction or purification steps of biological samples, enabling cytokinin analysis directly from crude bacterial supernatants. For the first time, the production of cytokinin was shown in the well-known plant pathogenic bacteria Erwinia amylovora and was also revealed in human pathogens Staphylococcus aureus and Streptococcus agalactiae. Importantly, this biosensor was shown to be an efficient tool for unraveling certain steps in cytokinin biosynthesis by micro-organisms since this it was successfully used to unveil the role of ygdH22, a LOG-like gene, that is probably involved in cytokinin biosynthesis pathway in Escherichia coli. Overall, we demonstrated that our biosensor displays several advantages including time- and cost-effectiveness by allowing a rapid and specific detection of cytokinins in bacterial supernatants These results also support its scalability to high-throughput formats.
Assuntos
Técnicas Biossensoriais , Citocininas/metabolismo , Histidina Quinase/genética , Proteínas de Plantas/genética , Saccharomyces cerevisiae/genética , Bactérias/metabolismo , MalusRESUMO
Cytokinin signaling is a key regulatory pathway of many aspects in plant development and environmental stresses. Herein, we initiated the identification and functional characterization of the five CHASE-containing histidine kinases (CHK) in the economically important Malus domestica species. These cytokinin receptors named MdCHK2, MdCHK3a/MdCHK3b, and MdCHK4a/MdCHK4b by homology with Arabidopsis AHK clearly displayed three distinct profiles. The three groups exhibited architectural variations, especially in the N-terminal part including the cytokinin sensing domain. Using a yeast complementation assay, we showed that MdCHK2 perceives a broad spectrum of cytokinins with a substantial sensitivity whereas both MdCHK4 homologs exhibit a narrow spectrum. Both MdCHK3 homologs perceived some cytokinins but surprisingly they exhibited a basal constitutive activity. Interaction studies revealed that MdCHK2, MdCHK4a, and MdCHK4b homodimerized whereas MdCHK3a and MdCHK3b did not. Finally, qPCR analysis and bioinformatics approach pointed out contrasted expression patterns among the three MdCHK groups as well as distinct sets of co-expressed genes. Our study characterized for the first time the five cytokinin receptors in apple tree and provided a framework for their further functional studies.
RESUMO
Elucidation of the monoterpene indole alkaloid biosynthesis has recently progressed in Apocynaceae through the concomitant development of transcriptomic analyses and reverse genetic approaches performed by virus-induced gene silencing (VIGS). While most of these tools have been primarily adapted for the Madagascar periwinkle (Catharanthus roseus), the VIGS procedure has scarcely been used on other Apocynaceae species. For instance, Rauwolfia sp. constitutes a unique source of specific and valuable monoterpene indole alkaloids such as the hypertensive reserpine but are also well recognized models for studying alkaloid metabolism, and as such would benefit from an efficient VIGS procedure. By taking advantage of a recent modification in the inoculation method of the Tobacco rattle virus vectors via particle bombardment, we demonstrated that the biolistic-mediated VIGS approach can be readily used to silence genes in both Rauwolfia tetraphylla and Rauwolfia serpentina. After establishing the bombardment conditions minimizing injuries to the transformed plantlets, gene downregulation efficiency was evaluated at approximately a 70% expression decrease in both species by silencing the phytoene desaturase encoding gene. Such a gene silencing approach will thus constitute a critical tool to identify and characterize genes involved in alkaloid biosynthesis in both of these prominent Rauwolfia species.
Assuntos
Oxirredutases/genética , Proteínas de Plantas/genética , Rauwolfia/genética , Biolística , Expressão Gênica , Regulação da Expressão Gênica de Plantas , Inativação Gênica , Vetores Genéticos , Oxirredutases/metabolismo , Proteínas de Plantas/metabolismo , Vírus de Plantas/genética , Rauwolfia/enzimologiaRESUMO
Substituted 3-[2-(diethoxyphosphoryl)propyl]oxazolo[4,5-b]pyridine-2(3H)-ones were obtained by functionalization at 6-position with various substituents (aryl, vinyl, carbonyl chains) via reactions catalysed with palladium. We found that these new fosmidomycin analogues inhibited the accumulation of ajmalicine, a marker of monoterpenoid indole alkaloids production in plant cells. Some of them have greater inhibitory effect than fosmidomycin and fully inhibit alkaloid accumulation at the concentration of 100 microM.
