RESUMEN
Starch, a major storage metabolite in plants, positively affects the agricultural yield of a number of crops. Its biosynthetic reactions use adenosine diphosphate glucose (ADPGlc) as a substrate; ADPGlc pyrophosphorylase, the enzyme involved in ADPGlc formation, is regulated by allosteric effectors. Evidence that this plastidial enzyme catalyzes a rate-limiting reaction in starch biosynthesis was derived by expression in plants of a gene that encodes a regulatory variant of this enzyme. Allosteric regulation was demonstrated to be the major physiological mechanism that controls starch biosynthesis. Thus, plant and bacterial systems for starch and glycogen biosynthesis are similar and distinct from yeast and mammalian systems, wherein glycogen synthase has been demonstrated to be the rate-limiting regulatory step.
RESUMEN
The herbicide glyphosate is a potent inhibitor of the enzyme 5-enolpyruvylshikimate- 3-phosphate (EPSP) synthase in higher plants. A complementary DNA (cDNA) clone encoding EPSP synthase was isolated from a complementary DNA library of a glyphosate-tolerant Petunia hybrida cell line (MP4-G) that overproduces the enzyme. This cell line was shown to overproduce EPSP synthase messenger RNA as a result of a 20-fold amplification of the gene. A chimeric EPSP synthase gene was constructed with the use of the cauliflower mosaic virus 35S promoter to attain high level expression of EPSP synthase and introduced into petunia cells. Transformed petunia cells as well as regenerated transgenic plants were tolerant to glyphosate.
RESUMEN
In many economically important plant species, the chemical composition of one or more non-protein compounds determines the value of the plant and may have an important role in protecting the plant from environmental stress, including pests, drought, salt, temperature and light. A number of potential opportunities exist whereby the range or amount of such valuable compounds can be increased by genetic engineering.
Asunto(s)
Ingeniería Genética , Plantas Modificadas Genéticamente/genética , Carbohidratos/biosíntesis , Aceites de Plantas/metabolismo , Plantas Modificadas Genéticamente/química , Plantas Modificadas Genéticamente/metabolismo , Polímeros/metabolismo , Terpenos/metabolismoRESUMEN
Effective and sustained control of fungal pathogens and nematodes is an important issue for all agricultural systems. Global losses caused by pathogens are estimated to be 12% of the potential crop production [1], despite the continued release of new resistant cultivars and pesticides. Furthermore, fungi are continually becoming resistant to existing resistance genes and fungicides, and a few of the pesticides are being withdrawn from the market for environmental reasons. In addition to reducing crop yield, fungal diseases often lower crop quality by producing toxins that affect humans and human health. Additional methods of disease control are therefore highly desirable. Breeding programs based on plant disease-resistance genes are being optimized by incorporating molecular marker techniques and biotechnology. These efforts can be expected to result in the first launches of new disease-resistant crops within the next five years.
