RESUMEN
Although Populus has become the model genus for molecular genetics and genomics research on forest trees, genetic and phylogenetic relationships within this genus have not yet been comprehensively studied at the molecular level. By using 151 AFLP (AFLP is a registered trademark of Keygene) markers, 178 accessions belonging to 25 poplar species and three interspecific hybrids were analyzed, using three accessions belonging to two willow species as outgroups. The genetic and phylogenetic relationships were generally consistent with the known taxonomy, although notable exceptions were observed. A dendrogram as well as a single most parsimonious tree, ordered the Populus sections from the oldest Leuce to the latest Aigeiros, a pattern consistent with their known evolutionary relationships. A close relationship between Populus deltoides of the Aigeiros section and species of the Tacamahaca section was observed and, with the exception of Populus wilsonii, between the species of the Leucoides, Tacamahaca, and Aigeiros sections. Populus nigra was clearly separated from its consectional P. deltoides, and should be classified separately from P. deltoides. The AFLP profiles pointed out to the lack of divergence between some species and revealed that some accessions corresponded with interspecific hybrids. This molecular study provides useful information about genetic relationships among several Populus species and, together with morphological descriptions and crossability, it may help review and update systematic classification within the Populus genus.
Asunto(s)
Evolución Molecular , Marcadores Genéticos/genética , Hibridación Genética , Filogenia , Populus/clasificación , Populus/genética , Análisis por Conglomerados , Técnicas de Amplificación de Ácido Nucleico , Polimorfismo de Longitud del Fragmento de Restricción , Especificidad de la EspecieRESUMEN
A reproducible Agrobacterium tumefaciens-mediated genetic transformation method that delivers fertile and morphologically normal transgenic plants was developed for cultivated tepary bean (Phaseolus acutifolius L. Gray). Factors contributing to higher transformation efficiencies include (1) a low initial concentration of bacteria coupled with a longer cocultivation period with callus, (2) an initial selection of callus on a medium containing low levels of the selectable agent, (3) omission of the selectable agent from the medium during callus differentiation to shoots and (4) the efficient conversion of transgenic shoots into fertile plants. All plants regenerated with this procedure (T0) were stably transformed, and the introduced foreign genes were inherited in a Mendelian fashion in most of the 33 independent transformants. Integration, stable transmission and high expression levels of the transgenes were observed in the T1 and/or T3 progenies of the transgenic lines. The binary transformation vectors contained the beta-glucuronidase reporter gene, the neomycin phosphotransferase II selectable marker gene and either an arcelin 1 or an arcelin 5 gene. Arcelins are seed proteins that are very abundant in some wild P. vulgaris L. genotypes showing resistance to the storage insect Zabrotes subfasciatus (Boheman) (Coleoptera, Bruchidae). Transgenic beans from two different cultivated P. acutifolius genotypes with high arcelin levels were infested with Z. subfasciatus, but they were only marginally less susceptible to infestation than the non-transgenic P. acutifolius. Hence, the arcelin genes tested here are not major determinants of resistance against Z. subfasciatus.
Asunto(s)
Técnicas de Transferencia de Gen , Glicoproteínas/metabolismo , Phaseolus/genética , Lectinas de Plantas/metabolismo , Transformación Genética/genética , Agrobacterium tumefaciens , Animales , Southern Blotting , Escarabajos/fisiología , Vectores Genéticos/genética , Glucuronidasa/genética , Glucuronidasa/metabolismo , Glicoproteínas/genética , Inmunidad Innata/genética , Kanamicina Quinasa/genética , Kanamicina Quinasa/metabolismo , Phaseolus/crecimiento & desarrollo , Phaseolus/parasitología , Lectinas de Plantas/genética , Plantas Modificadas Genéticamente , Transgenes/genéticaRESUMEN
Sedentary endoparasitic nematodes of plants are obligate parasites of roots in which they complete their sophisticated life cycle. To study the plant/root-knot nematode interaction, a screening was performed for mutants of Arabidopsis thaliana that were less susceptible to nematode infection. Ethyl methanesulfonate-mutagenized A. thaliana M2 seeds (13,000) were germinated and the seedlings were screened in vitro for nematode susceptibility. Another 5,000 plants were screened in a sand mixture under glasshouse conditions. Ten mutants that were at least a 2-fold less susceptible than the wild-type were retained and analyzed in more detail in vitro as well as in the greenhouse. All mutants with a fairly normal morphology had only a slightly, but reproducibly, decreased susceptibility to nematodes. One mutant (AMi 2) with a 17-fold lower susceptibility to nematode infection and an aberrant phenotype could be rescued by tryptophan.