Assuntos
Catharanthus/fisiologia , Fosfomicina/análogos & derivados , Alcaloides Indólicos/antagonistas & inibidores , Indóis/antagonistas & inibidores , Terpenos/metabolismo , Catharanthus/efeitos dos fármacos , Relação Dose-Resposta a Droga , Fosfomicina/farmacologia , CinéticaRESUMO
Calreticulin (CALR) mutations have recently been reported in 70-84% of JAK2V617F-negative myeloproliferative neoplasms (MPN), and this detection has become necessary to improve the diagnosis of MPN. In a large single-centre cohort of 298 patients suffering from Essential Thrombocythemia (ET), the JAK2V617F, CALR and MPL mutations were noted in 179 (60%), 56 (18.5%) and 13 (4.5%) respectively. For the detection of the CALR mutations, three methods were compared in parallel: high-resolution melting-curve analysis (HRM), product-sizing analysis and Sanger sequencing. The sensitivity for the HRM, product-sizing analysis and Sanger sequencing was 96.4%, 98.2% and 89.3% respectively, whereas the specificity was 96.3%, 100% and 100%. In our cohort, the product-sizing analysis was the most sensitive method and was the easiest to interpret, while the HRM was sometimes difficult to interpret. In contrast, when large series of samples were tested, HRM provided results more quickly than did the other methods, which required more time. Finally, the sequencing method, which is the reference method, had the lowest sensitivity but can be used to describe the type of mutation precisely. Altogether, our results suggest that in routine laboratory practice, product-sizing analysis is globally similar to HRM for the detection of CALR mutations, and that both may be used as first-line screening tests. If the results are positive, Sanger sequencing can be used to confirm the mutation and to determine its type. Product-sizing analysis provides sensitive and specific results, moreover, with the quantitative measurement of CALR, which might be useful to monitor specific treatments.
Assuntos
Neoplasias da Medula Óssea/diagnóstico , Calreticulina/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Neoplasias da Medula Óssea/genética , Feminino , Mutação da Fase de Leitura/genética , Humanos , Mutação INDEL/genética , Masculino , Pessoa de Meia-Idade , Mutação/genética , Sensibilidade e Especificidade , Análise de Sequência/métodos , Adulto JovemRESUMO
In Catharanthus roseus, accumulating data highlighted the existence of a coordinated transcriptional regulation of structural genes that takes place within the secoiridoid biosynthetic branch, including the methyl erythritol phosphate (MEP) pathway and the following steps leading to secologanin. To identify transcription factors acting in these pathways, we performed a yeast one-hybrid screening using as bait a promoter region of the hydroxymethylbutenyl 4-diphosphate synthase (HDS) gene involved in the responsiveness of C. roseus cells to hormonal signals inducing monoterpene indole alkaloid (MIA) production. We identified that ZCT2, one of the three members of the zinc finger Catharanthus protein (ZCT) family, can bind to a HDS promoter region involved in hormonal responsiveness. By trans-activation assays, we demonstrated that ZCT1 and ZCT2 but not ZCT3 repress the HDS promoter activity. Gene expression analyses in C. roseus cells exposed to methyljasmonate revealed a persistence of induction of ZCT2 gene expression suggesting the existence of feed-back regulatory events acting on HDS gene expression in correlation with the MIA production.