Asunto(s)
Agricultura/métodos , Productos Agrícolas/genética , Genes de Plantas/genética , Ingeniería Genética/métodos , Enfermedades de las Plantas , Animales , Productos Agrícolas/microbiología , Regulación de la Expresión Génica de las Plantas , Técnicas de Transferencia de Gen , Predisposición Genética a la Enfermedad , Patentes como Asunto , Plantas/genética , Plantas/microbiología , Plantas Modificadas Genéticamente/genética , Transgenes/genéticaRESUMEN
Hepatitis is common in the Stann Creek District of southern Belize. To determine the etiologies, incidence, and potential risk factors for acute jaundice, we conducted active surveillance for cases. Cases of jaundice diagnosed by a physician within the previous 6 weeks were enrolled. Evaluation included a questionnaire and laboratory tests for hepatitis A, B, C, D, and E, a blood film for malaria, and a serologic test for syphilis. Etiologies of jaundice among 62 evaluable patients included acute hepatitis A, 6 (9.7%), acute hepatitis B, 49 (79.0%), hepatitis non-A-E, 2 (3.2%), and malaria, 5 (8.1%). There were no cases of acute hepatitis E. One patient each with antibody to hepatitis C and D were detected. The annualized incidence of hepatitis A was 0.26 per 1,000. All cases of hepatitis A were in children 4-16 years of age. The annualized incidence of hepatitis B, 2.17 per 1,000, was highest in adults aged 15-44 years (4.4 per 1,000) and was higher in men (36 cases; 3.09 per 1,000) than women (13 cases; 1.19 per 1,000). Four (31%) of the women with hepatitis B were pregnant. The annualized incidence was significantly higher in Mestizo (6.18 per 1000) and Maya (6.79 per 1,000) than Garifuna (0.38 per 1,000) or Creole (0.36 per 1,000). Persons with hepatitis B were significantly more likely to be born outside of Belize (82%), had been in Belize < 5 years (73%), and lived and worked in rural areas (96%) than was the general population. Of those > or = 14 years of age with hepatitis B, only 36% were married. Few persons admitted to transfusions, tattoos, IV drug use, multiple sexual partners, visiting prostitutes, or sexually transmitted diseases. Only 1 of 49 had a reactive test for syphilis. Six patients were hospitalized (including 3 with acute hepatitis B and one with hepatitis A), and none to our knowledge died. Acute hepatitis B is the most common cause of viral hepatitis in the Stann Creek District, but the modes of transmission remain obscure. Infants, women attending prenatal clinics, and new workers are potential targets for immunization with hepatitis B vaccine.
Asunto(s)
Hepatitis/epidemiología , Ictericia/diagnóstico , Enfermedad Aguda , Adolescente , Adulto , Belice/epidemiología , Niño , Preescolar , Transmisión de Enfermedad Infecciosa/prevención & control , Femenino , Hepatitis/etiología , Hepatitis/inmunología , Hepatitis B/epidemiología , Hepatitis B/inmunología , Hepatitis B/prevención & control , Vacunas contra Hepatitis B , Humanos , Incidencia , Ictericia/etiología , Malaria/complicaciones , Malaria/epidemiología , Masculino , Persona de Mediana Edad , Vigilancia de la Población , Embarazo , Pruebas Serológicas , Encuestas y CuestionariosRESUMEN
Kynureninase from Pseudomonas marginalis has been shown to catalyze the elimination of beta-functionalities of beta-substituted amino acids such as beta-chloro-L-alanine, resulting in the formation of aminoacrylate-pyridoxal phosphate-enzyme complex. This intermediate can be processed further to produce either pyruvate, ammonia, and active enzyme or an inactive enzyme complex. Approximately 1 in 500 turnovers leads to inactivation of the enzyme. The mechanism of inactivation appears to involve nucleophilic addition of a carboxylate group at the active site to the beta-carbon of the aminoacrylate complex. Both subunits of kynureninase have been shown to be catalytically competent although the native enzyme contains only one pyridoxal phosphate per dimer. Since both aspartate beta-decarboxylase and kynureninase catalyze mechanistically similar reactions, these results further support the notion that the two active sites may have several common features.
Asunto(s)
Aminoácidos/farmacología , Hidrolasas/antagonistas & inhibidores , Pseudomonas/enzimología , Sitios de Unión , Radioisótopos de Carbono , Cinética , Relación Estructura-Actividad , beta-Alanina/análogos & derivados , beta-Alanina/farmacologíaRESUMEN
While the last 50 years of agriculture have focused on meeting the food, feed, and fiber needs of humans, the challenges for the next 50 years go far beyond simply addressing the needs of an ever-growing global population. In addition to producing more food, agriculture will have to deal with declining resources like water and arable land, need to enhance nutrient density of crops, and achieve these and other goals in a way that does not degrade the environment. Biotechnology and other emerging life sciences technologies offer valuable tools to help meet these multidimensional challenges. This paper explores the possibilities afforded through biotechnology in providing improved agronomic "input" traits, differentiated crops that impart more desirable "output" traits, and using plants as green factories to fortify foods with valuable nutrients naturally rather than externally during food processing. The concept of leveraging agriculture as green factories is expected to have tremendous positive implications for harnessing solar energy to meet fiber and fuel needs as well. Widespread adaptation of biotech-derived products of agriculture should lay the foundation for transformation of our society from a production-driven system to a quality and utility-enhanced system.
