RESUMEN
The plant cell wall is an important factor for determining cell shape, function and response to the environment. Secondary cell walls, such as those found in xylem, are composed of cellulose, hemicelluloses and lignin and account for the bulk of plant biomass. The coordination between transcriptional regulation of synthesis for each polymer is complex and vital to cell function. A regulatory hierarchy of developmental switches has been proposed, although the full complement of regulators remains unknown. Here we present a protein-DNA network between Arabidopsis thaliana transcription factors and secondary cell wall metabolic genes with gene expression regulated by a series of feed-forward loops. This model allowed us to develop and validate new hypotheses about secondary wall gene regulation under abiotic stress. Distinct stresses are able to perturb targeted genes to potentially promote functional adaptation. These interactions will serve as a foundation for understanding the regulation of a complex, integral plant component.
Asunto(s)
Arabidopsis/genética , Arabidopsis/metabolismo , Pared Celular/metabolismo , Regulación de la Expresión Génica de las Plantas/genética , Redes Reguladoras de Genes/genética , Factores de Transcripción/metabolismo , Arabidopsis/crecimiento & desarrollo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , ADN de Plantas/genética , ADN de Plantas/metabolismo , Factores de Transcripción E2F/metabolismo , Retroalimentación , Regulación del Desarrollo de la Expresión Génica/genética , Deficiencias de Hierro , Especificidad de Órganos , Regiones Promotoras Genéticas/genética , Reproducibilidad de los Resultados , Salinidad , Factores de Tiempo , Xilema/genética , Xilema/crecimiento & desarrollo , Xilema/metabolismoRESUMEN
The Arabidopsis Ca(2+)/calmodulin (CaM)-binding transcription factor SIGNAL RESPONSIVE1 (AtSR1/CAMTA3) was previously identified as a key negative regulator of plant immune responses. Here, we report a new role for AtSR1 as a critical component of plant defense against insect herbivory. Loss of AtSR1 function impairs tolerance to feeding by the generalist herbivore Trichoplusia ni as well as wound-induced jasmonate accumulation. The susceptibility of the atsr1 mutant is associated with decreased total glucosinolate (GS) levels. The two key herbivory deterrents, indol-3-ylmethyl (I3M) and 4-methylsulfinylbutyl (4MSOB), showed the most significant reductions in atsr1 plants. Further, changes in AtSR1 transcript levels led to altered expression of several genes involved in GS metabolism including IQD1, MYB51 and AtST5a. Overall, our results establish AtSR1 as an important component of plant resistance to insect herbivory as well as one of only three described proteins involved in Ca(2+)/CaM-dependent signaling to function in the regulation of GS metabolism, providing a novel avenue for future investigations of plant-insect interactions.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/fisiología , Glucosinolatos/metabolismo , Mariposas Nocturnas/fisiología , Enfermedades de las Plantas/inmunología , Proteínas Serina-Treonina Quinasas/metabolismo , Animales , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Señalización del Calcio , Calmodulina/metabolismo , Ciclopentanos/metabolismo , Resistencia a la Enfermedad , Regulación de la Expresión Génica de las Plantas , Herbivoria , Mutación , Oxilipinas/metabolismo , Enfermedades de las Plantas/parasitología , Reguladores del Crecimiento de las Plantas/metabolismo , Plantas Modificadas Genéticamente , Proteínas Serina-Treonina Quinasas/genética , ARN de Planta/genética , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Heridas y LesionesRESUMEN
HYDROPEROXIDE LYASE (HPL) genes encode enzymes that catalyze the cleavage of fatty acid hydroperoxides into aldehydes and oxoacids. There are three HPLs in rice (Oryza sativa), designated OsHPL1 through OsHPL3. To explore the possibility of differential functional activities among these genes, we have examined their expression patterns and biochemical properties of their encoded products. Transcript analysis indicates that these genes have distinct patterns and levels of expression. OsHPL1 is ubiquitously expressed, OsHPL2 is expressed in the leaves and leaf sheaths, whereas OsHPL3 is wound inducible and expressed exclusively in leaves. OsHPLs also differ in their substrate preference as determined by in vitro enzyme assays using 9-/13-hydroperoxy linolenic and 9-/13-hydroperoxy linoleic acids as substrates. OsHPL1 and OsHPL2 metabolize 9-/13-hydroperoxides, whereas OsHPL3 metabolizes 13-hydroperoxy linolenic acid exclusively. Sequence alignments of the HPL enzymes have identified signature residues potentially responsible for the substrate specificity/preference of these enzymes. All three OsHPLs are chloroplast localized as determined by chloroplast import assays and green fluorescent protein (GFP) fusion studies. Aldehyde measurements in transgenic Arabidopsis (Arabidopsis thaliana) plants overexpressing individual OsHPL-GFP fusions indicate that all rice HPLs are functional in a heterologous system, and each of them generates a distinct signature of the metabolites. Interestingly, these aldehydes were only detectable in leaves, but not in roots, despite similar levels of OsHPL-GFP proteins in both tissues. Similarly, there were undetectable levels of aldehydes in rice roots, in spite of the presence of OsHPL1 transcripts. Together, these data suggest that additional tissue-specific mechanism(s) beyond transcript and HPL enzyme abundance, regulate the levels of HPL-derived metabolites.