Assuntos
Catharanthus/enzimologia , Catharanthus/genética , Regulação da Expressão Gênica de Plantas , Proteínas de Plantas/genética , Regiões Promotoras Genéticas , Fatores de Transcrição/genética , Enzimas/genética , Enzimas/metabolismo , Eritritol/análogos & derivados , Eritritol/metabolismo , Dados de Sequência Molecular , Proteínas de Plantas/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Análise de Sequência de DNA , Fatores de Transcrição/metabolismoRESUMO
In plant cytokinin (CK) signaling, type-B response regulators (RRs) act as major players in orchestrating the transcriptome changes in response to CK. However, their direct targets are poorly known. The identification of putative type-ARR1 motifs located within the promoter of the CK-responsive hydroxyl methyl butenyl diphosphate synthase (HDS) gene from the methyl erythritol phosphate (MEP) pathway prompted us to investigate the ability of a previously isolated periwinkle type-B RR (CrRR5) that presents high homologies with ARR1 to interact with the promoter. Electrophoretic mobility shift assays (EMSAs) demonstrated that the CrRR5 DNA-binding domain binds specifically type-ARR1 motifs within the HDS promoter. We also established through yellow fluorescent protein (YFP) imaging the targeting of CrRR5 into cell nucleus in accordance with its putative function of transcription factor. In transient assays performed on periwinkle cells cultivated with CK, overexpression of the full-length CrRR5 or a truncated CrRR5 engineering a constitutive active form (35S:ΔDDK) did not affect the HDS promoter activity that reached a threshold. By contrast, in absence of CK, overexpression of CrRR5ΔDDK enhanced promoter activity up to the threshold level observed in cells grown with CK. Our results strongly suggest that CrRR5 directly transactivates the HDS promoter. CrRR5 is the first identified transcription factor mediating the CK signaling that targets a gene from the MEP pathway involved in isoprenoid metabolism. Moreover, CrRR5 could play a role in a regulatory mechanism controlling CK homeostasis in periwinkle cells.
Assuntos
Catharanthus/enzimologia , Citocininas/metabolismo , Enzimas/genética , Enzimas/metabolismo , Eritritol/biossíntese , Reguladores de Crescimento de Plantas/metabolismo , Fatores de Transcrição/metabolismo , Catharanthus/genética , Regulação da Expressão Gênica de Plantas , Genes de Plantas , Homeostase/fisiologia , Regiões Promotoras GenéticasRESUMO
CaaX-prenyltransferases (CaaX-PTases) catalyse the covalent attachment of isoprenyl groups to conserved cysteine residues located at the C-terminal CaaX motif of a protein substrate. This post-translational modification is required for the function and/or subcellular localization of some transcription factors and components of signal transduction and membrane trafficking machinery. CaaX-PTases, including protein farnesyltransferase (PFT) and type-I protein geranylgeranyltransferase (PGGT-I), are heterodimeric enzymes composed of a common alpha subunit and a specific beta subunit. We have established RNA interference cell lines targeting the beta subunits of PFT and PGGT-I, respectively, in the Catharanthus roseus C20D cell line, which synthesizes monoterpenoid indole alkaloids in response to auxin depletion from the culture medium. In both types of RNAi cell lines, expression of a subset of genes involved in the early stage of monoterpenoid biosynthetic pathway (ESMB genes), including the MEP pathway, is strongly decreased. The role of CaaX-PTases in ESMB gene regulation was confirmed by using the general prenyltransferase inhibitor s-perillyl alcohol (SP) and the specific PFT inhibitor Manumycin A on the wild type line. Furthermore, supplementation of SP inhibited cells with monoterpenoid intermediates downstream of the steps encoded by the ESMB genes restores monoterpenoid indole alkaloids biosynthesis. We conclude that protein targets for both PFT and PGGT-I are required for the expression of ESMB genes and monoterpenoid biosynthesis in C. roseus, this represents a non previously described role for protein prenyltransferase in plants.