Asunto(s)
Biotecnología/métodos , Abastecimiento de Alimentos , Fenómenos Fisiológicos de la Nutrición , Agricultura/métodos , Agricultura/tendencias , Biotecnología/tendencias , Países Desarrollados , Países en Desarrollo , Humanos , Plantas ComestiblesRESUMEN
In the presence of NADH and O2, 2-methyl-3-hydroxypyridine-5-carboxylate oxygenase (EC 1.14.12.4) from Pseudomonas sp. MA-1 catalyzes reductive oxygenation of 2-methyl-3-hydroxypyridine-5-carboxylate (Cpd I) to yield alpha-N-(acetylaminomethylene)succinic acid (Cpd A). Steady state kinetic data and studies with alternate substrates are consistent only with an ordered mechanism in which Cpd I binds first, followed by NADH; the first product, NAD+, is then released. This event is followed by oxygen binding, and finally release of the oxygenated and reduced cleavage product, Cpd A. This kinetic mechanism was confirmed by studying inhibition by NAD+, which binds competitively with oxygen, but not with NADH. The kinetic mechanism of this reaction resembles that proposed for bacterial flavin monooxygenases that catalyze hydroxylation of aromatic homocyclic compounds.
Asunto(s)
Oxigenasas de Función Mixta/metabolismo , Pseudomonas/enzimología , Flavoproteínas/metabolismo , Cinética , Matemática , NAD , Oxígeno , Piridinas/metabolismo , Especificidad por SustratoRESUMEN
The bacterium Pseudomonas PG2982 metabolizes glyphosate (N-(phosphonomethyl)glycine) by converting it to glycine, a one-carbon unit, and phosphate. Here we show that this conversion involves the intermediate formation of sarcosine. When cells are incubated with [14C]glyphosate, the 14C can be entrapped in glycine or sarcosine. With added sarcosine, 14C from all three carbons of glyphosate is recovered solely in sarcosine. In experiments with glycine, radioactivity from the carboxymethyl moiety of glyphosate is trapped in glycine as well as serine, whereas radioactivity from the phosphonomethyl carbon is only incorporated into serine. These results are consistent with a pathway involving the conversion of glyphosate to sarcosine by cleavage of its carbon-phosphorus (C-P) bond, followed by the oxidation of sarcosine to glycine and formaldehyde.
Asunto(s)
Glicina/análogos & derivados , Pseudomonas/metabolismo , Sarcosina/metabolismo , Escherichia coli/metabolismo , Glicina/metabolismo , Modelos Químicos , GlifosatoRESUMEN
Import of the precursor to 5-enolpyruvylshikimate-3-phosphate synthase (pEPSPS) into chloroplasts is inhibited by the herbicide glyphosate. Inhibition of import is maximal at glyphosate concentrations of >/=10 mum and occurs only when pEPSPS is present as a ternary complex of enzyme-shikimate-3-phosphate-glyphosate. Glyphosate alone had no effect on the import of pEPSPS since it is not known to interact with the enzyme in the absence of shikimate-3-phosphate. Experiments with wild-type and glyphosate-resistant mutant forms of pEPSPS show that inhibition of import is directly proportional to the binding constants for glyphosate. Inhibition of import is thus a direct consequence of glyphosate binding to the enzyme-shikimate-3-phosphate complex. The potential for non-specific effects of glyphosate on the chloroplast transport mechanism has been discounted by showing that import of another chloroplast-designated protein was unaffected by high concentrations of glyphosate and shikimate-3-phosphate. The mechanism of import inhibition by glyphosate is consistent with a precursor unfolding/refolding model.