Asunto(s)
Aldehído-Liasas/metabolismo , Aldehídos/metabolismo , Arabidopsis/genética , Sistema Enzimático del Citocromo P-450/metabolismo , Oryza/enzimología , Proteínas de Plantas/metabolismo , Aldehído-Liasas/química , Aldehído-Liasas/genética , Secuencia de Aminoácidos , Arabidopsis/anatomía & histología , Arabidopsis/ultraestructura , Cloroplastos/enzimología , Sistema Enzimático del Citocromo P-450/química , Sistema Enzimático del Citocromo P-450/genética , Regulación de la Expresión Génica de las Plantas , Proteínas Fluorescentes Verdes/análisis , Proteínas Fluorescentes Verdes/metabolismo , Datos de Secuencia Molecular , Familia de Multigenes/fisiología , Oryza/anatomía & histología , Pisum sativum/genética , Pisum sativum/ultraestructura , Filogenia , Hojas de la Planta/anatomía & histología , Hojas de la Planta/metabolismo , Proteínas de Plantas/química , Proteínas de Plantas/genética , Raíces de Plantas/anatomía & histología , Raíces de Plantas/metabolismo , Plantas Modificadas Genéticamente/anatomía & histología , Plantas Modificadas Genéticamente/metabolismo , Plantas Modificadas Genéticamente/ultraestructura , ARN Mensajero/metabolismo , Proteínas Recombinantes de Fusión/análisis , Proteínas Recombinantes de Fusión/metabolismo , Alineación de SecuenciaRESUMEN
A cDNA coding for 3-ketoacyl-acyl-carrier protein (ACP) synthase III (KAS III) from spinach (Spinacia oleracea; So KAS III) was used to isolate two closely related KAS III clones (Ch KAS III-1 and Ch KAS III-2) from Cuphea hookeriana. Both Ch KAS IIIs are expressed constitutively in all tissues examined. An increase in the levels of 16:0 was observed in tobacco (Nicotiana tabacum, WT-SR) leaves overexpressing So KAS III when under the control of the cauliflower mosaic virus-35S promoter and in Arabidopsis and rapeseed (Brassica napus) seeds overexpressing either of the Ch KAS IIIs driven by napin. These data indicate that this enzyme has a universal role in fatty acid biosynthesis, irrespective of the plant species from which it is derived or the tissue in which it is expressed. The transgenic rapeseed seeds also contained lower levels of oil as compared with the wild-type levels. In addition, the rate of lipid synthesis in transgenic rapeseed seeds was notably slower than that of the wild-type seeds. The results of the measurements of the levels of the acyl-ACP intermediates as well as any changes in levels of other fatty acid synthase enzymes suggest that malonyl-ACP, the carbon donor utilized by all the 3- ketoacyl-ACP synthases, is limiting in the transgenic plants. This further suggests that malonyl-coenzyme A is a potential limiting factor impacting the final oil content as well as further extension of 16:0.