Asunto(s)
Arabidopsis/genética , Arabidopsis/parasitología , Mutación , Enfermedades de las Plantas/genética , Enfermedades de las Plantas/parasitología , Raíces de Plantas/parasitología , Tylenchoidea/fisiología , Animales , Susceptibilidad a Enfermedades , Femenino , Interacciones Huésped-Parásitos , Fenotipo , Raíces de Plantas/genética , Semillas/genética , Semillas/parasitologíaRESUMEN
Three glycine-rich protein genes of Arabidopsis thaliana (Atgrp-6, Atgrp-7, and Atgrp-8) that correspond to putative genes coding for pollenins (AtolnB;2, AtolnB;3, and AtolnB;4, respectively) are expressed predominantly in the anthers and, more specifically, in the tapetum layer. Tapetal cells are responsible for nutrition of developing pollen grains and show some functional similarities to nematode feeding sites (NFS) induced in plant roots by sedentary parasitic nematodes. The aim of this study was to analyze promoter activity of the Atgrp genes in NFS. Transformed Arabidopsis plants containing a promoter-ss-glucuronidase (gus) fusion of the Atgrp-7 gene were inoculated with the root-knot nematode Meloidogyne incognita and the cyst nematode Heterodera schachtii. GUS assays were performed at different time points after infection. Histochemical analysis revealed an up-regulation of Atgrp-7-gus expression 3 days after inoculation in the feeding sites of both nematodes. Maximal Atgrp-7-gus staining levels in NFS were observed 1 week after nematode infection.
RESUMEN
The cell wall polymer lignin is believed to be condensed by specific cell wall-localized oxidoreductases. In many plants species, including poplar, the peroxidase-directed oxidation of the lignin analogue syringaldazine (SYR) has been localized to cells that undergo secondary wall formation, a process that includes lignification. As a first step to analyse the corresponding peroxidases. we have isolated previously two anionic isoenzymes (PXP 3-4 and PXP 5) from poplar xylem (Populus trichocarpa), which use SYR as a substrate. Here, we demonstrate that these enzymes are responsible for the visualized SYR oxidation in the developing xylem. The cDNA that corresponds to PXP 3-4 was isolated and the deduced protein was found closely related to the other SYR-oxidizing peroxidase PXP 5 (ca. 98% of identity). PXP 3-4 was expressed in a baculovirus expression system yielding high levels of active peroxidase (3 mg/l medium). The heterologously produced protein showed characteristics similar to those of the corresponding protein from poplar xylem (enzymatic properties, isoelectric point, and migration in a native gel). PXP 3-4 was expressed in the stem and in the root xylem. The data demonstrate that PXP 3-4 (and/or PXP 5) are present in differentiating xylem. supporting a function in secondary cell wall formation.
Asunto(s)
Hidrazonas/metabolismo , Peroxidasas/genética , Proteínas de Plantas , Árboles/genética , Secuencia de Aminoácidos , ADN Complementario/química , ADN Complementario/genética , ADN Complementario/aislamiento & purificación , Regulación Enzimológica de la Expresión Génica , Regulación de la Expresión Génica de las Plantas , Isoenzimas/genética , Isoenzimas/metabolismo , Datos de Secuencia Molecular , Oxidación-Reducción , Peroxidasas/metabolismo , Filogenia , ARN Mensajero/genética , ARN Mensajero/metabolismo , Alineación de Secuencia , Análisis de Secuencia de ADN , Homología de Secuencia de Aminoácido , Distribución Tisular , Árboles/enzimologíaRESUMEN
The first step in the biosynthetic pathway of vitamin C in plants is the formation, at the level of sugar nucleotide, of l-galactosyl residues, catalyzed by a largely unknown GDP-d-mannose 3",5"-epimerase. By using combined conventional biochemical and mass spectrometry methods, we obtained a highly purified preparation of GDP-d-mannose 3",5"-epimerase from an Arabidopsis thaliana cell suspension. The native enzyme is an 84-kDa dimer, composed of two apparently identical subunits. In-gel tryptic digestion of the enzyme subunit, followed by peptide sequencing and a blast search, led to the identification of the epimerase gene. The closest homolog of the plant epimerase is the BlmG gene product of Streptomyces sp., a putative NDP-d-mannose 5"-epimerase. The plant GDP-d-mannose 3",5"-epimerase is, to our knowledge, a novel member of the extended short-chain dehydrogenase/reductase family. The enzyme was cloned and expressed in Escherichia coli cells.