RESUMEN
5-Enolpyruvyl shikimate 3-phosphate synthase catalyzes the reversible condensation of phosphoenolpyruvate and shikimate 3-phosphate to yield 5-enolpyruvyl shikimate 3-phosphate and inorganic phosphate. The enzyme is a target for the nonselective herbicide glyphosate (N-phosphonomethylglycine). In order to determine the role of lysine residues in the mechanism of action of this enzyme as well as in its inhibition by glyphosate, chemical modification studies with pyridoxal 5'-phosphate were undertaken. Incubation of the enzyme with the reagent in the absence of light resulted in a time-dependent loss of enzyme activity. The inactivation followed pseudo first-order and saturation kinetics with Kinact of 45 microM and a maximum rate constant of 1.1 min-1. The inactivation rate increased with increase in pH, with a titratable pK of 7.6. Activity of the inactive enzyme was restored by addition of amino thiol compounds. Reaction of enzyme with pyridoxal 5'-phosphate was prevented in the presence of substrates or substrate plus glyphosate, an inhibitor of the enzyme. Upon 90% inactivation, approximately 1 mol of pyridoxal 5'-phosphate was incorporated per mol of enzyme. The azomethine linkage between pyridoxal 5'-phosphate and the enzyme was reduced by NaB3H4. Tryptic digestion followed by reverse phase chromatographic separation resulted in the isolation of a peptide which contained the pyridoxal 5'-phosphate moiety as well as 3H label. By amino acid sequencing of this peptide, the modified residue was identified as Lys-22. The amino acid sequence around Lys-22 is conserved in bacterial, fungal, as well as plant enzymes suggesting that this region may constitute a part of the enzyme's active site.
Asunto(s)
Transferasas Alquil y Aril , Escherichia coli/enzimología , Lisina , Transferasas/antagonistas & inhibidores , 3-Fosfoshikimato 1-Carboxiviniltransferasa , Secuencia de Aminoácidos , Sitios de Unión , Cromatografía Líquida de Alta Presión , Cinética , Fragmentos de Péptidos/análisis , Fosfoenolpiruvato/farmacología , Ácido Shikímico/análogos & derivados , Ácido Shikímico/metabolismo , Transferasas/metabolismo , Tripsina/metabolismoRESUMEN
A highly specific inducible membrane-bound 4-pyridoxic acid dehydrogenase has been solubilized and purified to apparent homogeneity from Pseudomonas MA-1 grown with pyridoxine as a sole source of carbon and nitrogen. The undenatured enzyme migrates as a single band on gel electrophoresis; denatured preparations show two barely resolved bands (Mr = 63,000 and 61,000). Undenatured preparations aggregate readily, as evidenced by Mr values of 148,000, 470,000, and greater than 670,000 obtained by density gradient centrifugation or by gel filtration under various conditions. The enzyme contains FAD but no Fe or acid-labile S; an average minimum molecular weight of 131,000 was calculated from the FAD content. In the presence of 2,6-dichloroindophenol, the enzyme dehydrogenates 4-pyridoxic acid to the corresponding aldehyde; this reaction is not inhibited by CN-. At the pH optimum of 8.0, a Vm of approximately 7.0 mumol min-1 mg-1 and a Km of 9 microM were obtained. 2,6-Dichloroindophenol, phenazine methosulfate, and menadione are effective electron acceptors; ubiquinones are less active, while NAD, FAD, and O2 are inactive. However, in membrane fractions, oxygen supports 4-pyridoxic acid oxidation via a CN--sensitive electron transport chain, indicating that the dehydrogenase probably is coupled to ATP generation in such preparations.