Assuntos
Alquil e Aril Transferases/genética , Catharanthus/genética , Regulação da Expressão Gênica de Plantas/genética , Monoterpenos/metabolismo , Alquil e Aril Transferases/antagonistas & inibidores , Alquil e Aril Transferases/metabolismo , Northern Blotting , Catharanthus/citologia , Catharanthus/metabolismo , Técnicas de Cultura de Células , Processos de Crescimento Celular/genética , Regulação para Baixo/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Dados de Sequência Molecular , Monoterpenos/farmacologia , Proteínas de Plantas/antagonistas & inibidores , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Prenilação de Proteína/efeitos dos fármacos , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Interferência de RNA/fisiologia , RNA de Plantas/genética , RNA de Plantas/metabolismo , Alcaloides de Triptamina e Secologanina/metabolismo , Fatores de TempoRESUMO
In higher plants, isopentenyl diphosphate (IPP) is synthesised both from the plastidic 2-C-methyl-d-erythritol 4-phosphate (MEP) and from the cytosolic mevalonate (MVA) pathways. Primary metabolites, such as phytol group of chlorophylls, carotenoids and the plant hormones abscisic acid (ABA) and gibberellins (GAs) are derived directly from the MEP pathway. Many secondary metabolites, such as monoterpene indole alkaloids (MIAs) in Catharanthus roseus, are also synthesised from this source of IPP. Using Northern blot and in situ hybridisation experiments, we show that three MEP pathway genes (1-deoxy-d-xylulose 5-phosphate synthase (DXS), 1-deoxy-d-xylulose 5-phosphate reductoisomerase (DXR) and 2C-methyl-d-erythritol 2,4-cyclodiphosphate synthase (MECS)) and the gene encoding geraniol 10-hydroxylase (G10H), a cytochrome P450 monooxygenase involved in the first committed step in the formation of iridoid monoterpenoids display identical cell-specific expression patterns. The co-localisation of these four transcripts to internal phloem parenchyma of young aerial organs of C. roseus adds a new level of complexity to the multicellular nature of MIA biosynthesis. We predict the translocation of pathway intermediates from the internal phloem parenchyma to the epidermis and, ultimately, to laticifers and idioblasts during MIA biosynthesis. Similarly, the translocation of intermediates from the phloem parenchyma is probably also required during the biosynthesis of hormones and photosynthetic primary metabolites derived from the MEP pathway.
Assuntos
Catharanthus/genética , Catharanthus/metabolismo , Sistema Enzimático do Citocromo P-450/genética , Eritritol/análogos & derivados , Eritritol/metabolismo , Genes de Plantas , Alcaloides Indólicos/metabolismo , Oxigenases de Função Mista/genética , Fosfatos Açúcares/metabolismo , Aldose-Cetose Isomerases/genética , Catharanthus/citologia , Expressão Gênica , Hibridização In Situ , Modelos Biológicos , Complexos Multienzimáticos/genética , Oxirredutases/genética , Fósforo-Oxigênio Liases/genética , Terpenos/metabolismo , Transferases/genéticaRESUMO
1,3 Dipolar cycloadditions of nitrile oxides generated in situ in the presence of a variety of olefins provided 4,5-dihydroisoxazoles. The whole procedure could be performed in a practical and efficient one-pot operation. The products are of excellent purity (95%) and are isolated in 60-83% yields. Some of them enhanced the accumulation of indole alkaloids in periwinkle cell cultures.
Assuntos
Catharanthus/efeitos dos fármacos , Isoxazóis/síntese química , Nitrilas/síntese química , Óxidos/síntese química , Catharanthus/crescimento & desenvolvimento , Relação Dose-Resposta a Droga , Avaliação Pré-Clínica de Medicamentos/métodos , Isoxazóis/farmacologia , Nitrilas/farmacologia , Óxidos/farmacologiaRESUMO
Fosmidomycin, 3-(N-formyl-N-hydroxyamido) propylphosphonic acid sodium salt, is an efficient inhibitor of 1-deoxy-D-xylulose-5-phosphate (DOXP) reductoisomerase, the second enzyme of the 2C-methyl-D-erythritol-4-phosphate (MEP) pathway notably present in Plasmodium species. We have synthesized a new series of analogues of fosmidomycin, containing a benzoxazolone, benzoxazolethione or oxazolopyridinone ring. As the MEP pathway is involved in the biosynthesis of all isoprenoids, accumulation of ajmalicine in Catharanthus roseus cells was chosen as a marker of monoterpenoid indole alkaloid (MIA) production. None of the twelve studied phosphonic esters 3 and phosphonic acids 4 affected periwinkle cell growth, but some of them (3c, 3e, 3g and 3h) showed a significant inhibition of ajmalicine accumulation: 45-85% at 125 microM. Surprisingly, this effect disappeared by conversion of 3c and 3g into the corresponding acids 4c and 4g, respectively.