RESUMEN
Self-incompatibility (SI) is genetically determined reproductive barrier preventing inbreeding and thereby providing the maintenance of plant species diversity. At present, active studies of molecular bases of SI mechanisms are underway. S-RNAse-based SI in Petunia hybrida L. is a self-/non-self recognition system that allows the pistil to reject self pollen and to accept non-self pollen for outcrossing. In the present work, using fluorescent methods including the TUNEL method allowed us to reveal the presence of markers of programmed cell death (PCD), such as DNA fragmentation, in growing in vivo petunia pollen tubes during the passage of the SI reaction. The results of statistical analysis reliably proved that PCD is the factor of S-RNAse-based SI. It was found that preliminary treatment before self-pollination of stigmas of petunia self-incompatible line with aminooxyacetic acid (AOA), inhibitor of ACC synthesis, led to stimulation of pollen tubes growth when the latter did not exhibit any hallmarks of PCD. These data argue in favor of assumption that ethylene controls the passage of PCD in incompatible pollen tubes in the course of S-RNAse-based SI functioning. The involvement of the hormonal regulation in SI mechanism in P. hybrida L. is the finding observed by us for the first time.
Asunto(s)
Aminoácidos Cíclicos/biosíntesis , Ácido Aminooxiacético/farmacología , Apoptosis/efectos de los fármacos , Petunia/citología , Petunia/fisiología , Tubo Polínico/citología , Autoincompatibilidad en las Plantas con Flores/efectos de los fármacos , Fragmentación del ADN/efectos de los fármacos , Petunia/efectos de los fármacos , Petunia/ultraestructura , Tubo Polínico/efectos de los fármacos , Tubo Polínico/ultraestructura , Ribonucleasas/metabolismoRESUMEN
It is known that plant cells can contain multiple distinct vacuoles; however, the abundance of multivacuolar cells and the mechanisms underlying vacuolar differentiation and communication among different types of vacuoles remain unknown. PH1 and PH5 are tonoplast P-ATPases that form a heteromeric pump that hyper-acidifies the central vacuole (CV) of epidermal cells in petunia petals. Here, we show that the sorting of this pump and other vacuolar proteins to the CV involves transit through small vacuoles: vacuolinos. Vacuolino formation is controlled by transcription factors regulating pigment synthesis and transcription of PH1 and PH5. Trafficking of proteins from vacuolinos to the central vacuole is impaired by misexpression of vacuolar SNAREs as well as mutants for the PH1 component of the PH1-PH5 pump. The finding that PH1-PH5 and these SNAREs interact strongly suggests that structural tonoplast proteins can act as tethering factors in the recognition of different vacuolar types.
Asunto(s)
Petunia/enzimología , Proteínas de Plantas/fisiología , ATPasas de Translocación de Protón Vacuolares/fisiología , Vacuolas/enzimología , Flores/citología , Flores/enzimología , Fusión de Membrana , Petunia/citología , Epidermis de la Planta/citología , Transporte de ProteínasRESUMEN
As established by us earlier, ethylene behaves as a regulator of germination, development, and growth of male gametophyte during the progamic phase of fertilization. However, the mechanisms of the regulation of these processes remain so far unstudied. It is believed that the main factor providing variety of the ethylene responses is its interaction with other phytohormones. According to our working hypothesis, ethylene controls germination of pollen grains (PGs) and growth of pollen tubes (PTs) by interacting with auxin, which, as the available data indicate, is likely a key regulator of plant cell polarization and morphogenesis and one of the factors modulating the biosynthesis of ethylene at the level of ACC-synthase gene expression. In the present work, on germinating in vitro male gametophyte and the pollen-stigma system for petunia (Petunia hybrida L.) effects of phytohormones (ethylene and IAA) and known blockers repressing ethylene reception (1-methylcyclopropene, 1-MCP), the synthesis of ACC (amino oxyacetic acid, AOA) and transport IAA (triyodbenzoynaya acid, TYBA) on PGs germination, PTs growth and the synthesis of ACC were investigated. According to the data obtained, exogenous ethylene and IAA stimulated both PGs germination and PTs growth. 1-MCP and TYBA completely inhibited the first process, whereas IAA abolished the inhibitory action of 1-MCP and AOA on both the above processes. Etrel only partially weakened the inhibitory effect of TYBA. Examination of ACC synthesis modulation with AOA showed that IAA does not affect the level of ACC in germinating in vitro male gametophyte and nonpollinated stigmas, while this phytohormone insignificantly raised the level of ACC and abolished the inhibitory effect of AOA on its synthesis in the pollenstigma system. Pollination of stigmas with the pollen preliminarily treated with 1-MCP led to 2.5-fold decline in both the rate of PT growth and the level of ACC. At the same time, IAA abolished the inhibitory action of 1-MCP recovering the synthesis of ACC and growth of PTs to the control values. All these results, taken together, provide evidence for the interaction of the signal transduction pathways of ethylene and auxin at the level of ACC biosynthesis in the course of germination and growth of petunia male gametophyte during the progamic phase of fertilization.