Asunto(s)
3-Oxoacil-(Proteína Transportadora de Acil) Sintasa/metabolismo , Regulación de la Expresión Génica de las Plantas/genética , Lípidos/biosíntesis , Nicotiana/enzimología , Plantas/enzimología , 3-Oxoacil-(Proteína Transportadora de Acil) Sintasa/química , 3-Oxoacil-(Proteína Transportadora de Acil) Sintasa/genética , Secuencia de Aminoácidos , Secuencia de Bases , Cartilla de ADN , ADN Complementario/genética , Regulación Enzimológica de la Expresión Génica/genética , Cinética , Datos de Secuencia Molecular , Aceites de Plantas/metabolismo , Proteínas de Plantas/química , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Plantas Modificadas Genéticamente/enzimología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Nicotiana/genéticaRESUMEN
Condensing enzymes, catalyzing the formation of carbon-carbon bonds in several biosynthetic pathways, have lately been recognized as potential drug targets against cancer and tuberculosis, as crucial for combinatorial biosynthesis of antibiotics and related compounds, and as determinants of plant oil composition. beta-Ketoacyl-ACP synthases (KAS) are the condensing enzymes present in the fatty acid biosynthesis pathway and are able to elongate an acyl chain bound to either co-enzyme A (CoA) or acyl carrier protein (ACP) with a two-carbon unit derived from malonyl-ACP. Several isoforms of KAS with different substrate specificity are present in most species. We have determined the crystal structure of KAS II from Synechocystis sp. PCC 6803 to 1.54 A resolution giving a detailed description of the active site geometry. In order to analyze the structure-function relationships in this class of enzymes in more detail, we have compared all presently known three-dimensional structures of condensing enzymes from different pathways. The comparison reveals that these enzymes can be divided into three structural and functional classes. This classification can be related to variations in the catalytic mechanism and the set of residues in the catalytic site, e.g. due to differences in the nature of the second substrate providing the two-carbon elongation unit. The variation in the acyl-carrier (ACP or CoA) specificity might also be connected to this classification and residues involved in ACP binding in structure class 2 can be suggested based on the comparison. Finally, the two subunits in the dimer contribute differently to formation of the substrate binding-pocket in the three structural classes.
Asunto(s)
3-Oxoacil-(Proteína Transportadora de Acil) Sintasa/química , Cianobacterias/enzimología , 3-Oxoacil-(Proteína Transportadora de Acil) Sintasa/clasificación , 3-Oxoacil-(Proteína Transportadora de Acil) Sintasa/metabolismo , Secuencia de Aminoácidos , Sitios de Unión , Catálisis , Secuencia Conservada , Cristalografía por Rayos X , Dimerización , Enlace de Hidrógeno , Modelos Moleculares , Datos de Secuencia Molecular , Filogenia , Estructura Cuaternaria de Proteína , Estructura Secundaria de Proteína , Subunidades de Proteína , Alineación de Secuencia , Electricidad Estática , Relación Estructura-ActividadRESUMEN
Although sequences representing members of the phytochrome (phy) family of photoreceptors have been reported in numerous species across the phylogenetic spectrum, relatively few phytochrome genes (PHY) have been fully characterized. Using rice, we have cloned and characterized the first PHYC gene from a monocot. Comparison of genomic and cDNA PHYC sequences shows that the rice PHYC gene contains three introns in the protein-coding region typical of most angiosperm PHY genes, in contrast to Arabidopsis PHYC, which lacks the third intron. Mapping of the transcription start site and 5'-untranslated region of the rice PHYC transcript indicates that it contains an unusually long, intronless, 5'-untranslated leader sequence of 715 bp. PHYC mRNA levels are relatively low compared to PHYA and PHYB mRNAs in rice seedlings, and are similar in dark- and light-treated seedlings, suggesting relatively low constitutive expression. Genomic mapping shows that the PHYA, PHYB, and PHYC genes are all located on chromosome 3 of rice, in synteny with these genes in linkage group C (sometimes referred to as linkage group A) of sorghum. Phylogenetic analysis indicates that rice phyC is closely related to sorghum phyC, but relatively strongly divergent from Arabidopsis phyC, the only full-length dicot phyC sequence available.