Asunto(s)
Oxidorreductasas de Alcohol/metabolismo , Pseudomonas/enzimología , Piridoxina/metabolismo , Flavina-Adenina Dinucleótido/análisis , Cinética , Peso Molecular , Octoxinol , Polietilenglicoles/análisis , Ácido Piridóxico/metabolismo , Espectrofotometría , Especificidad por SustratoRESUMEN
The gene encoding an isoamylase with neutral pH optimum (iam) from a Flavobacterium species was cloned using a PCR probe generated from highly conserved regions of amylolytic enzymes. Active isoamylase was expressed from a 4.9-kb Pst I fragment in Escherichia coli, and was detected in the extracellular medium by a plate assay. The iam nucleotide sequence has an open reading frame of 2334 nucleotides (778 amino acids) with a GC content of 69%. Sequence analysis suggests that transcriptional control of the Flavobacterium sp. iam gene is mediated through the product of a malT regulatory gene. The deduced amino acid sequence of iam contained an N-terminal signal peptide of 32 amino acids, and was 61% homologous with Pseudomonas amyloderamosa isoamylase. The mature enzyme, which was engineered for overexpression in E. coli and purified to homogeneity, has a relative molecular mass of 83 kDa, a pH optimum of 6-7, and a highest rate of hydrolysis for glycogen (but did not cleave pullulan). Polyclonal antiserum generated from purified donor isoamylase cross-reacted with crude and purified recombinant isoamylase from E. coli. This is the first report of the cloning, characterization, and sequence of an novel isoamylase that has a neutral pH optimum. A comparison of the sequence of Flavobacterium sp. iam with acidic isoamylase from Pseudomonas sp. identified putative residues which may be associated with the pH for optimal activity of isoamylases.
Asunto(s)
Flavobacterium/genética , Genes Bacterianos/genética , Isoamilasa/genética , Isoamilasa/metabolismo , Secuencia de Aminoácidos , Secuencia de Bases , Clonación Molecular , Secuencia Conservada , Escherichia coli/genética , Flavobacterium/enzimología , Regulación Bacteriana de la Expresión Génica , Concentración de Iones de Hidrógeno , Isoamilasa/química , Isoamilasa/aislamiento & purificación , Datos de Secuencia Molecular , Peso Molecular , Proteínas Recombinantes de Fusión/aislamiento & purificación , Proteínas Recombinantes de Fusión/metabolismo , Análisis de Secuencia de ADN , Homología de Secuencia de AminoácidoRESUMEN
Lyophilized samples of Pseudomonas sp. PG2982 grown on 13C- and 15N-labeled glyphosate have been analyzed by single and double cross-polarization 13C NMR. Both the carbon and nitrogen metabolism of glyphosate are significantly influenced by the nitrogen source used for the growth of the organism. When ammonium sulfate is the source of nitrogen, the glycyl moiety of glyphosate is utilized intact for the biosynthesis of purines and proteins. But when the organism is grown on glycine as the source of nitrogen, the carbons and nitrogen of glyphosate are scrambled, consistent with incorporation into serine and pyruvate, and hence participation in general metabolism. When both ammonium and glycine are present in the growth medium, regulation of the metabolic fluxes along each of the two major pathways appears to be determined by the intracellular glycine concentration.
Asunto(s)
Glicina/análogos & derivados , Glicina/metabolismo , Pseudomonas/metabolismo , Amoníaco/metabolismo , Carbono/metabolismo , Espectroscopía de Resonancia Magnética , Nitrógeno/metabolismo , Piruvatos/metabolismo , Ácido Pirúvico , Serina/metabolismo , GlifosatoRESUMEN
The metabolism of glyphosate [N-(phosphonomethyl)glycine] in a bacterium tentatively identified as an Arthrobacter sp., capable of growth on this herbicide as its sole phosphorus source, has been investigated using solid-state NMR techniques as well as radiotracer analysis. The pathway involves the conversion of glyphosate to glycine, a C1 unit and phosphate. The phosphonomethyl carbon is specifically incorporated into the amino acids serine, cysteine, methionine, and histidine, as well as into purine bases and thymine, indicating the involvement of tetrahydrofolate in single-carbon transfer reactions. Glycine derived from glyphosate is utilized in purine and protein biosynthesis. This pathway for glyphosate degradation in a gram-positive bacterium is similar to that previously reported for Pseudomonas sp. PG2982 [Jacob et al. (1985) J. Biol. Chem. 260, 5899-5905] and is distinct from that reported for soil metabolism of glyphosate where aminomethylphosphonic acid has been shown to be a major metabolite. Preliminary evidence is presented which indicates that the conversion of glyphosate to glycine and the C1 unit involves the intermediate formation of sarcosine. Thus, the primary event in glyphosate degradation by Arthrobacter sp. GLP-1 is the cleavage of its C-P bound. This report constitutes the first demonstration of the metabolism of glyphosate in a gram-positive bacterium.