Asunto(s)
Ácido Aminooxiacético/metabolismo , Ciclopropanos/farmacología , Ácidos Indolacéticos/farmacología , Petunia/metabolismo , Tubo Polínico/metabolismo , Petunia/citología , Tubo Polínico/citologíaRESUMEN
The WD40 proteins ANTHOCYANIN11 (AN11) from petunia (Petunia hybrida) and TRANSPARENT TESTA GLABRA1 (TTG1) from Arabidopsis thaliana and associated basic helix-loop-helix (bHLH) and MYB transcription factors activate a variety of differentiation processes. In petunia petals, AN11 and the bHLH protein AN1 activate, together with the MYB protein AN2, anthocyanin biosynthesis and, together with the MYB protein PH4, distinct genes, such as PH1 and PH5, that acidify the vacuole. To understand how AN1 and AN11 activate anthocyanin biosynthetic and PH genes independently, we isolated PH3. We found that PH3 is a target gene of the AN11-AN1-PH4 complex and encodes a WRKY protein that can bind to AN11 and is required, in a feed-forward loop, together with AN11-AN1-PH4 for transcription of PH5. PH3 is highly similar to TTG2, which regulates hair development, tannin accumulation, and mucilage production in Arabidopsis. Like PH3, TTG2 can bind to petunia AN11 and the Arabidopsis homolog TTG1, complement ph3 in petunia, and reactivate the PH3 target gene PH5. Our findings show that the specificity of WD40-bHLH-MYB complexes is in part determined by interacting proteins, such as PH3 and TTG2, and reveal an unanticipated similarity in the regulatory circuitry that controls petunia vacuolar acidification and Arabidopsis hair development.
Asunto(s)
Antocianinas/metabolismo , Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , Petunia/genética , Proteínas de Plantas/metabolismo , Arabidopsis/citología , Arabidopsis/crecimiento & desarrollo , Proteínas de Arabidopsis/genética , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Flores/citología , Flores/genética , Flores/fisiología , Regulación de la Expresión Génica de las Plantas , Redes Reguladoras de Genes , Homeostasis , Concentración de Iones de Hidrógeno , Petunia/citología , Petunia/fisiología , Filogenia , Proteínas de Plantas/genética , Plantas Modificadas Genéticamente , Vacuolas/metabolismoRESUMEN
KEY MESSAGE: Site-directed mutagenesis of nitrate reductase genes using direct delivery of purified Cas9 protein preassembled with guide RNA produces mutations efficiently in Petunia × hybrida protoplast system. The clustered, regularly interspaced, short palindromic repeat (CRISPR)-CRISPR associated endonuclease 9 (CRISPR/Cas9) system has been recently announced as a powerful molecular breeding tool for site-directed mutagenesis in higher plants. Here, we report a site-directed mutagenesis method targeting Petunia nitrate reductase (NR) gene locus. This method could create mutations efficiently using direct delivery of purified Cas9 protein and single guide RNA (sgRNA) into protoplast cells. After transient introduction of RNA-guided endonuclease (RGEN) ribonucleoproteins (RNPs) with different sgRNAs targeting NR genes, mutagenesis at the targeted loci was detected by T7E1 assay and confirmed by targeted deep sequencing. T7E1 assay showed that RGEN RNPs induced site-specific mutations at frequencies ranging from 2.4 to 21 % at four different sites (NR1, 2, 4 and 6) in the PhNR gene locus with average mutation efficiency of 14.9 ± 2.2 %. Targeted deep DNA sequencing revealed mutation rates of 5.3-17.8 % with average mutation rate of 11.5 ± 2 % at the same NR gene target sites in DNA fragments of analyzed protoplast transfectants. Further analysis from targeted deep sequencing showed that the average ratio of deletion to insertion produced collectively by the four NR-RGEN target sites (NR1, 2, 4, and 6) was about 63:37. Our results demonstrated that direct delivery of RGEN RNPs into protoplast cells of Petunia can be exploited as an efficient tool for site-directed mutagenesis of genes or genome editing in plant systems.