Asunto(s)
Arabidopsis/enzimología , Ácido Ascórbico/metabolismo , Carbohidrato Epimerasas/aislamiento & purificación , Secuencia de Aminoácidos , Arabidopsis/metabolismo , Secuencia de Bases , Carbohidrato Epimerasas/química , Carbohidrato Epimerasas/metabolismo , Cromatografía por Intercambio Iónico , Cartilla de ADN , Datos de Secuencia Molecular , Proteínas Recombinantes/química , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Homología de Secuencia de Aminoácido , Espectrometría de Masa por Ionización de Electrospray , Espectrometría de Masa por Láser de Matriz Asistida de Ionización DesorciónRESUMEN
Rhodococcus fascians infects a wide range of plants, initiating the formation of leafy galls that consist of centers of shoot amplification and shoot growth inhibition. R. fascians is an epiphyte but it also can establish endophytic populations. Bacterial signals involved in symptom development initiate de novo cell division and shoot meristem formation in differentiated tissues. The R. fascians signals exert activities that are distinct from mere cytokinin effects, and the evidence points to a process that adopted cytokinin biosynthetic enzymes to form derivatives with unique activity. Genes implicated in leafy gall formation are located on a linear plasmid and are subject to a highly controlling, complex regulatory network, integrating autoregulatory compounds and environmental signals. Leafy galls are considered as centers with specific metabolic features, a niche where populations of R. fascians experience a selective advantage. Such "metabolic habitat modification" might be universal for gall-inducing bacteria.
Asunto(s)
Magnoliopsida/microbiología , Tumores de Planta/microbiología , Rhodococcus/genética , Magnoliopsida/metabolismo , Enfermedades de las Plantas/genética , Enfermedades de las Plantas/microbiología , Reguladores del Crecimiento de las Plantas/metabolismo , Hojas de la Planta/metabolismo , Hojas de la Planta/microbiología , Tumores de Planta/genética , Rhodococcus/metabolismo , Rhodococcus/patogenicidad , Virulencia/genéticaRESUMEN
Poly(ADP-ribos)ylation is a reversible protein modification that in higher plants is catalyzed by two structurally different poly(ADP-ribose) polymerases, App and Zap. In vivo imaging of green-fluorescent protein (GPF) fusions showed that both Zap and App were associated with chromatin through the cell cycle progression. The in vivo behaviour of the App-GFP protein fusions can be attributed to the activity of two NASA motifs that mediate protein-protein interactions and nucleic acid binding. Expression of Zap deletion variants revealed that both Zn fingers and helix-turn-helix domains contributed to the association with chromosomes, whereas the localization in the nucleoplasm was mostly determined by the Zn fingers. The results highlight novel properties of protein sequences found in plant poly(ADP-ribose) polymerases and suggest important functions for this class of nuclear enzymes in chromosome dynamics.
Asunto(s)
Arabidopsis/enzimología , Proteínas de Plantas/genética , Poli(ADP-Ribosa) Polimerasas/metabolismo , Secuencia de Aminoácidos , Arabidopsis/genética , Técnicas de Cultivo de Célula , Cromatina , Clonación Molecular , Vectores Genéticos , Proteínas Fluorescentes Verdes , Proteínas Luminiscentes , Microscopía Confocal , Datos de Secuencia Molecular , Proteínas de Plantas/química , Poli Adenosina Difosfato Ribosa/química , Poli(ADP-Ribosa) Polimerasas/química , Estructura Terciaria de Proteína , Proteínas Recombinantes de Fusión , Análisis de Secuencia de ADN , Análisis de Secuencia de ProteínaRESUMEN
The ability of Rhodococcus fascians strain D188 to provoke leafy gall formation on a variety of plant species is correlated with the linear plasmid pFiD188, on which different pathogenicity loci were identified. The att locus affects the severity of symptom development on tobacco, whereas the fas locus is essential for virulence. To gain insight into the function of the att locus, sequence and expression analyses were performed. The att locus contains nine open reading frames homologous to arginine and beta-lactam biosynthetic genes. att gene expression is transcriptionally induced by leafy gall extracts, but not by extracts of uninfected plants, and depends on the attR gene that encodes a LysR-type transcriptional regulator. The att locus proves to be essential for the formation of inducing factors (IFs) that are present in gall extracts. Because the induction of the fas locus also requires the presence of IFs in gall extracts, the att locus is proposed to play an important role in regulating the expression of the virulence loci of R. fascians.