Asunto(s)
Arthrobacter/metabolismo , Glicina/análogos & derivados , Arthrobacter/crecimiento & desarrollo , Cromatografía en Capa Delgada , Glicina/metabolismo , Espectroscopía de Resonancia Magnética/métodos , Compuestos Organofosforados/metabolismo , Sarcosina/farmacología , GlifosatoRESUMEN
In order to detect covalent reaction intermediates in the 5-enolpyruvylshikimate-3-phosphate (EPSP) synthase reaction, we have investigated the interaction of EPSP synthase with the reaction product EPSP. An exchange of EPSP-methylene protons could be demonstrated by incubating EPSPS with EPSP in D2O. Since trace amounts of contaminating Pi would lead to reversal of EPSPS reaction and hence methylene proton exchange, we added pyruvate kinase, ADP, Mg++ and K+. Under these conditions, any contaminating Pi that is converted to PEP is trapped as ATP. No exchange of EPSP protons with those of the solvent could be detected in the presence of this trap system, suggesting that enzyme-bound EPSP is unable to form a covalent tetrahedral complex. Incorporation of [14C] from [14C]-S3P and [14C]-PEP into EPSP could be detected, but only in the absence of a PEP (or Pi) trap system. This indicates that for the exchange reaction, Pi is required, and also indicates the absence of a covalent intermediate, unless the carboxyvinyl-enzyme-bound S3P is completely restricted from exchange.
Asunto(s)
Transferasas Alquil y Aril , Transferasas/metabolismo , 3-Fosfoshikimato 1-Carboxiviniltransferasa , Catálisis , Escherichia coli/enzimología , Espectroscopía de Resonancia Magnética , Fosfatos , ProtonesRESUMEN
Reaction of Petunia hybrida 5-enol-pyruvylshikimate-3-phosphate synthase (EPSPS) with the arginine reagents phenylglyoxal (PGO) and p-hydroxyphenylglyoxal (HPGO) leads to inactivation of the enzyme. Inactivation with HPGO leads to modification of approximately 3 mol of arginine per mole of enzyme. The modification reaction follows pseudo-first-order kinetics with a t1/2 of 1 min at 5 mM p-hydroxyphenylglyoxal in 0.1 M triethanolamine HCl, pH 7.8. By titration of HPGO-modified enzyme with 5,5'-bis(dithio-2-nitrobenzoic acid), the possibility of cysteine modification by the arginine reagent was ruled out. While shikimate 3-phosphate (S3P) afforded partial protection to the enzyme against inactivation by HPGO, complete protection could be obtained by using a mixture of S3P and glyphosate. Under the latter conditions, only 1 mol arginine was modified per mole of enzyme. This pattern of reactivity suggests that two arginines may be involved in the binding of S3P and glyphosate to EPSP synthase. A third reactive arginine appears to be nonessential for EPSPS activity. Labeling of EPSP synthase with [14C]phenylglyoxal, peptic digestion, HPLC mapping, and amino acid sequencing indicate that Arg-28 and Arg-131 are two of the reactive arginines labeled with [14C]PGO.