Asunto(s)
Sistemas CRISPR-Cas/genética , Mutagénesis Sitio-Dirigida/métodos , Petunia/genética , Protoplastos/metabolismo , Ribonucleoproteínas/genética , Secuencia de Bases , Ingeniería Genética/métodos , Genoma de Planta/genética , Secuenciación de Nucleótidos de Alto Rendimiento , Microscopía Fluorescente , Modelos Genéticos , Petunia/citología , Proteínas de Plantas/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Ribonucleoproteínas/metabolismo , Homología de Secuencia de Ácido NucleicoRESUMEN
Embryo infection is important for efficient seed transmission of viroids. To identify the major pattern of seed transmission of viroids, we used in situ hybridization to histochemically analyze the distribution of Potato spindle tuber viroid (PSTVd) in each developmental stage of petunia (flowering to mature seed stages). In floral organs, PSTVd was present in the reproductive tissues of infected female × infected male and infected female × healthy male but not of healthy female × infected male before embryogenesis. After pollination, PSTVd was detected in the developed embryo and endosperm in all three crosses. These findings indicate that PSTVd is indirectly delivered to the embryo through ovule or pollen during the development of reproductive tissues before embryogenesis but not directly through maternal tissues as cell-to-cell movement during embryogenesis.
Asunto(s)
Petunia/virología , Enfermedades de las Plantas/virología , Solanum lycopersicum/virología , Viroides/fisiología , Flores/citología , Flores/crecimiento & desarrollo , Flores/fisiología , Flores/virología , Hibridación in Situ , Meristema/citología , Meristema/crecimiento & desarrollo , Meristema/fisiología , Meristema/virología , Petunia/citología , Petunia/crecimiento & desarrollo , Petunia/fisiología , Brotes de la Planta/citología , Brotes de la Planta/crecimiento & desarrollo , Brotes de la Planta/fisiología , Brotes de la Planta/virología , Tubérculos de la Planta/virología , Polen/citología , Polen/crecimiento & desarrollo , Polen/fisiología , Polen/virología , Reproducción , Semillas/citología , Semillas/crecimiento & desarrollo , Semillas/fisiología , Semillas/virologíaRESUMEN
Calreticulin (CRT) is a highly conserved and ubiquitously expressed Ca²âº-binding protein in multicellular eukaryotes. As an endoplasmic reticulum-resident protein, CRT plays a key role in many cellular processes including Ca²âº storage and release, protein synthesis, and molecular chaperoning in both animals and plants. CRT has long been suggested to play a role in plant sexual reproduction. To begin to address this possibility, we cloned and characterized the full-length cDNA of a new CRT gene (PhCRT) from Petunia. The deduced amino acid sequence of PhCRT shares homology with other known plant CRTs, and phylogenetic analysis indicates that the PhCRT cDNA clone belongs to the CRT1/CRT2 subclass. Northern blot analysis and fluorescent in situ hybridization were used to assess PhCRT gene expression in different parts of the pistil before pollination, during subsequent stages of the progamic phase, and at fertilization. The highest level of PhCRT mRNA was detected in the stigma-style part of the unpollinated pistil 1 day before anthesis and during the early stage of the progamic phase, when pollen is germinated and tubes outgrow on the stigma. In the ovary, PhCRT mRNA was most abundant after pollination and reached maximum at the late stage of the progamic phase, when pollen tubes grow into the ovules and fertilization occurs. PhCRT mRNA transcripts were seen to accumulate predominantly in transmitting tract cells of maturing and receptive stigma, in germinated pollen/growing tubes, and at the micropylar region of the ovule, where the female gametophyte is located. From these results, we suggest that PhCRT gene expression is up-regulated during secretory activity of the pistil transmitting tract cells, pollen germination and outgrowth of the tubes, and then during gamete fusion and early embryogenesis.