Asunto(s)
Genes Bacterianos , Nicotiana/microbiología , Tumores de Planta/microbiología , Rhodococcus/genética , Rhodococcus/patogenicidad , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Regulación Bacteriana de la Expresión Génica , Histidina/metabolismo , Mutación , Plantas/metabolismo , Rhodococcus/metabolismo , Nicotiana/fisiologíaRESUMEN
Owing to their sessile lifestyle, it is crucial for plants to acquire stress tolerance. The function of heat-shock proteins, including small heat-shock proteins (smHSPs), in stress tolerance is not fully explored. To gain further knowledge about the smHSPs, the gene that encoded the cytosolic class II smHSP in Arabidopsis thaliana (At-HSP17.6A) was characterized. The At-HSP17.6A expression was induced by heat and osmotic stress, as well as during seed development. Accumulation of At-HSP17.6A proteins could be detected with heat and at a late stage of seed development, but not with osmotic stress, suggesting stress-induced post-transcriptional regulation of At-HSP17.6A expression. Overproduction of At-HSP17.6A could increase salt and drought tolerance in Arabidopsis. The chaperone activity of At-HSP17.6A was demonstrated in vitro.
Asunto(s)
Proteínas de Arabidopsis/fisiología , Arabidopsis/fisiología , Proteínas de Choque Térmico/fisiología , Chaperonas Moleculares/fisiología , Proteínas de Plantas/fisiología , Adaptación Biológica , Citosol , Desecación , Regulación de la Expresión Génica , Genes de Plantas , Calor , Presión Osmótica , Proteínas Recombinantes , Semillas/fisiologíaRESUMEN
To analyse experimentally the correlation between transgene silencing and the presence of an inverted repeat in transgenic Arabidopsis thaliana plants, expression of the beta-glucuronidase (gus) gene was studied when present as a convergently transcribed inverted repeat or as a single copy in otherwise isogenic lines. In transformants containing two invertedly repeated gus genes separated by a 732 bp palindromic sequence, gus expression was low, as exemplified by the expression levels in the parental line KH15. The parental KH15 locus could induce efficiently in trans silencing of gus copies at allelic and non-allelic positions. In transformants containing two invertedly repeated gus genes separated by a 826 bp non-repetitive spacer region, gus expression was high or intermediate, especially in hemizygous state and at late developmental stages, as demonstrated in detail for line KHsb67. Removal of one of the gus copies by Cre recombinase resulted in all cases in constitutively high gus expression in hemizygous as well as in homozygous state. The derived deletion lines could no longer induce in trans silencing of homologous gus copies. The results show that convergent transcription of transgenes in an inverted repeat is an important parameter to trigger their silencing and that co-transformation of two T-DNAs with identical transgenes can be used to obtain inverted repeats and targeted co-suppression of the homologous endogenes. Moreover, the data suggest that the spacer region in between the inverted genes plays a role in the efficiency of initiating and maintaining silencing.
Asunto(s)
Eliminación de Gen , Silenciador del Gen , Secuencias Repetitivas de Ácidos Nucleicos/genética , Transgenes/genética , Arabidopsis/genética , Arabidopsis/crecimiento & desarrollo , Regulación del Desarrollo de la Expresión Génica , Regulación Enzimológica de la Expresión Génica , Regulación de la Expresión Génica de las Plantas , Glucuronidasa/genética , Plantas Modificadas Genéticamente/genética , Plantas Modificadas Genéticamente/crecimiento & desarrolloRESUMEN
Two novel homologous Arabidopsis proteins, DIP1 and DIP2, interact with the DNA-binding domain of plant poly(ADP-ribose) polymerase (PARP) in the yeast two-hybrid system and in vitro. Their homology to the transcriptional coactivator ALY suggests that plant PARP may play a role in the regulation of transcription.