Asunto(s)
Proteínas de Arabidopsis , Oryza/genética , Células Fotorreceptoras , Fitocromo/genética , Factores de Transcripción , Secuencia de Aminoácidos , Southern Blotting , Mapeo Cromosómico , Clonación Molecular , ADN de Plantas/química , ADN de Plantas/genética , Grano Comestible/genética , Evolución Molecular , Exones , Regulación de la Expresión Génica de las Plantas , Genes de Plantas/genética , Intrones , Datos de Secuencia Molecular , Filogenia , Fitocromo A , Fitocromo B , Alineación de Secuencia , Análisis de Secuencia de ADN , Homología de Secuencia de Aminoácido , Transcripción Genética , Zea mays/genéticaAsunto(s)
3-Oxoacil-(Proteína Transportadora de Acil) Sintasa/química , 3-Oxoacil-(Proteína Transportadora de Acil) Sintasa/metabolismo , 3-Oxoacil-(Proteína Transportadora de Acil) Sintasa/genética , Escherichia coli/enzimología , Escherichia coli/genética , Isoenzimas/química , Isoenzimas/genética , Isoenzimas/metabolismo , Modelos Moleculares , Conformación Proteica , Ingeniería de Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Especificidad por SustratoRESUMEN
In a database search for homologs of acyl-coenzyme A oxidases (ACX) in Arabidopsis, we identified a partial genomic sequence encoding an apparently novel member of this gene family. Using this sequence information we then isolated the corresponding full-length cDNA from etiolated Arabidopsis cotyledons and have characterized the encoded recombinant protein. The polypeptide contains 675 amino acids. The 34 residues at the amino terminus have sequence similarity to the peroxisomal targeting signal 2 of glyoxysomal proteins, including the R-[I/Q/L]-X5-HL-XL-X15-22-C consensus sequence, suggesting a possible microsomal localization. Affinity purification of the encoded recombinant protein expressed in Escherichia coli followed by enzymatic assay, showed that this enzyme is active on C8:0- to C14:0-coenzyme A with maximal activity on C12:0-coenzyme A, indicating that it has medium-chain-specific activity. These data indicate that the protein reported here is different from previously characterized classes of ACX1, ACX2, and short-chain ACX (SACX), both in sequence and substrate chain-length specificity profile. We therefore, designate this new gene AtACX3. The temporal and spatial expression patterns of AtACX3 during development and in various tissues were similar to those of the AtSACX and other genes expressed in glyoxysomes. Currently available database information indicates that AtACX3 is present as a single copy gene.
Asunto(s)
Proteínas de Arabidopsis , Arabidopsis/enzimología , Oxidorreductasas/genética , Acil-CoA Oxidasa , Secuencia de Aminoácidos , Secuencia de Bases , Mapeo Cromosómico , Clonación Molecular , Cartilla de ADN , ADN Complementario , Escherichia coli/genética , Datos de Secuencia Molecular , Oxidorreductasas/metabolismo , Homología de Secuencia de Aminoácido , Especificidad por SustratoRESUMEN
The composition and positional distribution of lipids in developing and mature transgenic Brassica napus seeds accumulating up to 7 mol% of caprylate (8:0), 29 mol% caprate (10:0) or 63 mol% of laurate (12:0) were examined. The accumulation of 8:0 and 10:0 resulted from over-expression of the medium-chain-specific thioesterase (Ch FatB2) alone or together with the respective chain-length-specific condensing enzyme (Ch KASIV). Seeds containing high levels of 12:0 were obtained from plants expressing bay thioesterase (BTE) alone or crossed with a line over-expressing the coconut lysophosphatidic acid acyltransferase (LPAAT), an enzyme responsible for the increase in acylation of 12:0 at the sn-2 position. In all instances, 10:0 and 12:0 fatty acids were present in substantial amounts in phosphatidylcholine during seed development with a drastic decrease of 80-90% in mature seeds. At all stages of seed development however, 8:0 was barely detectable in this membrane lipid. Altogether, these results indicate that these transgenic seeds exclude and/or remove the medium-chain fatty acids from their membrane and that this mechanism(s) is more effective with the shorter-chain fatty acids. Furthermore, seeds of 8:0- and 10:0-producing lines had only negligible levels of these fatty acids present in the sn-2 position of the triacylglycerols. In contrast, all 12:0-producing seeds had a substantial amount of this fatty acid in the sn-2 position of the triacylglycerols, suggesting that the endogenous LPAAT is able to acylate 12:0 if no other acyl-CoA species are available.