Asunto(s)
Calcio/metabolismo , Calreticulina/genética , Regulación de la Expresión Génica de las Plantas , Petunia/genética , Secuencia de Aminoácidos , Secuencia de Bases , Proteínas de Unión al Calcio/genética , Proteínas de Unión al Calcio/metabolismo , Calreticulina/metabolismo , ADN Complementario/química , ADN Complementario/genética , ADN de Plantas/química , ADN de Plantas/genética , Flores/citología , Flores/genética , Flores/fisiología , Expresión Génica , Homeostasis , Datos de Secuencia Molecular , Petunia/citología , Petunia/fisiología , Filogenia , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Polen/citología , Polen/genética , Polen/fisiología , Polinización , Estructura Terciaria de Proteína , Alineación de Secuencia , Análisis de Secuencia de ADNRESUMEN
In which cells of the flower volatile biosynthesis takes place is unclear. In rose and snapdragon, some enzymes of the volatile phenylpropanoid/benzenoid pathway have been shown to be present in the epidermal cells of petals. It is therefore generally believed that the production of these compounds occurs in these cells. However, whether the entire pathway is active in these cells and whether it is exclusively active in these cells remains to be proven. Cell-specific transcription factors activating these genes will determine in which cells they are expressed. In petunia, the transcription factor EMISSION OF BENZENOIDS II (EOBII) activates the ODORANT1 (ODO1) promoter and the promoter of the biosynthetic gene isoeugenol synthase (IGS). The regulator ODO1 in turn activates the promoter of the shikimate gene 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS). Here the identification of a new target gene of ODO1, encoding an ABC transporter localized on the plasma membrane, PhABCG1, which is co-expressed with ODO1, is described. PhABCG1 expression is up-regulated in petals overexpressing ODO1 through activation of the PhABCG1 promoter. Interestingly, the ODO1, PhABCG1, and IGS promoters were active in petunia protoplasts originating from both epidermal and mesophyll cell layers of the petal, suggesting that the volatile phenylpropanoid/benzenoid pathway in petunia is active in these different cell types. Since volatile release occurs from epidermal cells, trafficking of (volatile) compounds between cell layers must be involved, but the exact function of PhABCG1 remains to be resolved.
Asunto(s)
Flores/citología , Flores/genética , Regulación de la Expresión Génica de las Plantas , Genes de Plantas/genética , Odorantes , Petunia/citología , Petunia/genética , Epidermis de la Planta/citología , Transportadoras de Casetes de Unión a ATP/química , Transportadoras de Casetes de Unión a ATP/genética , Transportadoras de Casetes de Unión a ATP/metabolismo , Secuencia de Aminoácidos , Membrana Celular/metabolismo , Espacio Intracelular/metabolismo , Datos de Secuencia Molecular , Peso Molecular , Especificidad de Órganos/genética , Epidermis de la Planta/genética , Proteínas de Plantas/química , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Regiones Promotoras Genéticas/genética , Transporte de Proteínas , Protoplastos/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Fracciones Subcelulares/metabolismoRESUMEN
Petunia is an excellent model system, especially for genetic, physiological and molecular studies. Thus far, however, genome-wide expression analysis has been applied rarely because of the lack of sequence information. We applied next-generation sequencing to generate, through de novo read assembly, a large catalogue of transcripts for Petunia axillaris and Petunia inflata. On the basis of both transcriptomes, comprehensive microarray chips for gene expression analysis were established and used for the analysis of global- and organ-specific gene expression in Petunia axillaris and Petunia inflata and to explore the molecular basis of the seed coat defects in a Petunia hybrida mutant, anthocyanin 11 (an11), lacking a WD40-repeat (WDR) transcription regulator. Among the transcripts differentially expressed in an11 seeds compared with wild type, many expected targets of AN11 were found but also several interesting new candidates that might play a role in morphogenesis of the seed coat. Our results validate the combination of next-generation sequencing with microarray analyses strategies to identify the transcriptome of two petunia species without previous knowledge of their genome, and to develop comprehensive chips as useful tools for the analysis of gene expression in P. axillaris, P. inflata and P. hybrida.