Asunto(s)
Proteínas de Arabidopsis , Arabidopsis/metabolismo , Proteínas de Drosophila , Proteínas Nucleares/metabolismo , Proteínas de Plantas/metabolismo , Poli(ADP-Ribosa) Polimerasas/metabolismo , Factores de Transcripción/metabolismo , Agrobacterium tumefaciens/genética , Secuencia de Aminoácidos , Animales , Arabidopsis/genética , Sitios de Unión , Núcleo Celular/metabolismo , Clonación Molecular , Escherichia coli/genética , Regulación de la Expresión Génica de las Plantas , Proteínas Fluorescentes Verdes , Proteínas Luminiscentes , Datos de Secuencia Molecular , Proteínas Nucleares/genética , Proteínas de Plantas/genética , Plantas Modificadas Genéticamente , Reacción en Cadena de la Polimerasa , Saccharomyces cerevisiae/genética , Homología de Secuencia de Aminoácido , Factores de Transcripción/química , Factores de Transcripción/genética , Transcripción Genética , Dedos de Zinc/genéticaRESUMEN
Cyclin-dependent kinases (CDKs) control the key transitions in the eukaryotic cell cycle. All the CDKs known to control G(2)/M progression in yeast and animals are distinguished by the characteristic PSTAIRE motif in their cyclin-binding domain and are closely related. Higher plants contain in addition a number of more divergent non-PSTAIRE CDKs with still obscure functions. We show that a plant-specific type of non-PSTAIRE CDKs is involved in the control of the G(2)/M progression. In synchronized tobacco BY-2 cells, the corresponding protein, accumulated in a cell cycle-regulated fashion, peaking at the G(2)/M transition. The associated histone H1 kinase activity reached a maximum in mitosis and required a yet unidentified subunit to be fully active. Down-regulation of the associated kinase activity in transgenic tobacco plants using a dominant-negative mutation delayed G(2)/M transition. These results provide the first evidence that non-PSTAIRE CDKs are involved in the control of the G(2)/M progression in plants.
Asunto(s)
Proteínas de Arabidopsis , Quinasas Ciclina-Dependientes/genética , Quinasas Ciclina-Dependientes/fisiología , Fase G2 , Mitosis , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Unión Competitiva , Ciclo Celular , Núcleo Celular , Células Cultivadas , Cromatografía en Gel , Regulación hacia Abajo , Citometría de Flujo , Genes Dominantes , Histonas/metabolismo , Immunoblotting , Datos de Secuencia Molecular , Mutación , Plantas Modificadas Genéticamente/metabolismo , Plantas Tóxicas , Plásmidos/metabolismo , Pruebas de Precipitina , Unión Proteica , Proteínas Quinasas/metabolismo , Estructura Terciaria de Proteína , Homología de Secuencia de Aminoácido , Factores de Tiempo , Nicotiana/citología , TransgenesRESUMEN
The steady-state level of transcripts coding for the pyrroline-5-carboxylate reductase of Arabidopsis (At-P5R) increased under salt and heat stress, mainly because of an enhanced mRNA stability. However, the At-P5R protein level was not induced, and its translation was inhibited at initiation stage and probably also at later stages. Replacement of the 5' untranslated region (5'UTR) and beta-glucuronidase (gus) fusion analysis revealed that the first 92 bp region of the At-P5R 5'UTR was sufficient to mediate transcript stabilization and translation inhibition during salt and heat stresses. Furthermore, the first 92 bp region of the At-P5R 5'UTR was also involved in transcription efficiency in a promoter-dependent manner. The results demonstrated that the stress regulation of At-P5R is complex and involves the 5'UTR which acts at three levels, partly in opposing directions.