Asunto(s)
Transferasas Alquil y Aril , Arginina , Transferasas , 3-Fosfoshikimato 1-Carboxiviniltransferasa , Secuencia de Aminoácidos , Fenómenos Químicos , Química , Cromatografía Líquida de Alta Presión , Cinética , Datos de Secuencia Molecular , Fenilglioxal/farmacología , Plantas , Compuestos de Sulfhidrilo , Transferasas/antagonistas & inhibidoresRESUMEN
The cells of higher plants contain distinct subcellular compartments (organelles) that perform specialized functions such as photosynthesis, carbohydrate and lipid metabolism, and so forth. The majority of the protein constituents of plant organelles are formed as cytosolic precursors with N-terminal extensions that direct transport across one or more membrane bilayers in a post- or co-translational fashion. Since the majority of proteins in plant cells are products of nuclear gene expression, there must be precise sorting mechanisms in the cytoplasm that direct proteins to their correct cellular locations. Based on recent studies of protein targeting to chloroplasts and vacuoles, the details of these intracellular sorting mechanisms are becoming clear. The ability to direct proteins to specific compartments within cells provides new opportunities for improvement of plants by genetic manipulation.
RESUMEN
A bacterial phosphonate monoester hydrolase was evaluated in plants as a conditional lethal gene useful for cell ablation and negative selection. Glyphosate is a potent herbicide whereas its phosphonate monoester derivative, glyceryl glyphosate, is approximately 50-fold less active. A phosphonate monoesterase gene (pehA) encoding an enzyme that hydrolyzes phosphonate esters including glyceryl glyphosate to glyphosate and glycerol was cloned from the glyphosate metabolizing bacterium, Burkholderia caryophilli PG2982. Constitutive expression of the pehA gene in Escherichia coli and Arabidopsis thaliana RLD had no observable phenotypic effects on growth and development. However, cells and plants expressing the pehA gene were killed when treated with glyceryl glyphosate. The phytotoxicity resulted from the hydrolysis of glyceryl glyphosate to glyphosate and subsequent inhibition of aromatic amino acid biosynthesis. As an example of tissue-specific cell ablation, floral sterility without vegetative toxicity was demonstrated by expressing the pehA gene using a tapetal-specific promoter and treating the mature plants with glyceryl glyphosate. A chromogenic phosphonate ester substrate, 5-bromo-4-chloro-indolyl phenylphosphonate, was used to monitor in situ expression of the pehA gene. The general utility of the pehA gene as a heterologous conditional lethal gene in plants is discussed.
Asunto(s)
Burkholderia/enzimología , Burkholderia/genética , Genes Letales , Hidrolasas/genética , Plantas/genética , Arabidopsis/genética , Mapeo Cromosómico , Clonación Molecular , Escherichia coli/genética , Genes Bacterianos , Glicina/análogos & derivados , Glicina/metabolismo , Herbicidas/metabolismo , Hidrolasas/metabolismo , Datos de Secuencia Molecular , Fenotipo , Plásmidos/genética , GlifosatoRESUMEN
Synthesis of the phytohormone ethylene is believed to be essential for many plant developmental processes. The control of ripening in climacteric fruits and vegetables is among the best characterized of these processes. One approach to reduce ethylene synthesis in plants is metabolism of its immediate precursor, 1-aminocyclopropane-1-carboxylic acid (ACC). Soil bacteria containing an enzyme, ACC deaminase, were identified by their ability to grow on ACC as a sole nitrogen source. The gene encoding ACC deaminase was cloned and introduced into tomato plants. Reduction in ethylene synthesis in transgenic plants did not cause any apparent vegetative phenotypic abnormalities. However, fruits from these plants exhibited significant delays in ripening, and the mature fruits remained firm for at least 6 weeks longer than the nontransgenic control fruit. These results indicated that ACC deaminase is useful for examining the role of ethylene in many developmental and stress-related processes in plants as well as for extending the shelf life of fruits and vegetables whose ripening is mediated by ethylene.