RESUMEN
Molecular characterization has been accomplished for five members of the peroxidase gene family in French bean. The most important of these, designated FBPI, corresponds to the isoform believed to be responsible for the apoplastic oxidative burst demonstrated by suspension-cultured cells in response to fungal elicitor. Identification was made by a complete match of six peptide sequences derived from the native protein to the translated sequence of the cDNA. Modelling of the surface structure in comparison with two other members of the peroxidase family did not reveal any unusual features which might account for its role in the oxidative burst. However, FBP1 when expressed in Pichia pastoris generated H2O2 using cysteine at pH 7.2, a specific property of the native protein when isolated from suspension-cultured cells. FBP1, together with other members of the family, were all induced in cell cultures by elicitor action although they all showed some expression in non-induced cultured cells. They were also expressed in all tissues examined with varying levels of intensity of detection in northern blots. This was confirmed by in situ hybridization and FBP1 expression was confirmed in tissues where it has been previously detected by immunolocalization methods. Assigning roles to individual peroxidases is an important goal and molecular identification of the oxidative burst peroxidase allows further exploration of the relative roles of the different systems involved in generating reactive oxygen species.
Asunto(s)
Oxígeno/metabolismo , Peroxidasa/genética , Phaseolus/genética , Secuencia de Aminoácidos , Clonación Molecular , ADN Complementario/química , ADN Complementario/genética , ADN de Plantas/química , ADN de Plantas/genética , Regulación Enzimológica de la Expresión Génica , Regulación de la Expresión Génica de las Plantas , Peróxido de Hidrógeno/metabolismo , Concentración de Iones de Hidrógeno , Hibridación in Situ , Datos de Secuencia Molecular , Peroxidasa/metabolismo , Phaseolus/citología , Phaseolus/enzimología , ARN Mensajero/genética , ARN Mensajero/metabolismo , Alineación de Secuencia , Análisis de Secuencia de ADN , Homología de Secuencia de Aminoácido , Distribución TisularRESUMEN
A cell suspension culture of a tobacco (Nicotiana tabacum L. cv. Petit Havana) cell line derived from a cultivar transformed with the Tcyt gene from Agrobacterium, which leads to high endogenous levels of cytokinin, has been established. This cell line shows increased cell aggregation, elongated cells and a 5-fold increase in wall thickness. If allowed to carry on growing it can form a single mass without shedding cells into the medium. When analysed at an earlier growth stage, these cultures were found to produce improved levels of vascular nodule formation than in other systems that employ exogenous cytokinin. This differentiation was optimised with respect to sucrose and auxin signals in order to induce maximum production of cells with thickened walls and a morphology characteristic of fibre cells and tracheids, in addition to cells that remain meristematic. In order to establish the validity of this system for studying secondary wall formation, the walls and associated biosynthetic changes were analysed in these cells by chemical analysis of the walls, changes in activities of enzymes of xylan and monolignol synthesis, and expression of mRNAs coding for enzymes of lignin biosynthesis. The wall composition of the transformed cells was compared with that determined for primary walls from a typical untransformed tobacco cell line. Recovery of wall material was 50% greater in the transformed culture. In this material a major difference was found in the pectin fraction where there was a distinct difference in size distribution together with a lower level of methylation for the transformed line, which may be related to increased adhesiveness. There were increased amounts of xylan, although the ratio of xyloglucan to xylan content was not substantially different due to the mixture of cell types. There was also an increase in cellulose and phenolic components. Increased activity of enzymes involved in the synthesis of xylan as a marker for the secondary wall occurred around the time of tracheid differentiation and coincided with a broad peak of cinnamyl alcohol dehydrogenase activity. The expression of mRNAs coding for enzymes of the general phenylpropanoid pathway, phenylalanine ammonia-lyase, cinnamate 4-hydroxylase, catechol O-methyl transferase was relatively constitutive in the cultures while transcripts of ferulate 5-hydroxylase, cinnamoyl CoA-reductase, cinnamyl alcohol dehydrogenase and lignin peroxidase were induced. The walls of the transformed cells also showed considerable differences in the subset of extractable proteins from that found in primary walls of tobacco when these were subjected to proteomic analysis. Many of these proteins appear to be novel and not present in primary walls. However an Mr-32,000 chitinase, an Mr-34,000 peroxidase, an Mr-65,000 polyphenoloxidase/laccase and possibly an Mr-68,000 xylanase could be identified as well as structural proteins.