Asunto(s)
Regiones no Traducidas 5' , Arabidopsis/genética , Regulación Enzimológica de la Expresión Génica , Regulación de la Expresión Génica de las Plantas , Proteínas de Plantas/genética , Pirrolina Carboxilato Reductasas/genética , Secuencia de Bases , Northern Blotting , Glucuronidasa/genética , Calor , Datos de Secuencia Molecular , Polirribosomas/genética , Regiones Promotoras Genéticas , Biosíntesis de Proteínas , ARN , Procesamiento Postranscripcional del ARN , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN de Planta , Proteínas Recombinantes de Fusión , Cloruro de Sodio , Factores de Transcripción/metabolismo , Transcripción Genética , delta-1-Pirrolina-5-Carboxilato ReductasaRESUMEN
A member of the cellulose synthase-like (subfamily D) gene family of Arabidopsis, AtCSLD3, has been identified by T-DNA tagging. The analysis of the corresponding mutant, csld3-1, showed that the AtCSLD3 gene plays a role in root hair growth in plants. Root hairs grow in phases: First a bulge is formed and then the root hair elongates by polarized growth, the so-called "tip growth." In the mutant, root hairs were initiated at the correct position and grew into a bulge, but their elongation was severely reduced. The tips of the csld3-1 root hairs easily leaked cytoplasm, indicating that the tensile strength of the cell wall had changed at the site of the tip. Based on the mutant phenotype and the functional conservation between CSLD3 and the genuine cellulose synthase proteins, we hypothesized that the CSLD3 protein is essential for the synthesis of polymers for the fast-growing primary cell wall at the root hair tip. The distinct mutant phenotype and the ubiquitous expression pattern indicate that the CSLD3 gene product is only limiting at the zone of the root hair tip, suggesting particular physical properties of the cell wall at this specific site of the root hair cell.
Asunto(s)
Proteínas de Arabidopsis , Arabidopsis/genética , Proteínas de Plantas/genética , Raíces de Plantas/crecimiento & desarrollo , Secuencia de Aminoácidos , Secuencia de Bases , ADN Bacteriano , Regulación de la Expresión Génica de las Plantas , Datos de Secuencia Molecular , Mutación , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Homología de Secuencia de AminoácidoRESUMEN
Populus deltoides, P. nigra, and P. trichocarpa are the most important species for poplar breeding programs worldwide. In addition, Populus has become a model for fundamental research on trees. Linkage maps were constructed for these three species by analyzing progeny of two controlled crosses sharing the same female parent, Populus deltoides cv. S9-2 x P. nigra cv. Ghoy and P. deltoides cv. S9-2 x P. trichocarpa cv. V24. The two-way pseudotestcross mapping strategy was used to construct the maps. Amplified fragment length polymorphism (AFLP) markers that segregated 1:1 were used to form the four parental maps. Microsatellites and sequence-tagged sites were used to align homoeologous groups between the maps and to merge linkage groups within the individual maps. Linkage analysis and alignment of the homoeologous groups resulted in 566 markers distributed over 19 groups for P. deltoides covering 86% of the genome, 339 markers distributed over 19 groups for P. trichocarpa covering 73%, and 369 markers distributed over 28 groups for P. nigra covering 61%. Several tests for randomness showed that the AFLP markers were randomly distributed over the genome.
Asunto(s)
Genes de Plantas , Ligamiento Genético , Repeticiones de Microsatélite , Polimorfismo Genético , Mapeo Cromosómico , Cruzamientos Genéticos , ADN/metabolismo , Marcadores Genéticos/genética , Heterocigoto , Modelos Genéticos , Modelos Estadísticos , Lugares Marcados de Secuencia , Árboles/genéticaRESUMEN
Transposon Display is a high-resolution method that was used here to visualize simultaneously individual members of the dTph1 transposable element family of Petunia hybrida. The method provides a tool for accurate analyses of copy numbers and insertion frequencies, and a means to study the behavior of a family of elements as a whole. Somatic insertion events can be identified and insertion events in a cell of the L2 apical lineage can be distinguished unequivocally from those in a cell outside this lineage. In sublines of the high-copy-number line W137, an average insertion rate equivalent to transposition of 10% of the total number of element copies in each generation was measured, copy number increases of over 20% in four generations were recorded, and element position turnover was analyzed. Insertion events are detected essentially randomly both in time and space. The general applicability of the technique for the analysis of the transpositional behavior of element systems is discussed.