Asunto(s)
Brassica/metabolismo , Caprilatos/metabolismo , Ácidos Decanoicos/metabolismo , Ácidos Láuricos/metabolismo , Metabolismo de los Lípidos , Ácidos Grasos/metabolismo , Plantas Modificadas Genéticamente , Semillas/metabolismoRESUMEN
In the biosynthesis of fatty acids, the beta-ketoacyl-acyl carrier protein (ACP) synthases catalyze chain elongation by the addition of two-carbon units derived from malonyl-ACP to an acyl group bound to either ACP or CoA. The enzyme is a possible drug target for treatment of certain cancers and for tuberculosis. The crystal structure of the complex of the enzyme from Escherichia coli, and the fungal mycotoxin cerulenin reveals that the inhibitor is bound in a hydrophobic pocket formed at the dimer interface. Cerulenin is covalently attached to the active site cysteine through its C2 carbon atom. The fit of the inhibitor to the active site is not optimal, and there is thus room for improvement through structure based design.
Asunto(s)
3-Oxoacil-(Proteína Transportadora de Acil) Sintasa/química , Cerulenina/química , Isoenzimas/química , 3-Oxoacil-(Proteína Transportadora de Acil) Sintasa/metabolismo , Antifúngicos/química , Antifúngicos/metabolismo , Sitios de Unión , Cerulenina/metabolismo , Escherichia coli , Isoenzimas/metabolismo , Datos de Secuencia Molecular , Unión Proteica , Conformación Proteica , Especificidad por SustratoRESUMEN
cDNA clones encoding a novel 3-ketoacyl-ACP synthase (KAS) have been isolated from Cuphea. The amino acid sequence of this enzyme is different from the previously characterized classes of KASs, designated KAS I and III, and similar to those designated as KAS II. To define the acyl chain specificity of this enzyme, we generated transgenic Brassica plants over-expressing the cDNA encoded protein in a seed specific manner. Expression of this enzyme in transgenic Brassica seeds which normally do not produce medium chain fatty acids does not result in any detectable modification of the fatty acid profile. However, co-expression of the Cuphea KAS with medium chain specific thioesterases, capable of production of either 12:0 or 8:0/10:0 fatty acids in seed oil, strongly enhances the levels of these medium chain fatty acids as compared with seed oil of plants expressing the thioesterases alone. By contrast, co-expression of the Cuphea KAS along with an 18:0/18.1-ACP thioesterase does not result in any detectable modification of the fatty acids. These data indicate that the Cuphea KAS reported here has a different acyl-chain specificity to the previously characterized KAS I, II and III. Therefore, we designate this enzyme KAS IV, a medium chain specific condensing enzyme.
Asunto(s)
3-Oxoacil-(Proteína Transportadora de Acil) Sintasa/metabolismo , Plantas/enzimología , 3-Oxoacil-(Proteína Transportadora de Acil) Sintasa/genética , Secuencia de Bases , Brassica/enzimología , Brassica/genética , Clonación Molecular , Cartilla de ADN/genética , ADN Complementario/genética , ADN de Plantas/genética , Ácidos Grasos/química , Ácidos Grasos/metabolismo , Expresión Génica , Datos de Secuencia Molecular , Plantas/genética , Plantas Modificadas Genéticamente , Reacción en Cadena de la Polimerasa , Semillas/enzimología , Especificidad por SustratoRESUMEN
In the biosynthesis of fatty acids, the beta-ketoacyl-acyl carrier protein (ACP) synthases catalyze chain elongation by the addition of two-carbon units derived from malonyl-ACP to an acyl group bound to either ACP or CoA. The crystal structure of beta-ketoacyl synthase II from Escherichia coli has been determined with the multiple isomorphous replacement method and refined at 2.4 A resolution. The subunit consists of two mixed five-stranded beta-sheets surrounded by alpha-helices. The two sheets are packed against each other in such a way that the fold can be described as consisting of five layers, alpha-beta-alpha-beta-alpha. The enzyme is a homodimer, and the subunits are related by a crystallographic 2-fold axis. The two active sites are located near the dimer interface but are approximately 25 A apart. The proposed nucleophile in the reaction, Cys163, is located at the bottom of a mainly hydrophobic pocket which is also lined with several conserved polar residues. In spite of very low overall sequence homology, the structure of beta-ketoacyl synthase is similar to that of thiolase, an enzyme involved in the beta-oxidation pathway, indicating that both enzymes might have a common ancestor.