Asunto(s)
Pared Celular/química , Nicotiana/metabolismo , Proteínas de Plantas/análisis , Plantas Tóxicas , Proteoma , Oxidorreductasas de Alcohol/metabolismo , Secuencia de Aminoácidos , Pared Celular/metabolismo , Pared Celular/ultraestructura , Células Cultivadas , Celulosa/biosíntesis , Sistema Enzimático del Citocromo P-450/metabolismo , Citocininas , Regulación de la Expresión Génica de las Plantas , Genoma de Planta , Lignina/biosíntesis , Oxigenasas de Función Mixta/metabolismo , Datos de Secuencia Molecular , Pentosiltransferasa/metabolismo , Fenoles/análisis , Fenotipo , Fenilanina Amoníaco-Liasa/metabolismo , Proteínas de Plantas/metabolismo , Polisacáridos/análisis , ARN Mensajero/metabolismo , Homología de Secuencia de Aminoácido , Nicotiana/genética , Nicotiana/ultraestructura , Transcinamato 4-Monooxigenasa , Transferasas/metabolismoRESUMEN
cDNAs showing high sequence similarity (>70%) over large stretches to plant CYP73A orthologues from other species were isolated from a cDNA library derived from mRNAs expressed in elicitor-treated suspension-cultured cells. These clones appear to code for a full-length 1554 bp open reading frame with a 78 bp 5'-untranslated region and a 140 bp 3'-untranslated region. The open reading frame, determined by sequence similarity, codes for a protein with a predicted Mr of 59229 and a pI of 8.8. It contains the conserved cysteine haem-binding site found in all cytochrome P450s. The protein encoded by this cDNA diverges however from other CYP73As in its N- and C-terminus and in four domains internally, so that overall sequence similarity is in the range 58-66%. Many clones contained an identical intron, which may be associated with a novel regulatory mechanism. Sequence similarity is sufficient for it to be classified as CYP73A15, although it is the least similar member of this family classified so far. The cDNA was expressed in yeast. Successful expression of cinnamate 4-hydroxylase activity required removal of the intron. High-level expression also required modification of the N-terminus to that of CYP73A1. Yeast did not process the intron at all and the leader sequence for A15 was not as compatible as that of A1. The mRNA for CYP73A15 was shown to be rapidly induced by elicitor treatment of suspension-cultured cells of French bean but induction was more transient than that of phenylalanine ammonia-lyase (PAL). In contrast, induction in cells undergoing xylogenesis was much more coordinate with PAL. The cloned cDNA may represent a cinnamate 4-hydroxylase isoform, whose expression is more related to differentiation than the responses to stress in which the majority of CYP73As cloned so far are involved.
Asunto(s)
Sistema Enzimático del Citocromo P-450/metabolismo , Fabaceae/enzimología , Expresión Génica/genética , Oxigenasas de Función Mixta/metabolismo , Plantas Medicinales , Levaduras/genética , Secuencia de Aminoácidos , Secuencia de Bases , Células Cultivadas , Cinamatos/metabolismo , Clonación Molecular , Sistema Enzimático del Citocromo P-450/genética , Fabaceae/citología , Fabaceae/genética , Fabaceae/crecimiento & desarrollo , Regulación de la Expresión Génica de las Plantas , Intrones/genética , Lignina/biosíntesis , Microsomas/metabolismo , Oxigenasas de Función Mixta/genética , Datos de Secuencia Molecular , Sistemas de Lectura Abierta/genética , Filogenia , Señales de Clasificación de Proteína/genética , Empalme del ARN , ARN Mensajero/análisis , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/metabolismo , Homología de Secuencia de Aminoácido , Transcinamato 4-Monooxigenasa , Xilosa/biosíntesis , Levaduras/metabolismoRESUMEN
The origin of the oxidative burst during plant-pathogen interactions remains controversial. A number of possibilities have been identified, which involve the protoplast, plasmalemma or apoplast. The apoplastic production of H2O2 requires three components, an extracellular peroxidase, ion fluxes leading to extracellular alkalinisation and release of a substrate. Fatty acids are the major compounds that appear in the apoplast following elicitation, which can activate H2O2 production by peroxidases in vitro. However, the reaction with peroxidases appears to be novel and is uncharacterised at present. The apoplastic mechanism also cannot be readily distinguished from the operation of a plasma membrane NADPH oxidase system by the use of the inhibitors diphenylene iodonium and N,N diethyl-dithiocarbamate since it is also inhibited by these. These inhibitors have often in the past been used to define the involvement of the latter in the oxidative burst. In common with the NADPH oxidase system, the peroxidase responsible has been cloned but unlike the NADPH oxidase it has been shown to function in vitro to generate H2O2. In vivo studies of the oxidative burst have shown that the alkalinisation is essential and the underlying ion fluxes may be regulated by cAMP. Calcium fluxes are also essential. Although the oxidative activity of peroxidase requires calcium the fluxes have obvious other function. These may include activation of release of substrate and through the activation of a CDPK, regulation of enzymes involved in phytoalexin and cell wall phenolic production such as PAL.
Asunto(s)
Plantas/metabolismo , Estallido Respiratorio , Calcio/metabolismo , Pared Celular/metabolismo , Concentración de Iones de Hidrógeno , Modelos Biológicos , Complejos Multienzimáticos/metabolismo , NADH NADPH Oxidorreductasas/metabolismo , Peroxidasas/metabolismo , Transducción de Señal , Especificidad por SustratoRESUMEN
Accumulation of mRNAs for the defense-related genes phenylalanine ammonia-lyase (PAL), chalcone synthase, chitinase (CHT), glucanase, and hydroxyproline-rich glycoprotein were examined in roots of dark red kidney bean (Phaseolus vulgaris L. cv Moncalm) colonized by the arbuscular mycorrhizal fungus Glomus intraradices Schenck & Smith. In three separate experiments the root length colonized ranged from 28 to 55% by 28 d after planting and inoculation. RNA blot analysis revealed little change in the accumulations of PAL, chalcone synthase, CHT, glucanase, and hydroxyproline-rich glycoprotein transcripts from the 28-d mycorrhizal roots compared to the uninoculated controls. Normalizing the ratios of defense-related transcript accumulation against RNA pools regarded as being constitutively expressed, actin mRNA, 25S rRNA, and 18S rRNA, indicated that changes in the ratios of up to 20% occur according to the RNA pool used for normalization. In situ hybridizations of colonized roots using probes for PAL and CHT showed that accumulations of both transcripts occurred only in arbusculated cells. Both young, finely branched arbuscules and older, clumped arbuscules displayed PAL and CHT message accumulations. The PAL and CHT mRNA accumulations were greater in cortical cells containing young arbuscules than in cells containing clumped arbuscules. Intercellular hyphae and vesicles elicited no response.