Asunto(s)
Elementos Transponibles de ADN/genética , Genes de Plantas/genética , Plantas/genética , Diferenciación Celular , ADN de Plantas/análisis , Endogamia , Mutagénesis Insercional , Desarrollo de la Planta , Plantas/metabolismoRESUMEN
The orchid Gastrodia elata depends on the fungus Armillaria mellea to complete its life cycle. In the interaction, fungal hyphae penetrate older, nutritive corms but not newly formed corms. From these corms, a protein fraction with in vitro activity against plant-pathogenic fungi has previously been purified. Here, the sequence of gastrodianin, the main constituent of the antifungal fraction, is reported. Four isoforms that encoded two different mature proteins were identified at the cDNA level. Another isoform was detected in sequenced peptides. Because the antifungal activity of gastrodianins produced in and purified from Escherichia coli and Nicotiana tabacum was comparable to that of gastrodianin purified from the orchid, gastrodianins are the active component of the antifungal fractions. Gastrodianin accumulation is probably an important part of the mechanism by which the orchid controls Armillaria penetration. Gastrodianin was found to be homologous to monomeric mannose-binding proteins of other orchids, of which at least one (Epipactis helleborine mannose-binding protein) also displayed in vitro antifungal activity. This establishes the gastrodianin-like proteins (GLIPs) as a novel class of antifungal proteins.
Asunto(s)
Antifúngicos/farmacología , Proteínas Portadoras/genética , Proteínas Portadoras/farmacología , Hongos/efectos de los fármacos , Magnoliopsida/genética , Lectinas de Unión a Manosa , Proteínas de Plantas/farmacología , Secuencia de Aminoácidos , Antifúngicos/química , Antifúngicos/aislamiento & purificación , Antifúngicos/metabolismo , Secuencia de Bases , Proteínas Portadoras/química , Proteínas Portadoras/aislamiento & purificación , Proteínas Portadoras/metabolismo , Clonación Molecular , Colectinas , ADN Complementario/aislamiento & purificación , ADN de Plantas , Escherichia coli/genética , Escherichia coli/metabolismo , Hongos/crecimiento & desarrollo , Lectinas/genética , Lectinas/metabolismo , Magnoliopsida/metabolismo , Mananos/genética , Mananos/metabolismo , Manosa/metabolismo , Espectrometría de Masas , Pruebas de Sensibilidad Microbiana , Datos de Secuencia Molecular , Lectinas de Plantas , Proteínas de Plantas/química , Proteínas de Plantas/genética , Proteínas de Plantas/aislamiento & purificación , Plantas Modificadas Genéticamente/genética , Plantas Modificadas Genéticamente/metabolismo , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/aislamiento & purificación , Isoformas de Proteínas/metabolismo , SimbiosisRESUMEN
In situ hybridization detection of mRNA is an essential tool for understanding regulation of gene expression in cells and tissues of different organisms. Over the years, numerous in situ protocols have been developed ranging from whole-mount techniques that allow fast transcript localization in intact organs to high-resolution methods based on the electron microscopic detection of mRNAs at the subcellular level. Here, we present a detailed protocol for the detection of mRNAs in plant tissues using radiolabeled single-stranded RNA probes. Hybridizations are carried out on tissue sections of paraffin- and plastic-embedded plant tissues. Although this in situ protocol is appropriate for plant tissues in general, it has been optimized for Arabidopsis thaliana. Variations on the procedure, required to obtain optimal results with different Arabidopsis tissues, are described.
Asunto(s)
Arabidopsis/metabolismo , Técnicas Genéticas , Hibridación in Situ/métodos , Metacrilatos/farmacología , Hibridación de Ácido Nucleico , Parafina/farmacología , ARN Mensajero/metabolismo , Radioisótopos de Azufre/metabolismoRESUMEN
In the compatible interaction between Arabidopsis thaliana and the endoparasitic nematode Meloidogyne incognita, galls are formed on the roots of the host plant. Differential display was used to identify alterations of gene expression in young A. thaliana root galls caused by M. incognita. Six genes were confirmed as plant genes by DNA gel blot hybridizations. Significant homology was found with a trypsin inhibitor, peroxidase, mitochondrial uncoupling protein, endomembrane protein, 20S proteasome alpha-subunit, and diaminopimelate decarboxylase. The cellular and temporal expression of each of the six genes was analyzed by mRNA in situ hybridizations.