Asunto(s)
3-Oxoacil-(Proteína Transportadora de Acil) Sintasa/química , Escherichia coli/enzimología , Acetil-CoA C-Acetiltransferasa/química , Secuencia de Aminoácidos , Sitios de Unión , Cristalografía por Rayos X , Cisteína/química , Dimerización , Modelos Químicos , Modelos Moleculares , Datos de Secuencia Molecular , Pliegue de Proteína , Estructura Secundaria de Proteína , Homología de Secuencia de Aminoácido , Levaduras/enzimologíaRESUMEN
Analysis of the beta-ketoacyl-ACP synthase (KAS) encoded by the fabF gene of Escherichia coli has been hampered by a reported instability of the cloned gene. Here we describe biochemical characterization of purified, active protein from the recombinant fabF gene. This enzyme has the properties ascribed to KAS II and not those of a putative KAS IV reported to be encoded by fabJ, a genomic clone with DNA sequence identical to that of fabF. We also characterize active protein from a recombinant fabB gene and suggest that this method may have a general utility for analysis of KAS enzymes.
Asunto(s)
3-Oxoacil-(Proteína Transportadora de Acil) Sintasa/genética , 3-Oxoacil-(Proteína Transportadora de Acil) Sintasa/metabolismo , Escherichia coli/enzimología , 3-Oxoacil-(Proteína Transportadora de Acil) Sintasa/química , 3-Oxoacil-(Proteína Transportadora de Acil) Sintasa/aislamiento & purificación , Cerulenina/farmacología , Clonación Molecular , Escherichia coli/genética , Genes Bacterianos , Vectores Genéticos , Proteínas Recombinantes/química , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Especificidad por Sustrato , TemperaturaRESUMEN
GT-2 is a novel DNA binding protein that interacts with a triplet functionally defined, positively acting GT-box motifs (GT1-bx, GT2-bx, and GT3-bx) in the rice phytochrome A gene (PHYA) promoter. Data from a transient transfection assay used here show that recombinant GT-2 enhanced transcription from both homologous and heterologous GT-box-containing promoters, thereby indicating that this protein can function as a transcriptional activator in vivo. Previously, we have shown that GT-2 contains separate DNA binding determinants in its N- and C-terminal halves, with binding site preferences for the GT3-bx and GT2-bx promoter motifs, respectively. Here, we demonstrate that the minimal DNA binding domains reside within dual 90-amino acid polypeptide segments encompassing duplicated sequences, termed trihelix regions, in each half of the molecule, plus 15 additional immediately adjacent amino acids downstream. These minimal binding domains retained considerable target sequence selectivity for the different GT-box motifs, but this selectivity was enhanced by a separate polypeptide segment farther downstream on the C-terminal side of each trihelix region. Therefore, the data indicate that the twin DNA binding domains of GT-2 each consist of a general GT-box recognition core with intrinsic differential binding activity toward closely related target motifs and a modified sequence conferring higher resolution reciprocal selectivity between these motifs.
Asunto(s)
Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/metabolismo , Genes de Plantas , Oryza/genética , Oryza/metabolismo , Fitocromo/genética , Regiones Promotoras Genéticas , Factores de Transcripción/química , Factores de Transcripción/metabolismo , Activación Transcripcional , Secuencia de Aminoácidos , Secuencia de Bases , Sitios de Unión , Células Cultivadas , Cartilla de ADN , Proteínas de Unión al ADN/biosíntesis , Cinética , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Fitocromo/biosíntesis , Fitocromo A , Plantas Tóxicas , Reacción en Cadena de la Polimerasa , Prolina , Estructura Secundaria de Proteína , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Mapeo Restrictivo , Especificidad por Sustrato , Nicotiana , Factores de Transcripción/biosíntesis , Transcripción Genética , TransfecciónRESUMEN
The Mexican shrub Cuphea hookeriana accumulates up to 75% caprylate (8:0) and caprate (10:0) in its seed oil. An acyl-ACP thioesterase cDNA from C. hookeriana, designated Ch FatB2, has been identified, which, when expressed in Escherichia coli, provides thioesterase activity specific for 8:0- and 10:0-ACP substrates. Expression of this clone in seeds of transgenic canola, an oilseed crop that normally does not accumulate any 8:0 and 10:0, resulted in a dramatic increase in the levels of these two fatty acids accompanied by a preferential decrease in the levels of linoleate (18:2) and linolenate (18:3). The Ch FatB2 differs from Ch FatB1, another Cuphea hookeriana thioesterase reported recently, in both substrate specificity and expression pattern. The Ch FatB1 has a broad substrate specificity with strong preference for 16:0-ACP and is expressed throughout the plant; whereas Ch FatB2 is specific for 8:0/10:0-ACP and its expression is confined to the seed. It is proposed that the amplified expression of Ch FatB2 in the embryo provides the hydrolytic enzyme specificity determining the fatty acyl composition of Cuphea hookeriana seed oil.