Asunto(s)
Petunia/genética , Proteínas de Plantas/genética , Proantocianidinas/biosíntesis , Transcriptoma , Secuencia de Bases , Secuencia de Consenso , Regulación hacia Abajo/genética , Flores/citología , Flores/genética , Flores/fisiología , Perfilación de la Expresión Génica , Regulación de la Expresión Génica de las Plantas/genética , Genoma de Planta/genética , Secuenciación de Nucleótidos de Alto Rendimiento , Anotación de Secuencia Molecular , Mutación , Análisis de Secuencia por Matrices de Oligonucleótidos , Petunia/química , Petunia/citología , Petunia/fisiología , Extractos Vegetales/química , Proteínas de Plantas/metabolismo , Proantocianidinas/análisis , ARN de Planta/genética , Plantones/citología , Plantones/genética , Plantones/fisiología , Semillas/química , Semillas/citología , Semillas/genética , Semillas/fisiología , Análisis de Secuencia de ADN , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Regulación hacia Arriba/genéticaAsunto(s)
Protoplastos/metabolismo , Proteínas de Arabidopsis/metabolismo , Flores/citología , Flores/metabolismo , Proteínas Fluorescentes Verdes/metabolismo , Petunia/citología , Petunia/metabolismo , Hojas de la Planta/citología , Hojas de la Planta/metabolismo , Plantas Modificadas Genéticamente , Protoplastos/citología , Proteínas Recombinantes de Fusión/metabolismoRESUMEN
Plastid engineering technique has been established only in Nicotiana tabacum, and the widespread application is severely limited so far. In order to exploit a method to transfer the genetically transformed plastomes already obtained in tobacco into other plant species, somatic cell fusion was conducted between a plastome transformant of tobacco and a cultivar of petunia (Petunia hybrida). A tobacco strain whose plastids had been transformed with aadA (a streptomycin/spectinomycin adenylyltransferase gene) and mdar [a gene for monodehydroascorbate reductase (MDAR)] and a petunia variety, 'Telstar', were used as cell fusion partners. An efficient regeneration system from the protoplasts of both the parents, and effectiveness of selection for the aadA gene with spectinomycin were established before the cell fusion. In addition, the influence of UV irradiation on the callus development from the protoplasts and shoot regeneration of tobacco was investigated. Protoplasts were cultured after cell fusion treatment with polyethylene glycol, and asymmetric somatic cybrids were selected using the aadA gene as a marker. Although many shoots of tobacco that had escaped the UV irradiation regenerated, several shoots possessing the morphology of petunia and the resistance to spectinomycin were obtained. Molecular analyses of the petunia type regenerants demonstrated that they had the nuclear and mitochondrial genomes derived from petunia besides the chloroplasts of tobacco transformed with aadA and mdar. Furthermore, it was ascertained that mdar was transcribed in the somatic cybrids. The results indicate the success in intergeneric transfer of transformed plastids of tobacco into petunia.
Asunto(s)
Cloroplastos/genética , Nicotiana/citología , Nicotiana/genética , Petunia/citología , Petunia/genética , Transgenes , Fusión Celular , Cloroplastos/metabolismo , Flores/anatomía & histología , Flores/genética , Flores/crecimiento & desarrollo , Flores/metabolismo , Regulación de la Expresión Génica de las Plantas , Hibridomas , NADH NADPH Oxidorreductasas/genética , NADH NADPH Oxidorreductasas/metabolismo , Nucleotidiltransferasas/genética , Nucleotidiltransferasas/metabolismo , Petunia/crecimiento & desarrollo , Petunia/metabolismo , Plantas Modificadas Genéticamente , Nicotiana/metabolismoRESUMEN
Adventitious root formation (ARF) in the model plant Petunia hybrida cv. Mitchell has been analysed in terms of anatomy, gene expression, enzymatic activities and levels of metabolites. This study focuses on the involvement of wound response and primary metabolism. Microscopic techniques were complemented with targeted transcript, enzyme and metabolite profiling using real time polymerase chain reaction (PCR), Northern blot, enzymatic assays, chromatography and mass spectrometry. Three days after severance from the stock plants, first meristematic cells appeared which further developed into root primordia and finally adventitious roots. Excision of cuttings led to a fast and transient increase in the wound-hormone jasmonic acid, followed by the expression of jasmonate-regulated genes such as cell wall invertase. Analysis of soluble and insoluble carbohydrates showed a continuous accumulation during ARF. A broad metabolite profiling revealed a strong increase in organic acids and resynthesis of essential amino acids. Substantial changes in enzyme activities and metabolite levels indicate that specific enzymes and metabolites might play a crucial role during ARF. Three metabolic phases could be defined: (i) sink establishment phase characterized by apoplastic unloading of sucrose and being probably mediated by jasmonates; (ii) recovery phase; and (iii) maintenance phase, in which a symplastic unloading occurs.
Asunto(s)
Petunia/metabolismo , Raíces de Plantas/metabolismo , Aminoácidos/metabolismo , Metabolismo de los Hidratos de Carbono/genética , Respiración de la Célula , Ciclo del Ácido Cítrico , Ciclopentanos/metabolismo , Ácidos Grasos Insaturados/metabolismo , Regulación de la Expresión Génica de las Plantas , Genes de Plantas , Glucólisis , Oxilipinas/metabolismo , Petunia/citología , Petunia/enzimología , Petunia/genética , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Raíces de Plantas/citología , Raíces de Plantas/enzimología , Raíces de Plantas/genética , ARN Mensajero/genética , ARN Mensajero/metabolismoRESUMEN
Double-stranded (ds) RNAs and imperfect hairpin RNAs of endogenous genes trigger post-transcriptional gene silencing (PTGS) and are cleaved by a Dicer-like nuclease into small interfering RNAs (siRNAs) and microRNs (miRNAs), respectively. Such small RNAs (siRNAs and miRNAs) then guide an RNA-induced silencing complex (RISC) for sequence-specific RNA degradation. While PTGS serves as an antiviral defense in plants, many plant viruses encode suppressors as a counter defense. Here we demonstrate that the PTGS suppressor (2b) of a severe strain (CM95R) of cucumber mosaic virus (CMV) can bind to in vitro synthesized siRNAs and even to long dsRNAs to a lesser extent. However, the 2b suppressor weakly bound to a miRNA (miR171) duplex in contrast to another small RNA-binding suppressor, p19 of tombusvirus that can effectively bind miRNAs. Because the 2b suppressor of an attenuated strain of CMV (CM95), which differs in a single amino acid from the 2b of CM95R, could barely bind siRNAs, we hypothesized that the weak suppressor activity of the attenuated strain resulted from a loss of the siRNA-binding property of 2b via a single amino acid change. Here we consider that 2b interferes with the PTGS pathway by directly binding siRNAs (or long dsRNA).