Asunto(s)
Caprilatos/metabolismo , Ácidos Decanoicos/metabolismo , Proteínas de Plantas , Plantas Modificadas Genéticamente/metabolismo , Plantas/enzimología , Tioléster Hidrolasas/metabolismo , Secuencia de Aminoácidos , Secuencia de Bases , Brassica/genética , Brassica/metabolismo , Clonación Molecular , ADN Complementario/genética , Ácidos Grasos Monoinsaturados/metabolismo , Hidrólisis , Datos de Secuencia Molecular , Plantas/genética , Plantas/metabolismo , Plantas Modificadas Genéticamente/enzimología , Aceite de Brassica napus , Proteínas Recombinantes/metabolismo , Análisis de Secuencia de ADN , Homología de Secuencia de Aminoácido , Especificidad por Sustrato , Tioléster Hidrolasas/genéticaRESUMEN
The seed oil of Cuphea palustris has an unusual fatty-acyl composition, whereby the principal fatty-acyl groups, myristate (64%) and caprylate (20%), differ by more than two methylenes. We have isolated two thioesterase (TE) cDNAs from C. palustris, encoding proteins designated Cp FatB1 and Cp FatB2, which, when expressed in Escherichia coli, have TE activities specific for 8:0/10:0- and 14:0/16:0-acyl carrier protein substrates, respectively. The specific activities of the recombinant affinity-purified enzymes indicate that Cp FatB2 is kinetically superior to Cp FatB1. This result is consistent with the predominance of 14:0 in the seed oil, despite apparently equal mRNA abundance of the two transcripts in the seed. In C. palustris the expression of both sequences is confined to the seed tissues. Based on these findings we propose that these two enzymes are major factors determining the bimodal chain-length composition of C. palustris oil. Analysis of the immature and mature seed oil by reverse-phase high-performance liquid chromatography confirmed that the principal triglycerides contain both 8:0 and 14:0. This result indicates that both fatty acids are synthesized at the same time and in the same cells at all developmental stages during oil deposition, suggesting that the two TEs act together in the same fatty acid synthesis system.
Asunto(s)
Ácidos Grasos/biosíntesis , Aceites de Plantas/química , Proteínas de Plantas , Plantas/genética , Semillas/química , Tioléster Hidrolasas/genética , Secuencia de Aminoácidos , Secuencia de Bases , Caprilatos/metabolismo , ADN Complementario/genética , Escherichia coli/genética , Biblioteca de Genes , Datos de Secuencia Molecular , Ácido Mirístico , Ácidos Mirísticos/metabolismo , Plantas/química , Plantas/enzimología , Estructura Secundaria de Proteína , ARN Mensajero/análisis , ARN de Planta/análisis , Proteínas Recombinantes/metabolismo , Análisis de Secuencia de ADN , Especificidad por Sustrato , Tioléster Hidrolasas/metabolismo , Distribución Tisular , Triglicéridos/químicaRESUMEN
GT-2 is a DNA-binding protein with high target-sequence specificity toward functionally defined, positively acting cis elements in the rice phytochrome A gene promoter. Using immunocytochemical procedures, it is shown here that GT-2 is localized to the nucleus, consistent with a function in transcriptional regulation. Immunoblot and immunocytochemical analyses show that rice shoots contain higher levels of GT-2 protein than roots, and that no photo-induced changes in GT-2 abundance or spatial distribution are detectable in these tissues, a result consistent with the proposed constitutive activity of GT-2. In both shoots and roots, GT-2 protein is undetectable in meristematic tissue but becomes expressed at later stages of cellular development, consistent with a role in contributing to the pattern of phytochrome A gene expression. By transfecting protoplasts with a series of constructs containing deletion derivatives of GT-2 fused to beta-glucuronidase (GUS), followed by in situ localization of GUS activity, two independent, functionally active nuclear localization sequences (NLSs) have been identified in GT-2. One NLS resides within each of a pair of previously identified, spatially separate, trihelix motifs in the protein. Sequence inversion and alanine-scanning mutagenesis has identified residues within these NLSs necessary for nuclear localization. Each NLS contains two basic domains separated by 10 amino acids, conforming to the bipartite class of NLS involved in the targeting of numerous other nuclear localized proteins.