Asunto(s)
Cucumovirus/metabolismo , Regulación de la Expresión Génica de las Plantas , Silenciador del Gen , MicroARNs/metabolismo , ARN Interferente Pequeño/metabolismo , Proteínas de Unión al ARN/metabolismo , Proteínas Virales/metabolismo , Secuencia de Aminoácidos , Secuencia de Bases , Línea Celular , Genes de Plantas/genética , MicroARNs/genética , Datos de Secuencia Molecular , Cebollas/citología , Cebollas/genética , Cebollas/virología , Petunia/citología , Petunia/genética , Petunia/virología , Plantas Modificadas Genéticamente , ARN Interferente Pequeño/genética , Proteínas de Unión al ARN/química , Nicotiana/genética , Proteínas Virales/químicaRESUMEN
Programmed cell death (PCD) was studied in the petals of Antirrhinum majus, Argyranthemum frutescens, and Petunia hybrida, using DNA degradation and changes in nuclear morphology as parameters. The petals exhibit loss of turgor (wilting) as a visible symptom of PCD. DNA degradation, as shown on agarose gels, occurred in all species studied, prior to visible wilting. The number of DNA masses in all the petals of a flower, determined by flow cytometry, markedly increased in Argyranthemum and Petunia, but decreased in Antirrhinum. Many small DNA masses were observed in Argyranthemum and Petunia. The surface of each small DNA mass stained with the lipophilic fluorochrome 3,3'-dihexyloxacarbocyanine iodide (DiOC6), indicating that these masses were surrounded by a membrane. In Antirrhinum, in contrast, the chromatin fragmented into several small spherical clumps that remained inside a large membranous structure. Nuclear fragmentation, therefore, did not occur in Antirrhinum, whereas nuclear fragmentation possibly was a cause of the small DNA masses in Argyranthemum and Petunia. It is concluded that at least two contrasting nuclear morphologies exist during PCD. In the first, the chromatin fragments inside the nucleus, not accompanied--or followed--by nuclear fragmentation. In the second, a large number of DNA masses were observed each enveloped by a membrane. The second type was probably due, at least partially, to nuclear fragmentation.
Asunto(s)
Apoptosis , Fragmentación del ADN , Flores/citología , Antirrhinum/citología , Antirrhinum/genética , Antirrhinum/ultraestructura , Asteraceae/citología , Asteraceae/genética , Asteraceae/ultraestructura , Núcleo Celular/ultraestructura , Citometría de Flujo , Flores/genética , Flores/ultraestructura , Lípidos de la Membrana/análisis , Petunia/citología , Petunia/genética , Petunia/ultraestructuraRESUMEN
The Petunia hybrida genes ANTHOCYANIN1 (AN1) and AN2 encode transcription factors with a basic-helix-loop-helix (BHLH) and a MYB domain, respectively, that are required for anthocyanin synthesis and acidification of the vacuole in petal cells. Mutation of PH4 results in a bluer flower color, increased pH of petal extracts, and, in certain genetic backgrounds, the disappearance of anthocyanins and fading of the flower color. PH4 encodes a MYB domain protein that is expressed in the petal epidermis and that can interact, like AN2, with AN1 and the related BHLH protein JAF13 in yeast two-hybrid assays. Mutation of PH4 has little or no effect on the expression of structural anthocyanin genes but strongly downregulates the expression of CAC16.5, encoding a protease-like protein of unknown biological function. Constitutive expression of PH4 and AN1 in transgenic plants is sufficient to activate CAC16.5 ectopically. Together with the previous finding that AN1 domains required for anthocyanin synthesis and vacuolar acidification can be partially separated, this suggests that AN1 activates different pathways through interactions with distinct MYB proteins.