Asunto(s)
Núcleo Celular/metabolismo , Proteínas de Unión al ADN/metabolismo , Proteínas Nucleares/metabolismo , Oryza/metabolismo , Factores de Transcripción/metabolismo , Secuencia de Aminoácidos , Western Blotting , Glucuronidasa/metabolismo , Datos de Secuencia Molecular , Homología de Secuencia de Aminoácido , Fracciones Subcelulares/metabolismoRESUMEN
We have determined the sequence of the phytochrome A gene (PHYA) and its flanking DNA from Arabidopsis thaliana and have identified transcription start sites for three nested transcripts of increasing length. The overall structure of the gene is similar as regards exon/intron organization to other angiosperm PHY genes characterized. The triple transcription start site arrangement is similar to that of pea PHYA but different from the single start site of oat, rice and maize PHYA genes, indicating a possible monocot-dicot difference. Comparison of the Arabidopsis PHYA promoter sequence with others available indicates that both pea and Arabidopsis promoters contain a DNA element with a core sequence motif identical to one conserved in all existing monocot PHYA sequences and defined by functional assay in the oat PHYA gene as repressor element, RE1, responsible for negative light regulation.
Asunto(s)
Arabidopsis/genética , Fitocromo/genética , Secuencia de Aminoácidos , Secuencia de Bases , ADN/genética , Genes de Plantas , Genes Reguladores , Datos de Secuencia Molecular , Regiones Promotoras Genéticas , Transcripción GenéticaRESUMEN
Complementary DNA clones encoding a DNA-binding factor have been obtained from Arabidopsis by DNA hybridization with a GT-2 factor cDNA clone from rice. The GT-2 gene appears to be present as a single copy in the Arabidopsis genome and is transcribed as a 2.1 kb mRNA which is not light-regulated. The longest open reading frame in the sequenced clones predicts a protein of 65 kDa, beginning with the first in-frame methionine. The protein contains basic, acidic, and proline/glutamine-rich motifs and has significant amino acid sequence homology to the rice GT-2 factor, including three regions of 50-75 amino acids each of greater than 60% identity. Two of these regions are predicted to form similar trihelix structures postulated to be involved in selective binding to specific variations of a GT-box motif DNA sequence found in the promoter regions of several plant genes. Except for weak similarity to a tobacco GT-box binding factor, GT-1a/B2F, Arabidopsis GT-2 has no similarity to other sequences in the databases. DNA-binding studies show that Arabidopsis GT-2 has binding characteristics similar to those of the rice GT-2 factor, but dissimilar to those of the tobacco GT-1a/B2F factor. The data indicate that a DNA-binding factor containing domains of similar structure and target-sequence specificity has been conserved between monocots and dicots.
Asunto(s)
Proteínas de Arabidopsis , Arabidopsis/genética , Proteínas de Unión al ADN/genética , Genes de Plantas , Proteínas de Plantas/genética , Secuencia de Aminoácidos , Secuencia de Bases , ADN Complementario/genética , Biblioteca de Genes , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , Estructura Secundaria de Proteína , Secuencias Reguladoras de Ácidos Nucleicos/genética , Análisis de Secuencia de ADN , Homología de Secuencia de Aminoácido , Transcripción GenéticaRESUMEN
hy8 long hypocotyl mutants of Arabidopsis defective in responsiveness to prolonged far-red light (the so-called "far-red high-irradiance response") are selectively deficient in functional phytochrome A. To define the molecular lesion in these mutants, we sequenced the phytochrome A gene (phyA) in lines carrying one or other of two classes of hy8 alleles. The hy8-1 and hy8-2 mutants that express no detectable phytochrome A each have a single nucleotide change that inserts a translational stop codon in the protein coding sequence. These results establish that phyA resides at the HY8 locus. The hy8-3 mutant that expresses wild-type levels of photochemically active phytochrome A has a glycine-to-glutamate missense mutation at residue 727 in the C-terminal domain of the phyA sequence. Quantitative fluence rate response analysis showed that the mutant phytochrome A molecule produced by hy8-3 exhibited no detectable regulatory activity above that of the phyA-protein-deficient hy8-2 mutant. This result indicates that glycine-727, which is invariant in all sequenced phytochromes, has a function important to the regulatory activity of phytochrome A but not to photoperception.