Asunto(s)
Antocianinas/metabolismo , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Petunia/metabolismo , Proteínas de Plantas/fisiología , Proteínas Proto-Oncogénicas c-myb/fisiología , Vacuolas/metabolismo , Secuencia de Aminoácidos , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/química , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Flores/anatomía & histología , Flores/citología , Flores/metabolismo , Regulación de la Expresión Génica de las Plantas , Secuencias Hélice-Asa-Hélice , Homeostasis , Concentración de Iones de Hidrógeno , Datos de Secuencia Molecular , Petunia/citología , Petunia/genética , Fenotipo , Filogenia , Proteínas de Plantas/química , Proteínas de Plantas/metabolismo , Estructura Terciaria de Proteína , Proteínas Proto-Oncogénicas c-myb/química , Proteínas Proto-Oncogénicas c-myb/metabolismo , ARN Mensajero/análisis , Alineación de Secuencia , Transducción de Señal , Técnicas del Sistema de Dos HíbridosAsunto(s)
Hemoglobinas/farmacología , Mitosis/efectos de los fármacos , Passiflora/fisiología , Petunia/fisiología , Protoplastos/citología , Técnicas de Cultivo de Célula/métodos , División Celular , Células Cultivadas , Passiflora/citología , Passiflora/efectos de los fármacos , Petunia/citología , Petunia/efectos de los fármacos , Protoplastos/efectos de los fármacos , Protoplastos/fisiología , RegeneraciónAsunto(s)
Antioxidantes/metabolismo , Fluorocarburos/farmacología , Consumo de Oxígeno/fisiología , Petunia/fisiología , Solanaceae/fisiología , Catalasa/metabolismo , Células Cultivadas , Mitosis/efectos de los fármacos , Petunia/citología , Petunia/efectos de los fármacos , Protoplastos/efectos de los fármacos , Protoplastos/fisiología , Solanaceae/citología , Solanaceae/efectos de los fármacosRESUMEN
TAZ1 (TAPETUM DEVELOPMENT ZINC FINGER PROTEIN1; renamed from PEThy; ZPT3-2) cDNA was first isolated as an anther-specific cDNA from petunia. Here, we report a functional characterization that includes analysis of spatial and temporal expression profiles and examination of anther phenotypes in TAZ1-silenced plants. TAZ1 showed a biphasic expression pattern. In the premeiotic phase, TAZ1 transcripts were found to accumulate in all cell types of the anther except the tapetum and gametophytic tissues, whereas the postmeiotic phase of anther development was characterized by expression exclusively in the tapetum. Silencing of TAZ1 by cosuppression resulted in aberrant development and precocious degeneration of the tapetum, followed by extensive microspore abortion that started soon after their release from pollen tetrads. A few pollen grains that survived showed reduced flavonol accumulation, defects in pollen wall formation, and poor germination rates. This study demonstrates an essential role for TAZ1 in the postmeiotic phase of tapetum development.
Asunto(s)
Flores/genética , Petunia/genética , Polen/genética , Factores de Transcripción/genética , Dedos de Zinc/genética , Supervivencia Celular/genética , Supervivencia Celular/fisiología , Pared Celular/fisiología , Clonación Molecular , Fertilidad , Flavonoides/metabolismo , Flavonoles , Flores/citología , Flores/crecimiento & desarrollo , Regulación del Desarrollo de la Expresión Génica , Regulación de la Expresión Génica de las Plantas , Silenciador del Gen , Glucuronidasa/genética , Glucuronidasa/metabolismo , Proteínas Fluorescentes Verdes , Hibridación in Situ , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Meiosis/genética , Meiosis/fisiología , Datos de Secuencia Molecular , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Petunia/citología , Petunia/crecimiento & desarrollo , Plantas Modificadas Genéticamente , Polen/crecimiento & desarrollo , Polen/metabolismo , Regiones Promotoras Genéticas , Factores de Transcripción/metabolismoRESUMEN
Petunia hybrida cell suspension cultures were exposed to ultrasonic standing wave fields at 2.43 MHz for 40 min with mean sound pressures (within homogenous sound fields) varying from 0 (control) to ca. 1.1 MPa. Mean (+/- s.d.; n =6-9) cell viability was reduced to 87+/-10% at 0.6 MPa and to 59 +/- 23% at 1.1 MPa, compared to an initial control value of 92 +/- 6% (P <0.05). Mean (n = 3) cell alkaline phosphatase concentration increased linearly with sound pressure from a control value of 0.006+/-0.001 to 0.02+/-0.01 Sigma-Units microg(-1) protein at 1.1 MPa (P<0.05). Similarly, mean cell catalase activity increased from a control value of 0.020 +/- 0.003 to 0.026 +/- 0.008 arbitrary units at 1.1 MPa. In contrast, mean cellular lactate dehydrogenase concentration was unchanged. These observations indicate that cellular repair processes associated with increased alkaline phosphatase activity might be triggered by physical cell damage caused by ultrasound. The observed increase in catalase activity suggests increasing production of free radicals and other sonochemicals, which warrants further study. The absence of changes in lactate dehydrogenase indicates that there was no major damage to respiratory pathways or to overall cellular integrity.